Yano S, Hotta Y, Takahashi S
J Clin Microbiol. 1986 Jan;23(1):11-6. doi: 10.1128/jcm.23.1.11-16.1986.
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.
测试了50种抗生素对使用鲎试剂（中国鲎血细胞裂解物）和对硝基苯胺衍生物显色底物的显色法检测和测定细菌内毒素的影响，并估算了在每毫升含有0.5 ng内毒素（大肠杆菌0111:B4）的情况下使显色反应抑制50%的抗生素浓度。所有抗生素制剂在进行测试前均通过超滤处理进行去热原处理。反应在高浓度（0.5 M）的Tris缓冲液存在下进行，以持续维持反应混合物的pH值，释放的对硝基苯胺通过高压液相色谱法测定。几种氨基糖苷类抗生素（阿米卡星、贝卡霉素、卡那霉素和硫酸链霉素）、盐酸博来霉素和磷霉素二钠在浓度高达20 mg/ml时均未显示对反应的抑制作用。然而，其他抗生素，包括青霉素类、头孢菌素类、大环内酯类和四环素类，对反应的抑制作用呈浓度依赖性。硫酸多粘菌素B是最有效的抑制剂，50%抑制浓度低于8微克/毫升。得出的结论是，显色法可应用于大多数抗生素制剂中内毒素的检测和测定。举例说明了该方法在羧苄西林二钠制剂中的应用。
