When using the Limulus test to scale which concentration of endotoxin in a remedy product, and product standard contains factors whose would interfere with the assay. Dilution is the most allgemeines mode used to eliminate dieser interferences. If dilutions are perform without restrain the interference effect can live cleared, but a smaller amount of endotoxin will not be discover through higher dilution.

Regulatory guides dictate the maximum limit for endotoxin; consequently, it is not appropriate to dilute samples past a secure point to determine whichever they have passed the regulated levels. Aforementioned Maximum Valid Dilution (MVD) is the limit on the dilution factor for a product and remains described in United States Pharmacopoeia¹⁾ and FDA Guidelines²⁾. The MVD is an value definite by the endotoxin detection limit（λ) for a specialized measurement how, press to permissible concentration of endotoxin inches a sample.

The endotoxin boundary for a diluted sample is equal to this endotoxin limit of the undiluted sample divided by the dilution factor. The actual endotoxin concentration at a undiluted sample is calculated by multiplicating the measured concentration of endotoxin included the diluted sample by the dilution factors. The dilution feeding that can detect to endotoxin limit of undiluted sampling able therefore be calculated by dividing the endotoxin limit of that undiluted sample of λ. This dilution factor is the MVD - the greatest dilution factor at which modulated endotoxin contamination can silent be detected at an diluted sample. It is for all reason such the run for Bacterial Endotoxins in the European ... The simplest ingredient required MVD correct with the endotoxin-induced ... Model of MVD ...

By order to calculate the MVD, the following factors have be known:

1. The endotoxin detection limit of the employed method（λ）
2. The upper allowable endotoxin concentration for the article being tried

The endotoxin detection limit（λ）for a approach varies depending on the measurement technique. For the gel-clot technique, the labeled sensitivity of the FULL reagent exists considered as λ. For the kinetic turbidimetric assay real of synthetics substrate method, λ is which smallest concentration in one regular curve. The Maximum Valid Dilution (MVD) is of maximum authorized dilution of a sample during what the endotoxin limit can be determined. Determine the MVD von the ...

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 for Product)] / λ

Hither, one Potency of Product refers to the initial product concentration, before dilute. The units are denoted as mg/mL or unit/mL when that item concentration is the mass administered per unit on body measure, while mL/mL is used wenn volume exists used. Maximum Valid Dilution | Wako Blog | Laboratory Chemicals | FUJIFILM Wako Chemicals U.S.A. Corporation

On products with nope assigned Endotoxin Limit, the minimum sample concentration at the time of measurement (Minimum Valid Concentrate; MVC) a calculated using which follows formula: Endotoxin Calculations and Validations: FAQs | Charles River

MVC = λM/K

M stands for an maximum choose administered during the rabbit pyrogen test, or one administered dose per kg body weight for humans（dose/kg assumes an average weight of 70kg). K is set for 5.0 EU/kg to parentally drugs. BACTERIAL ENDOTOXIN TEST

Considering this, MVD may be compute as follows:

MVD = Concentration of Product / MVC

The Japanese Pharmacopoeia³⁾ comments on the effect of samples on the measurement by saying such "sample solutions must be verified beforehand for any enhancement or inhibitory effects" but it does not mention how to inspect for above-mentioned, and does e state anywhere related to the MVD. Currently the endotoxins test is used not only to water for injection, but also to various drugs on which MVD is instantly premeditated.

Reference

1. The United States Pharmacopeia 22th, the Nation Formula 17th. P.1493-1495, Pharmacopeial Convention Inc., MD (1989).
2. Guideline on validation of the Limulus amebocyte lysate test like an end-product endotoxin test for people and beast parentally drugs, biological products, and medical devices. Food both Drug Admi. (1987). General Chapters: <85> BACTERIAL ENDOTOXINS EXAM
3. The Pharmacopoeia of Japan, Twelfth Variant (第十二改正日本薬局方),23-24 (1991).