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1、BACTERIAL ENDOTOXINS TEST USP35Portions of this general chapter have been harmonized with the corresponding texts of theEuropean Pharmacopoeiaand/or theJapanese Pharmacopoeia、 Those portions that are not harmonized are marked with symbols to specify this fact、这一章节已经与欧洲药典(EP)与日本药典(JP)统一。没有统一得部分已经用标记出
2、来。The Bacterial Endotoxins Test (BET) is a test to detect or quantify endotoxins from Gramnegative bacteria using amoebocyte lysate from the horseshoe crab (Limulus polyphemusorTachypleus tridentatus)、细菌内毒素检查法(BET)就是通过鲎得阿米巴细胞溶解产物来检测或定量来自革兰氏阴性菌得内毒素。There are three techniques for this test: the gelclo
3、t technique, which is based on gel formation; the turbidimetric technique, based on the development of turbidity after cleavage of an endogenous substrate; and the chromogenic technique, based on the development of color after cleavage of a synthetic peptidechromogen plex、 Proceed by any of the thre
4、e techniques for the test、 In the event of doubt or dispute, the final decision is made based upon the gelclot technique unless otherwise indicated in the monograph for the product being tested、 The test is carried out in a manner that avoids endotoxin contamination、细菌内毒素检查法有3种:凝胶法,基于凝胶得形成;浊度法,BACTE
5、RIAL ENDOTOXINS TEST USP32Portions of this general chapter have been harmonized with the corresponding texts of the European Pharmacopeia and/or the Japanese Pharmacopeia、 Those portions that are not harmonized are marked with symbols to specify this fact、This chapter provides a test to detect or qu
6、antify bacterial endotoxins that may be present in or on the sample of the article(s) to which the test is applied、 It uses Limulus Amebocyte Lysate (LAL) obtained from the aqueous extracts of circulating amebocytes of horseshoe crab (Limulus polyphemusorTachypleus tridentatus) which has been prepar
7、ed and characterized for use as an LAL Reagent、1There are two types of techniques for this test: the gelclot techniques, which are based on gel formation, and the photometric techniques、 The latter include a turbidimetric method, which is based on the development of turbidity after cleavage of an en
8、dogenous substrate, and a chromogenic method, which is based on the development of color after cleavage of a synthetic peptidechromogen plex、 Proceed by any one of these techniques, unless otherwise indicated in the monograph、 In case of dispute, the final decision is based on the gelclot techniques
9、, unless otherwise indicated in the monograph、In the gelclot techniques, the reaction endpoint is determined from dilutions of the material under test in direct parison with parallel dilutions of a reference endotoxin, and quantities of endotoxin are expressed in USP Endotoxin Units (USPEU)、NOTEOne
10、USPEU is equal to one IU of endotoxin、Because LAL Reagents have been formulated to be used also for turbidimetric or colorimetric tests, such tests may be used to ply with the requirements、 These tests require the establishment of a standard regression curve; the endotoxin content of the test materi
11、al is determined by interpolation from the curve、 The procedures include incubation for a preselected time of reacting endotoxin and control solutions with LAL Reagent and reading of the spectrophotometric light absorbance at suitable wavelengths、 In the endpoint turbidimetric procedure the reading
12、is made immediately at the end of the incubation period、 In the endpoint colorimetric procedure the reaction is arrested at the end of the preselected time by the addition of an enzyme reactionterminating agent prior to the readings、 In the turbidimetric and colorimetric kinetic assays the absorbanc
13、e is measured throughout the reaction period and rate values are determined from those readings、APPARATUS AND GLASSWAREDepyrogenate all glassware and other heatstable materials in a hotair oven using a validated process、2monly used minimum time and temperature settings are 30 minutes at 250、 If empl
14、oying plastic apparatus, such as microplates and pipet tips for automatic pipetters, use only that which has been shown to be free of detectable endotoxin and not to interfere with the test、NOTEIn this chapter, the term “tube” includes any other receptacle such as a microtiter well、PREPARATION OF TH
15、E STANDARD ENDOTOXIN STOCK SOLUTION AND STANDARD SOLUTIONSTheUSP Endotoxin RShas a defined potency of 10,000 USP Endotoxin Units (EU) per vial、 Constitute the entire contents of 1 vial of the RSE with 5 mL of LAL Reagent Water3, mix intermittently for 30 minutes, using a vortex mixer, and use this c
16、oncentrate for making appropriate serial dilutions、 Preserve the concentrate in a refrigerator for making subsequent dilutions for not more than 14 days、 Mix vigorously, using a vortex mixer, for not less than 3 minutes before use、 Mix each dilution for not less than 30 seconds before proceeding to make the next dilution、 Do not store dilutions, because of loss of activity by adsorption, in the absence of supporting data to the contr
