CN110632319A - Method for detecting bacterial endotoxin in glacial acetic acid - Google Patents
Method for detecting bacterial endotoxin in glacial acetic acid Download PDFInfo
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Abstract
The invention provides a method for detecting bacterial endotoxin in glacial acetic acid, which adopts a gel method for detection, and a test solution is prepared by a method of re-dissolving after volatilizing. The preparation method of the test solution comprises the following steps: firstly, a proper volume of glacial acetic acid solution is sucked into an evaporation dish for removing pyrogen, the mixture is heated to volatilize the solution under the condition of 15-90 ℃, then the mixture is redissolved by using a proper amount of bacterial endotoxin detection water, and the mixture is vortexed for 2-10min to be dissolved, and finally a test solution with a proper concentration is obtained. Compared with the prior art, the pretreatment method provided by the invention effectively solves the problem of false negative interference in endotoxin detection, is simple, convenient and reliable to operate, has strong reproducibility and high sensitivity, and has important significance in ensuring the safety of preparations.
Description
Technical Field
The invention belongs to the field of microbial detection, and relates to a method for detecting bacterial endotoxin as an auxiliary material of glacial acetic acid.
Technical Field
Glacial acetic acid, namely anhydrous acetic acid, is one of important organic acids, is non-toxic and non-irritant, and has antifungal, antiseptic, disinfectant and corrosive effects. Is often used in injection, and the main functions include acidity regulator, acidifier, antimicrobial agent, etc.
In the development of injection products, in order to ensure that patients do not cause adverse reactions such as fever after injecting drugs, the content of bacterial endotoxin in raw materials needs to be strictly controlled so as to ensure the safety of the products. Bacterial endotoxins are a component of the cell wall of many gram-negative bacteria called lipopolysaccharide and are the major component of pyrogens. A large amount of bacterial endotoxin enters blood to trigger thermal reaction, which causes fever, leucocyte reaction, endotoxin shock, diffusible intravascular coagulation and the like. When glacial acetic acid is used as an injection auxiliary material, the control of the bacterial endotoxin limit is one of the key control items of the product.
The gel method is one of the bacterial endotoxin detection methods recorded in the Chinese pharmacopoeia of 2015 edition, and has the characteristics of easy operation, good accuracy, high sensitivity, strong practicability and lower cost. When the bacterial endotoxin is detected by the gel method, firstly, the maximum effective dilution Multiple (MVD) of a sample is calculated according to the endotoxin control limit of the sample and the sensitivity of the selected limulus reagent, and the endotoxin limit is detected after the sample is diluted within the maximum effective dilution Multiple (MVD). However, the research finds that false negative interference still exists when the glacial acetic acid is diluted to the maximum dilution multiple, so that the positive control group of the test sample cannot be agglutinated, and the problems cannot be solved by selecting limulus reagents with different sensitivities, so that the direct dilution method cannot be used for detecting the endotoxin limit by the glacial acetic acid gel method.
CN10171870A discloses a method for detecting bacterial endotoxin, in which a sample solution is prepared and diluted to 2-8 times, and about 10 drops of phosphate standard buffer are respectively added to adjust the pH value to about 6.8, so as to eliminate false negative interference. In the research process, the pH value of glacial acetic acid test solution is adjusted to about 6-8 by using sodium carbonate or other commercial regulators with different concentrations, and false negative interference still exists in a test positive control group, which indicates that the interference problem cannot be solved by adding the regulators.
The research shows that when the bacterial endotoxin limit of the glacial acetic acid auxiliary material is detected by adopting a bacterial endotoxin detection method-gel method of Chinese pharmacopoeia 2015 edition, the interference can not be eliminated after the sample is diluted to the maximum effective dilution Multiple (MVD), and then sodium carbonate and other regulators are added to further remove the interference, but the result shows that the method is not suitable for controlling the bacterial endotoxin limit of the glacial acetic acid auxiliary material, so that a new method is needed for controlling the bacterial endotoxin limit of the glacial acetic acid auxiliary material.
Disclosure of Invention
In order to overcome the technical problem of false negative interference in the detection method of endotoxin in glacial acetic acid auxiliary materials in the prior art, the invention provides a novel detection method, which avoids the problem of false negative interference in the conventional method, and the method is simple and convenient to operate, high in accuracy and good in reproducibility and has very important significance for ensuring the safety of an injection.
The method for detecting endotoxin in glacial acetic acid adopts a gel method for detection, and a test solution is prepared by a method of re-dissolving after volatilizing.
According to the method for detecting endotoxin in glacial acetic acid, all glassware is dried to remove exogenous endotoxin interference, and the preferable drying condition is drying at 250 ℃ for more than 2 h.
The invention relates to a method for detecting endotoxin in glacial acetic acid, which comprises the following steps: sucking a proper volume of glacial acetic acid solution into an evaporation dish for removing pyrogen, heating at 15-90 ℃ to volatilize the glacial acetic acid solution, redissolving the glacial acetic acid solution by using a proper amount of water for checking bacterial endotoxin, and vortexing for 2-10min to dissolve the glacial acetic acid solution to obtain a test solution with a proper concentration.
