When using the Limulus test to measure the concentration of endotoxin in a drug product, the product normally contains factors which could interfere with the assay. Dilution is the most common method used to eliminate these interferences. If dilutions are performed without limit the interference effect can be eliminated, but a smaller amount of endotoxin will not be detected with higher dilution.

Regulatory guidelines dictate the maximum limit for endotoxin; therefore, it is not appropriate to dilute samples past a certain point to determine whether they have passed the regulated levels. The Maximum Valid Dilution (MVD) is the limit on the dilution factor for a product and is described in United States Pharmacopoeia¹⁾ and FDA Guidelines²⁾. The MVD is a value determined by the endotoxin detection limit（λ) for a particular measurement method, and the permissible concentration of endotoxin in a sample.

The endotoxin limit for a diluted sample is equal to the endotoxin limit of the undiluted sample divided by the dilution factor. The actual endotoxin concentration in an undiluted sample is calculated by multiplying the measured concentration of endotoxin in the diluted sample by the dilution factor. The dilution factor that can detect the endotoxin limit of undiluted sample can therefore be calculated by dividing the endotoxin limit of the undiluted sample by λ. This dilution factor is the MVD - the maximum dilution factor at which regulated endotoxin contamination can still be detected in a diluted sample.

In order to calculate the MVD, the following factors must be known:

1. The endotoxin detection limit of the employed method（λ）
2. The maximum allowable endotoxin concentration for the product being tested

The endotoxin detection limit（λ）for a method varies depending on the measurement technique. For the gel-clot technique, the labeled sensitivity of the LAL reagent is considered as λ. For the kinetic turbidimetric assay and the synthetic substrate method, λ is the smallest concentration in the standard curve.

FDA Guidelines²⁾ list the drugs which have assigned endotoxin limits, and the MVD for those drugs can be calculated using the following formula:

MVD = [(Endotoxin Limit) × (Potency of Product)] / λ

Here, the Potency of Product refers to the initial product concentration, before dilution. The units are denoted as mg/mL or unit/mL when the product concentration is the mass administered per unit of body mass, while mL/mL is used when volume is used.

For products with no assigned Endotoxin Limit, the minimum sample concentration at the time of measurement (Minimum Valid Concentration; MVC) is calculated using the following formula:

MVC = λM/K

M stands for the maximum dose administered during the rabbit pyrogen test, or the administered dose per kg body weight for humans（dose/kg assumes an average weight of 70kg). K is set at 5.0 EU/kg for parenteral drugs.

Given this, MVD may be calculated as follows:

MVD = Concentration of Product / MVC

The Japanese Pharmacopoeia³⁾ comments on the effect of samples on the measurement by saying that "sample solutions must be checked beforehand for any enhancement or inhibitory effects" but it does not mention how to check for these, nor does it state anything related to the MVD. Currently the endotoxins test is applied not only to water for injection, but also to various drugs for which MVD is now calculated.

Reference

1. The United States Pharmacopeia 22th, the National Formulary 17th. P.1493-1495, Pharmacopeial Convention Inc., MD (1989).
2. 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).
3. The Pharmacopoeia of Japan, Twelfth Edition (第十二改正日本薬局方),23-24 (1991).