1, reaction temperature 37℃ 1℃, time 60min 2min. The reaction time and temperature must be strictly controlled, otherwise wrong conclusions will be drawn. If the reaction temperature or time is insufficient, false positive will appear; At the same time, if the reaction temperature or time exceeds the requirements of pharmacopoeia, false negative will appear.
2. According to the Pharmacopoeia, the test should be carried out in the environment of 100 and in the area of 100. However, the requirements for laboratory temperature are not clearly specified under the inspection item, but according to Item 4 of Article 30 of China Pharmacopoeia, unless otherwise specified, 25℃ 2℃ shall prevail.
Personally, it is not necessary to control the test environment in the local area of 100 in the environment of 100, because this environment is easy to pollute endotoxin (reason: the polluted endotoxin solution in the previous test is easy to blow out from the tuyere after volatilization). But the laboratory temperature should be controlled below 20℃, which is an enzymatic reaction, and the temperature has a great influence on it.
3. The pH value of the test solution, the ion concentration, the sensitivity of the limulus reagent and the operating level of the operator will all affect the test results.
Therefore, China Pharmacopoeia (version 20 15) began to modify the "bacterial endotoxin test method" greatly:
① Test the sensitivity of limulus lysate. The purpose of this test is not only to check whether the sensitivity of limulus lysate is accurate, but also to check whether the operation method of inspectors is correct and whether the experimental conditions meet the requirements.
(2) "interference test", that is, when the new drug has "quality standard" or the raw material source, formula and production process of the production enterprise change, interference test should be carried out; The method of eliminating interference generally adopts BET water dilution or adding acid and alkali to adjust pH to eliminate interference. Its purpose is to confirm that the test sample has no interference with the reaction between limulus lysate and bacterial endotoxin under the test concentration and conditions.
③ When doing "bacterial endotoxin test" at ordinary times, do it alone: 2 tubes of samples; PPC2 tube; Positive control 2 tubes; Negative control 2 test tubes. It is also stipulated that the experiment can only be considered effective if the positive control tube and PPC tube are positive and the negative control tube is negative. Otherwise, the experimental results are invalid.
Reason: If the positive control tube is not positive, = = = The most likely reason for this problem lies in the first step "Preparation of bacterial endotoxin standard solution". Adding BET water to endotoxin standard or working standard is a key step, so it must be operated according to Pharmacopoeia, that is, after adding BET water, it should be oscillated on an oscillator for 15min, otherwise the endotoxin will not be mixed evenly. Secondly, after replacing the limulus lysate or BET water from different batches or manufacturers, the sensitivity test of limulus lysate must be done, otherwise we don't know whether the sensitivity of this batch of limulus lysate and the water for inspection meets the requirements. -
-The negative control tube is not negative, indicating that the BET water is polluted.
-PPC tube should be positive, but not positive, which means that the test article or its diluent will interfere with the test under the test conditions.
If you are interested in the bacterial endotoxin test, you can use "Baidu" to search my articles about bacterial endotoxin test published in professional magazines such as China Pharmacist, China Pharmacy and strait pharmaceutical journal Magazine in recent years. This paper discusses the establishment of bacterial endotoxin test for Delisheng injection, piracetam, vinpocetine and glucose injection, dexamethasone sodium phosphate, ribavirin and the interference of bacterial endotoxin test for vitamin C injection. In order to deepen the understanding of this issue.