The present invention provides a preferable heating temperature of 30 ℃ to 90 ℃, and further preferable temperature of 32 ℃ to 42 ℃.
According to the method for detecting endotoxin in glacial acetic acid, the limit value of bacterial endotoxin in glacial acetic acid auxiliary materials is not higher than L2.5 EU/mL, the maximum effective dilution Multiple (MVD) is L/lambda, and the MVD is calculated to be not more than 40 times according to the labeling sensitivity (lambda is 0.0625EU/mL) of a limulus reagent, namely the maximum effective dilution multiple of redissolution with water is not more than 40 times.
In addition, the invention also provides a method for detecting bacterial endotoxin in glacial acetic acid, which comprises the following steps:
a) taking a proper amount of glacial acetic acid solution, volatilizing at 15-90 ℃, and re-dissolving 5 times of volume of water for bacterial endotoxin detection to prepare a test sample stock solution;
b) the sensitivity of limulus reagent was checked by using bacterial endotoxin standards at concentrations of 2 λ, 0.5 λ and 0.25 λ, and the results were recorded.
c) Respectively and uniformly mixing the stock solution of the test sample of a) with the bacterial endotoxin standard substance according to a proportion; and diluted with water to final concentrations of 2 λ, 0.5 λ, 0.25 λ for bacterial endotoxin detection, vortexed for 1min, an interference experiment was performed, and results were recorded.
d) Diluting the stock solution of the test sample of a) to 40 times with water for bacterial endotoxin detection to obtain test solutions, reacting the test solutions with limulus reagents respectively, detecting the bacterial endotoxin of the test sample, and recording the result.
According to the method for detecting endotoxin in glacial acetic acid, a limulus reagent is reacted and cultured in a 37 ℃ test tube thermostat for 60min +/-2 min. After the culture is finished, the culture medium is gently taken out from the test tube thermostat, vibration is avoided, and the culture medium is slowly inverted for 180 degrees to observe.
The beneficial technical effects of the invention are as follows: the heating and volatilizing method is adopted to eliminate the interference of glacial acetic acid on the detection of the limit of endotoxin in gel bacteria, so that the problem of false negative interference in the prior art is avoided, the detection result is accurate and reliable, the operation is simple and convenient, the reproducibility is good, and the method has important significance for ensuring the safety of the injection.
Detailed Description
The following specific examples are given for a more complete understanding of the present invention, but the present invention is not limited to the following examples.
1. Experimental materials and instruments
Experimental materials:
limulus reagent: zhanjiang Andu, standard: 0.1 mL/count, sensitivity: 0.0625 EU/mL; bacterial endotoxin working standards: the titer of the Chinese drug biological product institute is as follows: 80 EU/Branch, batch number: 150601 + 201886; water for bacterial endotoxin test: china institute for testing biological products of drugs, batch number: 1803090.
the instrument comprises the following steps: a dry heat sterilization cabinet, an electric heating constant temperature water bath kettle, a water-proof constant temperature incubator, a vortex mixer and the like.
Comparative example 1:
the operation steps are as follows: 0.2g of sodium carbonate was dissolved in 2mL of water for endotoxin test, and a solution having a concentration of 100mg/mL was prepared as a pH adjuster. 0.5mL of glacial acetic acid solution is taken and mixed with 2mL of inspection water by vortex to prepare a stock solution of the test sample. Mixing the stock solution 0.15mL and the test water 0.15mL, adding sodium carbonate solution 0.3mL, standing for 2min, and vortexing for 6min to obtain the test solution. Mixing the stock solution 0.15mL and endotoxin with different concentrations 0.15mL, adding sodium carbonate solution 0.3mL, standing for 2min, and vortexing for 6min to obtain test solution containing endotoxin of 2 lambda, 0.5 lambda and 0.25 lambda. Then the prepared test solution and the test solution containing endotoxin of 2 lambda, 0.5 lambda and 0.25 lambda are respectively reacted with limulus reagent, and cultured in a test tube thermostat at 37 +/-2 ℃ for 60 +/-2 min, and the result is recorded as shown in the following table 1. Limulus reagent (λ 0.125 EU/mL): batch No. 1702101, Amios.
TABLE 1
The results in table 1 above illustrate that: when a sodium carbonate solution of 100mg/mL is used as a pH regulator for investigating a glacial acetic acid bacterial endotoxin method, false negative interference exists in an interference experiment, which indicates that the method is not suitable for controlling the limit of bacterial endotoxin in a glacial acetic acid auxiliary material.
