Bacterial endotoxin test
The bacterial endotoxin test is to determine whether the bacterial endotoxin in the test article meets the requirements. Endotoxin is a lipopolysaccharide of the cell wall of Gram-negative bacteria, and its toxic component is lipid A. The bacteria died after disintegration. Bacterial endotoxin testing involves two methods, a gel method and a photometric method, the latter including a turbidimetric method and a chromogenic substrate method. When testing the test, any one of the methods can be used for the test. When the measurement results are controversial, unless otherwise specified, the results of the gel method shall prevail.Basic Information
Specialist Category: Inspection Category: Pathogenic Microbiological Examination
Applicable gender: whether men and women apply fasting: fastingAnalysis results:
The gel does not remain intact and is negative from the wall of the tube.
Positive records that did not slip off the wall were positive.
The type and proportion of the flora in the body are normal, and the human body is in a dynamic balance.Clinical significance
The pyrogen is mainly an endotoxin released by bacteria. The pyrogen enters the blood circulation system of the body and causes a series of adverse reactions such as fever. Therefore, the injection pyrogen or bacterial endotoxin test is an important quality index to ensure the safety of the injection.
Clinically, when a large amount of infusion is given intravenously, the patient has symptoms of chills, high fever, sweating, fainting, vomiting within 0.5 to 1 hour due to the presence of pyrogens in the drug solution. The body temperature can reach 40 °C during high fever. They can even shock.Positive result may be disease: Bacillus licheniformis enteritis considerations
In the establishment of bacterial endotoxin test method, when calculating endotoxin limit L, MVD and MVC, it is necessary to pay attention to important parameters such as maximum human dose, product specifications, sensitivity of sputum reagent, etc., because calculation errors will lead to all subsequent efforts. All failed.
Common problems are:
1. The clinical dose is determined incorrectly. Only the conventional dose is used. The maximum possible dose and route of administration of the human body are not considered. In the limit calculation formula L=K/M, due to the definition of K value and M value. Blurring makes the assignment of K and M values inaccurate, which ultimately leads to incorrect calculation of the limits. For example, an injection of a clinical adult is administered 40 to 80 mg per intravenous infusion, and an infant (three months or more) is administered intravenously by 10 to 20 mg each time. The limit of the adult (body weight 60kg) dose is 3.75EU/mg, while the infant weight is only about 5kg, the calculated limit is about 1.25EU/mg, if the maximum dose per kilogram of body weight is not considered, the bacterial endotoxin limit There will be a big difference.
2. The bacterial endotoxin limit of some declared varieties is not calculated based on the clinical dosage, but is calculated based on the dose of the same type of pyrogen test, resulting in a limit error.
3. The bacterial endotoxin limit of some varieties was copied from the foreign Pharmacopoeia, and the difference between the clinical use dose of the domestic market and the weight of the Chinese and foreigners was not considered, and the limit was determined incorrectly.
4. False calculation of daily dosing dose (multiple dosing in one day).
5. When the variety specification (concentration) changes, the calculation MVD is not adjusted.
6. The maximum dose to be used in establishing the method has exceeded the dose used in the clinic.Inspection process
First, the titer determination
Bacterial endotoxin standards are calibrated as benchmarks to determine the equivalent potency of the base weight. The titer per 1 mg of working standard should be no less than 2 EU, no more than 50 EU, and experimental data with uniformity and stability. The titer is determined as follows: At least 4 samples of each batch of working standard are used, and the same batch of sputum reagent and the same standard are used. Each working standard is diluted with endotoxin test water* according to the weight and standard product. A series of 2-fold dilutions of endotoxin solution are prepared and reacted with hydrazine reagent. When the logarithm of the reaction end point of the four working standards When the standard deviation (S) of the mean is less than 0.365, the geometric mean (in ng/mg) of the reaction endpoints of the four working standards and the reaction endpoint (in EU/mg) of the standard are calculated.
Second, test preparation
1. The utensils used in this method are treated to remove any exogenous endotoxin that may be present. Dry roasting at 250 ° C for 30 minutes or 180 ° C for 2 hours may also be used. Other suitable methods may also be used. The test operation should prevent microbial contamination.
2. Check the sensitivity label value of the batch number 鲎 reagent before the test, which should meet the requirements. Add the amount of the hydrazine reagent solvent. After dissolution, it is a guanidine reagent solution and is ready for use.
3. Dilution of the test sample: When the sensitivity mark value of the batch number 鲎 reagent is less than the internal poison limit of the test sample, calculate it by the following formula, and use the endotoxin test water* to dilute the reagent for inspection.
Dilution factor of test sample = X / λ
X: Endotoxin limited endotoxin limit (EU/ml)
λ: sensitivity value of the test agent for the test agent (EU/ml)
4 × 75mm test tube (or 0.1ml / set of sputum reagent original ampoule) containing 0.1ml sputum reagent solution, 2 of which are added with 0.1ml for the test tube, and 1 for the 2 λ endotoxin working standard 0.1 ml was used as a positive control tube, and 0.1 ml of a sputum reagent solution was added as a negative control tube. After gently mixing the test tubes, the tube was closed and placed vertically in a 37 ± 1 ° C water bath for 60 ± 2 minutes. Be careful when holding the tube and taking the tube to avoid negative results due to vibration.
(4) Judgment of results
The tube was gently removed from the water bath and slowly inverted at 1800. The gel in the tube was not deformed. The positive one was not slipped from the tube wall. (+) The gel could not remain intact and was negative from the tube wall. (One). If the test tube 2 is (1), it shall be considered to be in compliance with the regulations, and the pyrogen test method (rabbit method) test shall not be carried out. If both tubes are (+), they should be considered not to comply with the regulations. For example, 1 tube in 2 tubes is (+), and 1 tube is (1). According to the method described, 4 tubes are retested. One tube in 4 tubes is (+), which means that it is not in conformity with the regulations. Except as otherwise provided in the text, the test articles that meet the requirements shall be tested by the pyrogen test method (the rabbit method) and judged according to the results.
The limit of bacterial endotoxin in the test article shall be in accordance with the provisions of the respective articles.
The positive control was (1) or the negative control was (+), and the test was ineffective.
Attachment: Bacterial endotoxin test for sterile water for injection, water for injection, sodium chloride injection, 5% and 10% glucose injection.Not suitable for the crowd
Generally no taboos.Adverse reactions and risks
Generally no harm.