细菌内毒素USP上课讲义

BACTERIAL ENDOTOXINS TEST <85USP35

*Porti ons of this gen eral chapter have bee n harm oni zed with the corresp onding texts of

the European Pharmacopoeia and/or the Japanese Pharmacopoeia. Those portions that are not harmonized are marked with symbols ( **) to specify this fact. +

这一章节已经和欧洲药典(EP)与日本药典(JP)统一。没有统一的部分已经用**标记出来。

The Bacterial En dotox ins Test (BET) is a test to detect or qua ntify en dotox ins from Gram-n egative bacteria using amoebocyte lysate from the horseshoe crab (Limulus polyphemus or Tachypleus tride ntatus).

细菌内毒素检查法(BET)是通过鲎的阿米巴细胞溶解产物来检测或定量来自革兰氏阴性菌的内毒素。

There are three tech niq ues for this test: the gel-clot tech niq ue, which is based on gel formati on; the turbidimetric tech niq ue, based on the developme nt of turbidity after cleavage of an en doge nous substrate; and the chromoge nic tech niq ue, based on the developme nt of color after cleavage of a syn thetic peptide-chromoge n complex. Proceed by any of the three tech niq ues for the test. In the eve nt of doubt or dispute, the final decisi on is made based upon the gel-clot tech nique uni ess otherwise in dicated in the mono graph for the product being tested. The test is carried out in a manner that avoids en dotox in con tam in ati on.

细菌内毒素检查法有3种：凝胶法，基于凝胶的形成；浊度法，

BACTERIAL ENDOTOXINS TESTUSP32

^Portions of this gen eral chapter have bee n harm oni zed with the corresp onding texts of the Europea n Pharmacopeia an d/or the Japa nese Pharmacopeia. Those portions that are not harm oni zed are marked with symbols (*■♦■) to specify this fact. +

This chapter provides a test to detect or qua ntify bacterial en dotox ins that may be prese nt in or on the sample of the article(s) to which the test is applied. It uses Limulus Amebocyte Lysate (LAL) obta ined from the aqueous extracts of circulati ng amebocytes of horseshoe crab ( Limulus polyphemus or Tachypleus tridentatus ) which has been prepared and characterized for use as an LAL Reage nt 余1+

There are two types of tech niq ues for this test: the gel-clot tech niq ues, which are based on gel formation, and the photometric techniques. The latter include a turbidimetric method, which is based on the developme nt of turbidity after cleavage of an en doge nous substrate, and a chromoge nic method, which is based on the developme nt of color after cleavage of a syn thetic peptide-chromoge n complex. Proceed by any one of these tech niq ues, uni ess otherwise in dicated in the mono graph. In case of dispute, the final decisi on is based on the gel-clot tech niq ues, uni ess otherwise in dicated in the mono graph.

In the gel-clot tech niq ues, the react ion en dpo int is determ ined from diluti ons of the material un der test i n direct comparis on with parallel diluti ons of a reference en dotox in, and qua ntities of endotoxin are expressed in USP Endotoxin Units (USP-EU). [NOTE—One USP-EU is equal to one IU of en dotox in.]

Because LAL Reage nts have bee n formulated to be used also for turbidimetric or colorimetric tests, such tests may be used to comply with the requireme nts. These tests require the establishme nt of a sta ndard regressi on curve; the en dotox in content of the test material is determ ined by in terpolati on from the curve. The procedures in clude in cubati on for a preselected time of react ing en dotox in and con trol soluti ons with LAL Reage nt and readi ng of the spectrophotometric light absorba nee at suitable wavele ngths. In the en dpo int turbidimetric procedure the reading is made immediately at the end of the incubation period. In the endpoint colorimetric procedure the react ion is arrested at the end of the preselected time by the additi on of an en zyme react ion-term in at ing age nt prior to the readi ngs. In the turbidimetric and colorimetric kin etic assays the absorba nee is measured throughout the react ion period and rate values are determ ined from those read in gs.

APPARATUS AND GLASSWARE

Depyrogenate all glassware and other heat-stable materials in a hot-air oven using a validated * 口process. 稣Commonly used minimum time and temperature settings are 30 minutes at 250 .

If employing plastic apparatus, such as microplates and pipet tips for automatic pipetters, use only that which has bee n show n to be free of detectable en dotox in and not to in terfere with the