Study On Bacterial Endotoxin Test

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.