Endotoxins are small, hydrophobic molecules that are part of the lipopolysaccharide complex that forms most of the outer membrane of Gram-negative bacteria. They are released when the bacteria die and their outer membranes disintegrate, triggering the toxicity mechanism of the fractionated lipopolysaccharides. To ensure patient safety, Endotoxin testing must be carried out, not only on end-product medical devices or injectables, but also on the raw materials that are used to make them.
Failure to detect endotoxins can have harmful, or even fatal, effects. When bacteria and endotoxin enter the bloodstream, they trigger an immune response, usually inflammation, the release of cytokines such as interleukin 1, and the production of tumor necrosis factor. The release of cytokine signals cause neutrophils to migrate toward the point of infection. This migration usually leads to phagocytosis of all associated organisms and proteins. When the host’s immune system is weak, or a high level of infection is encountered, the bacteria can cause sepsis and its associated risks.
The importance of Endotoxin Testing is clear when looking at how susceptible and sensitive humans are to even Minute amounts of endotoxin. Endotoxins may be introduced into the bloodstream through contaminated intravenous devices or medications. Exposure to the compounds results in fever, inflammation, and endotoxemia. When left untreated, it has the potential to cause sepsis (5), resulting in septic shock, which is responsible for 44,000 deaths in the United Kingdom (6) and approximately 200,000 deaths in the US (7) each year.
Ensuring that equipment and medication are free from endotoxin is particularly important when caring for vulnerable patients, including the elderly, those in intensive care, and infants. For example, sudden infant death syndrome (SIDS) has been linked to varying levels of endotoxin present in the bloodstream .
Endotoxin testing methods require interpretation by skilled laboratory technicians. Here, a technician performs an assay in the lab.
It has also been shown that endotoxin exposure can damage nerve axons directly or indirectly (9), suggesting that it is an essential factor in the pathogenesis of critical illness polyneuropathy (CIP) in sepsis.
This article describes and compares the Gel clot method used to detect bacterial endotoxins in sterile raw materials.
AMOXYCILLIN SODIUM RAW MATEIAL BET TEST
Reconstitution of Control Standard Endotoxin(CSE)
Add recommended volume of LRW to the vial of CSE. Record the date of reconstitution on the vial. Vortex the vial for 30 minutes with the help of vortex mixer. Check the Endotoxin Concentration on the certificate of analysis and label the final concentration. Store the reconstituted Endotoxin in this vial at 2-80C for up to 28 days in the refrigerator. Each day that the Endotoxin standard to be used, vortex the vial for 3 minutes.
Reconstitution of Lysate:
Pipette Recommended volume of LRW to the vial of Lysate. Record the date of reconstitution on the vial. Mix gently to dissolve Lysate avoid foaming. Check the compatibility Certificate of analysis of CSE and Lysate.
Reconstituted lysate should be used within 24 hours or else discard it.
The maximum valid dilution(MVD) is the maximum allowable dilution of a sample at which the Endotoxin limit can be determined.
The determination of MVD of the amoxicillin sodium sterile. The general equation to determine MVD is
MVD= Endotoxin Limit x Concentration / Lysate Sensitivity
Where:
Endotoxin Limit of Amoxycillin Sodium Sterile = 0.25 EU/mg
Concentration of Stock Solution= 100mg/ml
Lysate Sensitivity (λ)= 0.125 EU/ml
Now MVD= 0.25 EU/ml x 100 mg/ml / 0.125 EU/ml
= 200
MVD/2= 100
MVD/4=50
Preparation of MVD dilutions of the Product:
New weight 100mg of sample which is required to make stock solution in mg per ml. Transfer it into a dehydrogenated dilution tube and add 1 ml of LRW in it and vortex and mark it as A (100mg/ml)
Now take 900µl of LRW in dilution tube and add 100µl solution from tube A and vortex for 2 minutes and mark it as B.
Now take 800µl of LRW in dilution tube and add 200µl solution from tube B and vortex for 2 minutes and mark it as C. This is MVD/4 dilution.
Preparation of CSE dilutions:
Take the reconstituted CSE and vortex it for not less than 3 minutes. Now serial dilute it to 4 λ by using LRW as diluents in dehydrogenated dilution tubes. Each dilution should be mixed for not less than 30 seconds before proceeding to make the next dilution.
Consider the reconstituted CSE strength = 10 EU/ml
4 λ = 4 x 0.125 EU/ml(lysate sensitivity) =0.5 EU/ml
Dilution factor = 10 EU/ml // 0.5 EU/ml = 20
Then add 100µl CSE and 1900µl LRW - 4 λ dilution
Performance of LAL Gel Clot Test :
Prepare and label the 10x75mm pyrogen-free test tubes as below. All solutions must be vortex for 15 seconds prior to pipetting into test tubes. Pipette LRW, CSE(4 λ), and product solution diluted at MVD/4 and Lysate( λ) in the assay tubes as given below.
Mix the content of individual tube gently and place in a heating block which is switched on 30 minutes prior to test to gain 37±10C temperature. Incubate the reaction mixture in all the tubes for a constant period usually at 37±10C for 60±2 minutes, avoiding vibrations.
Interpretation of results;
After incubation period got over invert it through 1800in one smooth motion. If a firm gel formed that remains in place up on inversion, record the results as positive.
A result is negative if an intact gel is not formed. Results should be match with following table for passing the product under test for BET.
Blank always should be negative, positive control and PPC always be positive for a test to be valid.
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