Test description

 

We are using LIMULUS AMEBOCYTE LYSATE ENDOSAFE ENDOCHROME-K test (LAL reagent) manufactured by Charles River Lab Inc to determine the level of endotoxin. The test is based on the reaction of endotoxins with lysate of amebocytes from American horseshoe crab (Limulus Polyphemus). During manufacturing of LAL reagent, a colorless substrate is added to amebocytes lysate followed by lyophilization. Reconstituted LAL reagent is mixed with the sample containing endotoxins. In the presence of endotoxin, the enzymatic reaction will cause a yellow color to develop upon cleavage of the chromophore, p-nitroaniline (pNA). The liberation of chromophore can be measured spectrophotometrically. The time required before the appearance of color is inversely proportional to the amount of endotoxin present. The concentration of endotoxin in the sample is then calculated from a standard curve. The standard curve is constructed using Control Standard Endotoxin made from E.coli strain 055: B5.

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Sample collection and preparation for analysis

 

All materials or diluents coming in contact with specimen must be endotoxin-free. Use aseptic technique at all times. Since the LAL-endotoxin reaction is pH dependent, the sample should be at pH of 6.5 to 8.0.

 

Sample interference

 

A test method must be validated for each sample by demonstrating the absence of significant interference. Inhibition is usually concentration dependent and is overcome by dilution (typically with endotoxin-free water). There are two common sources of inhibition. One is interference with the enzyme reaction. And another is the alteration of the dispersion of the endotoxin.

Samples containing ß-glucans (food, eatables, plant, mold, carbohydrates, mold, starch, plant material, food) interfere with the assay resulting in reporting of erroneously high endotoxin level. Please inform us if your sample contains carbohydrates and/or glucan-like substance. The interference caused by glucans can be blocked using an endotoxin-specific buffer.

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Maximum Valid Dilution

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The U.S. Food and Drug Administration has established endotoxin limits of 5 EU/kg for intravenous drugs and 0.2 EU/kg for intrathecal drugs. Specific limits for compendial items also have been adopted. These limits may be used to determine the extent of dilution that may be used to overcome an interference problem without exceeding the limit endotoxin concentration. The Maximum Valid Dilution (MVD) is calculated by the following equation:

 

MVD = (Endotoxin Limit x Product Potency) / λ

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where λ is the lowest point on the standard curve.

For example, the limit for a substance is 0.17 EU/mg. If a standard curve with the lowest level of 0.05 EU/mL of endotoxin is used to test this product, where the potency is 20 mg/mL, the MVD equals 1:68. Thus, the sample may be diluted up to 1:68 to resolve potential inhibition.