Establishment And Application Of Techniques Of Bacterial Endotoxins Test For Injection Of Veterinary Drugs

Pyrogens are a group of chemically diverse substances that inducing hyperthermia in humans and animals. The most important pyrogenic substances in pharmaceutical industry are bacterial endotoxins. Bacterial endotoxins are part of the cell wall of gram negative bacteria. Their pyrogenic activity is much higher than that of most other pyrogenic substances. These endotoxins are lipopolysaaccharides. Although there are small number of pyrogen which possess a different structure, the conclusion is generally justified that the absence of bacterial endotoxinsin a product implies the absence of pyrogenic substances, provided the presence of non-endotoxin pyrogenic substances can be ruled out.The aim in this paper is to study and apply the leading Pharmacopoeia of the international harmonization of Bacterial Endotoxins Test for detecting bacterial endotoxins in veterinary drugs injection of China.There are two methods of detection: the Pyrogen test is based upon the measurement of body temperature of rabbits before and after injection of the specimen, the Limulus test is based upon the clotting reaction of an enzyme complex of cells of the horseshoe crab together with bacterial endotoxins(in vitro test). The Limulus test, which has been adopted as the Test for Bacterial Endotoxins in the Pharmacopeias, can detect or quantitate endotoxins of Gram-negative bacterial origin using blood corpuscle extracts(Limulus amebocyte lysate, LAL) of horseshoe crabs(Limulus polyphemus, Tachypleus tridentatus, etc.). It may be conducted by the gel-clot or spectrophotometric(turbidimetric and colorimetric) techniques, the former being based on gel formation due to the activation of LAL by endotoxins. The turbidimetric technique is based on the LAL turbidity change during the gel formation and the colorimetric technique on activation of peptide hydrolytic enzymes in LAL. The Limulus test has been officially utilized as a simple and highly sensitive method for the determination of endotoxins in drugs in lieu of the in vivo Pyrogen Test using rabbits. For the Bacterial Endotoxins Test of Pharmacopeias, the gel-clot technique and spectrophotometric techniques were adopted. In this paper, techniques of the application of the endotoxin test for veterinary drugs injection were investigated by two different limulus amebocyte lysate(LAL) test methods, gel-clot and kinetic turbidimetric methods, using two endotoxin-specific reagents. Although most veterinary drugs injection show inhibition or enhancement in practice, this test can be generally conducted by eliminating interfering effects through dilution of specimens by a factor not exceeding the maximum valid dilution(MVD) with water. Since MVD is dependent on the sensitivity of applied methodology, the turbidimetric, which is more sensitive than the gel-clot technique, has a distinct advantage. According to accuracy evaluation of test methods, the kinetic-turbidimetric and gel-clot assays were shown to allow more accurate measurement and, therefore, more sensitive detection of interference to the bacterial endotoxin test than the Pyrogen test. In total, four products were evaluated on their interfering effect. Although the test was shown to be applicable even to the group showing the strongest interference, it was assumed to be crucial to use appropriate reagents and an accurate test method for avoiding approval of a pyrogenic product. Based on our results, we can conclude that the LAL assay is a senstitve and specific assay for the detection of bacterial endotoxins from gram negative bacteria in veterinary drugs injection. A comparison of the Pyrogen test also indicates that the Limulus test is more senstitve and accurate.