One of the problems with the Limulus test is the effect of the sample itself on the measurement. For example, observed endotoxin levels may be abnormally lower (inhibition) or higher (enhancement) than expected. Although the effect of a sample on the Limulus test is complicated and difficult to untangle, it can be categorized broadly based on how the measurement is impacted. A sample can alter the activity of the LAL reagent, the physical properties of endotoxin molecules, and other parts of the measurement.

Below we describe some sample components and characteristics which may influence measurements.

1. Sample components which alter LAL reagent activity:
   Proteases, Protease inhibitors, chelating agents, protein-denaturing agents, surface active agents (surfactants), and heavy metals
2. Sample components which affect the physical properties of endotoxin molecules:
   Metal ions (iron, aluminum etc.), surfactants, etc.
3. Sample characteristics which impact the measurement in ways other than the main reaction:
   Turbidity, color, sulfa agents (for the synthetic substrate method with diazo coupling reaction) etc.

High concentrations of salt and sugar are also known to have inhibitory effects on the Limulus test. Also, Limulus reagent activation is caused by enzyme reactions, thus pH and temperature also have effects on the measurement.

The Japanese pharmacopoeia¹⁾ says that "sample solutions must be confirmed in advance for inhibitory or enhancing properties" but does not mention how to confirm these. Here, the United States Pharmacopoeia²⁾ and FDA Guidelines³⁾ which are used in the validation of the Limulus test could provide useful information. The basic idea mentioned in these guidelines is that tests should be carried out only after searching for and identifying conditions which could prevent any inhibition or enhancement caused by the sample.

According to the inhibition and enhancement test by gel-clot Technique in the United States Pharmacopoeia, the measurement is valid if the gelation endpoint of the sample solution spiked with endotoxin is within a two-fold dilution of the labeled sensitivity of LAL. According to the FDA Guideline³⁾ for the kinetic turbidimetric assay and the synthetic substrate methods, basic criteria for confirming validity of measurements are calibration curve linearity and a sample spike recovery test result of 100±25% (if the sample is spiked with a concentration 4 times larger than the minimum concentration of the calibration curve). A more detailed explanation of these methods will be introduced later.

The most commonly used method to eliminate sample interference in a measurement is dilution. Dilution is able to eliminate the effect of the sample, but may raise the endotoxin detection limit along with the increased dilution ratio. For products with a regulated endotoxin limit, it is necessary to limit the dilution ratio in order to detect the required limit of endotoxin concentration.

The maximum dilution ratio allowed for detection of the endotoxin limit is called the Maximum Valid Dilution (MVD) and is regulated in the United States Pharmacopoeia²⁾. MVD can be calculated by the allowable limit of endotoxin for each product, and the sensitivity of the LAL reagent used in each measurement method. The test product cannot be diluted above the MVD.

There are other methods such as chromatography and ultra-filtration which may eliminate the effects of the sample, but conditions must be set for each sample in order to apply these methods, and therefore these methods are not used often except in special cases.

References

1. The Japanese Pharmacopoeia, 12th edition, General Tests, "7. Endotoxins Test"
2. The United States Pharamacopeia 22th, The National Formulary 17th, p.1493-1495, Pharamacopeia Convention Inc., MD (1989).
3. Guideline on validation of the Limulus amebocyte lysate test as an end-product endotoxin test for human and animal parenteral drugs, biological products, and medical devices, Food and Drug Adm. (1987).