Determination of endotoxin in injectable antibiotic preparations by the chromogenic assay method using a Limulus reagent (Tachypleus hemocyte lysate) and a chromogenic substrate
Determination of endotoxin in injectable antibiotic preparations by the chromogenic assay method using a Limulus reagent (Tachypleus hemocyte lysate) and a chromogenic substrate
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使用鲎试剂（鲎血细胞裂解液）和显色底物显色测定法测定注射用抗生素制剂中的内毒素
DOI：
10.1128/jcm.23.1.11-16.1986
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发表时间：
1986
影响因子：
9.4
通讯作者：
S. Takahashi
中科院分区：
文献类型：
--
作者：
S. Yano;Y. Hotta;S. Takahashi
本文研究了50种抗生素对细菌内毒素检测的影响，并测定了细菌内毒素(E.coli0111：B4)浓度为0.5 ng/ml时，细菌内毒素的50%抑制浓度。所有的抗生素制剂在接受测试之前都经过了超滤处理来脱热。反应在高浓度(0.5M)的Tris缓冲液中进行，以保持反应混合物的pH值，并用高压液相色谱仪测定释放出来的对硝基苯胺。几种氨基糖苷类抗生素(丁胺卡那霉素、贝卡那霉素、卡那霉素和硫酸链霉素)、盐酸博莱霉素和磷霉素二钠在20 mg/ml以下对该反应无抑制作用，而青霉素类、头孢菌素类、大环内酯类和四环素类抗生素则呈浓度依赖性地抑制该反应。硫酸多粘菌素B的抑制作用最强，50%的抑制率小于8微克/毫升。结论：发色法可用于大多数抗生素制剂中内毒素的检测。并举例说明了该方法在甲苯西林二钠制剂中的应用。
The effects of 50 antibiotics on the detection and determination of bacterial endotoxins by the chromogenic method using a Limulus reagent (Tachypleus hemocyte lysate) and a chromogenic substrate of p-nitroaniline derivatives were tested, and the antibiotic concentration for 50% inhibition of the chromogenic reaction in the presence of 0.5 ng of endotoxin (Escherichia coli 0111:B4) per ml was estimated. All the antibiotic preparations were depyrogenized by ultrafiltration treatment before they were subjected to the test. The reaction was conducted in the presence of a high concentration (0.5 M) of Tris buffer to constantly maintain the pH of the reaction mixture, and liberated p-nitroaniline was determined by high-pressure liquid chromatography. Several aminoglycosides (amikacin, bekanamycin, kanamycin, and streptomycin sulfate), bleomycin hydrochloride, and fosfomycin disodium showed no inhibition of the reaction up to 20 mg/ml. However, other antibiotics, including penicillins, cephalosporins, macrolides, and tetracyclines, inhibited the reaction concentration dependently. Polymyxin B sulfate was the most potent inhibitor, with less than 8 micrograms/ml for 50% inhibition. It was concluded that the chromogenic method can be applied to the detection and determination of endotoxin in most of the antibiotic preparations. An application of this method to carbenicillin disodium preparations was exemplified.