【作者】 岳丽娜；

【导师】 贺高红；

【作者基本信息】 大连理工大学 ， 化学工程， 2005， 硕士

【摘要】 细菌内毒素是革兰氏阴性菌细胞壁外膜的脂多糖成分,能导致人体发热、休克甚至死亡。内毒素能与鲎试剂发生特异性的反应而被检测,主要的检测方法有:凝胶法、浊度法和比色法。其中凝胶检测法具有检测速度快、特异性好、操作方便等优点,被各国药典收录。并规定当各种内毒素检测方法得出的检测结果不一致时,以凝胶法为仲裁方法。然而凝胶法存在一些缺欠,如:结果判断有主观性影响,灵敏度差。为解决这些问题,论文考察了不同浓度的内毒素与鲎试剂在试管中的反应,采用传统的凝胶法观察凝胶的形成情况,同时在BET-32C内毒素测定仪上采用动态浊度法检测其浊度(由吸光度表示又称光密度,OD)值,建立两者之间的关系。凝胶形成时凝胶的OD值为0. 067±0. 012。我们选择下限0. 055为凝胶形成的临界值,将OD临界值作为凝胶检测的指标,采用内毒素检测仪器代替目视法观察凝胶的形成情况,建立了半自动凝胶检测法。使用19种鲎试剂对该临界OD值进行验证,其中18种鲎试剂的结果与药典规定的鲎试剂复核试验结果一致。此外,对克林霉素磷酸注射液和头孢拉定注射液(共7个批号)分别用传统的凝胶法和本论文提出的半自动凝胶检测法进行内毒素限量的测定对比,结果一致。在常规内毒素检测中,需要做药品阳性对照实验,目的在于证明被检查的供试品对检测不存在抑制因素。在检测克林霉素磷酸注射液中的内毒素时经常出现药品阳性对照试管假阴性结果的问题。本论文采用半自动凝胶检测法,对这一问题进行了研究,结果发现使用灵敏度0. 125EU/ml的鲎试剂,药品干扰仍然存在。稀释药品,使用更高灵敏度的鲎试剂可以解决这一问题。在使用浊度法检测内毒素时经常出现标准曲线斜率变化大的现象。为此论文研究了标准品的菌种纯度问题。采用气相色谱质谱联用法,对内毒素标准品和大肠杆菌、绿脓杆菌、环境内毒素样品中3-羟基脂肪酸种类进行了比较,结果显示,20EU/支的标准品中含有3-羟基十二烷酸,即其含有非大肠杆菌的细菌来源。这与药典规定不符。更多还原

【Abstract】 Endotoxins are lipopolysaccharides that form the outer cell membrane of gram-negative bacteria. Endotoxin contaminating pharmaceuticals can be estimated by the bacterial endotoxins test using Limulus Amebocyte Lysate (LAL) or Tachypleus Amebocyte Lysate (TAL). The traditional detecting methods involve gel-clot method, turbidimetric assay and colorimetric assay. The gel-clot assay is rapid, specific, easily to perform and it is the referee when there is an endotoxin content inconsistency between different endotoxin assays. But unfortumatly, it has some Limitations including the subjective endpoint and the relative lack of sensitivity. In order to overcome these limitations various concentrations of endotoxin mixed with TALs were monitored by the kinetic turbidimetric assay and observed by traditional gel-clot assay. The relevant turbidimetric values and the gel-clot formations while the reaction finished were recorded and compared. The turbidimetric value when the gel-clot formed was 0.067±0.012. The lower limit 0.055 was set as the critical turbidimetric value and defined as the criteria of the semiautomatic monitoring method. Nineteen kinds of TALs were tested to validate the critical value and eighteen of them coincided with the ideal situation of gel-clot formation according to the pharmacopoeia. Moreover clindamycin phosphate and cefradine for injection were tested to validate the semiautomatic monitoring method and the results of them coincided with the traditional gel-clot assay.When clindamycin phosphate was tested in routing assay, the result of PPC was always false negative. TALs with various sensitivities were used to perform the semiautomatic monitoring method for gel-clot endotoxin assay.And it is found that the problem of false negative can be avoided by using TALs with the sensitivity above 0.125EU/ml.To analyse the impurity of bacterium source of standard endotoxin 3-hydroxy fatty acid species in different endotoxin standards was determined by gas chromatography mass spectrometry. And the 3-hydroxy fatty acids in national standard endotoxins, escherichia coli, pseudomonas aerugjnosa and deionized water were compared to discuss the purity of bacterium sources of the standard endotoxin. It’s shown that there was 3-hydroxydodecanoic acid in 20 EU working standard endotoxin, which indicated the impurity of bacterium source.更多还原