Comparative example 2:
the operation steps are as follows: sodium carbonate 0.25g was dissolved in 4mL of endotoxin test water to prepare a solution having a concentration of 62.5mg/mL as a pH adjuster. 0.5mL of glacial acetic acid solution is taken and mixed with 2mL of inspection water by vortex to prepare a stock solution of the test sample. Mixing the stock solution 0.1mL and 0.1mL of water for examination, adding 0.2mL of water for examination, mixing, adding 0.4mL of sodium carbonate solution, standing for 2min, and vortexing for 6min to obtain the sample solution. Mixing test solution 0.1mL and endotoxin of different concentrations 0.1mL, adding 0.2mL of water for examination, mixing, adding 0.4mL of sodium carbonate solution, standing for 2min, and vortexing for 6min to obtain test solution containing endotoxin of 2 lambda, 0.5 lambda, and 0.25 lambda. The prepared test solution and the test solution containing endotoxin of 2 lambda, 0.5 lambda and 0.25 lambda are respectively reacted with limulus reagent, and are cultured in a 37 +/-2 ℃ test tube thermostat for 60 +/-2 min, and the results are recorded as shown in the following table 2. Limulus reagent (λ 0.125 EU/mL): batch No. 1702101, Amios.
TABLE 2
The results of table 2 above illustrate that: when a sodium carbonate solution of 62.5mg/mL is used as a pH regulator for investigating a glacial acetic acid bacterial endotoxin method, false negative interference exists in an interference experiment, which shows that the limit control of the bacterial endotoxin in the glacial acetic acid auxiliary material cannot be realized by adjusting the dosage of the sodium carbonate.
Example 1:
the operation steps are as follows: sucking 0.5mL glacial acetic acid solution into a pyrogen-removed evaporation dish, heating at 85 +/-5 ℃ to volatilize, redissolving with 2.5mL bacterial endotoxin detection water, and vortexing for 5min to dissolve the bacterial endotoxin to obtain a stock solution of a test sample. 0.1mL of the stock solution was mixed with 0.7mL of test water to obtain a test solution. Mixing the stock solution 0.1mL of the test sample with 0.1mL of endotoxin standard solution with concentration of 16 lambda and 8 lambda, adding 0.6mL of water for examination, and mixing to obtain test sample solution containing endotoxin of 2 lambda and lambda. The prepared test solution and the test solution containing 2 lambda and lambda endotoxin are respectively reacted with limulus reagent, cultured in a 37 +/-2 ℃ test tube thermostat for 60 +/-2 min, and the result is recorded as shown in the following table 3. Limulus reagent (λ 0.125 EU/mL): batch No. 1805071, Amios.
TABLE 3
The results in table 3 above illustrate that: when the method of heating and volatilizing at 85 +/-5 ℃ is adopted for investigating the glacial acetic acid bacterial endotoxin method, no interference exists in an interference experiment, and the method is suitable for controlling the limit of the bacterial endotoxin in the glacial acetic acid auxiliary material.
Example 2:
the operation steps are as follows: sucking 0.5mL glacial acetic acid solution into a pyrogen-removed evaporation dish, heating at 35 +/-5 ℃ to volatilize, redissolving with 2.5mL bacterial endotoxin detection water, and vortexing for 5min to dissolve the bacterial endotoxin solution to obtain a stock solution of a test sample. 0.1mL of the stock solution was mixed with 0.7mL of test water to obtain a test solution. Mixing the stock solution 0.1mL of the test sample with 0.1mL of endotoxin standard solution with concentration of 16 lambda and 8 lambda, adding 0.6mL of water for examination, and mixing to obtain test sample solution containing endotoxin of 2 lambda and lambda. The prepared test solution and the test solution containing 2 lambda and lambda endotoxin are respectively reacted with limulus reagent, cultured in a 37 +/-2 ℃ test tube thermostat for 60 +/-2 min, and the result is recorded as shown in the following table 4. Limulus reagent (λ 0.125 EU/mL): batch No. 1805071, Amios.
TABLE 4
The results of table 4 above illustrate that: when the method of heating and volatilizing at 35 +/-5 ℃ is adopted for investigating the glacial acetic acid bacterial endotoxin method, an interference experiment is passed, and the method is suitable for controlling the limit of the bacterial endotoxin in the glacial acetic acid auxiliary material.
In addition, the experimental steps of the embodiment 2 are repeated, the heating is carried out at 20 +/-5 ℃ or 60 +/-5 ℃ to volatilize the volatile matter, and the obtained experimental result is the same as the result of the embodiment 2, so that the analysis method has high accuracy and good reproducibility.
Claims (8)
1. A method for detecting bacterial endotoxin in glacial acetic acid is characterized by comprising the following steps: the gel method is adopted for detection, and the test solution is prepared by a method of re-dissolving after volatilizing.
2. The detection method according to claim 1, characterized in that: all glassware used in the detection by adopting the gel method is dried for more than 2h at 250 ℃, and exogenous endotoxin interference is removed.
3. The detection method according to claim 1, characterized in that: the test solution is prepared by a method of re-dissolving after volatilizing, and the method comprises the following steps:
firstly, a proper volume of glacial acetic acid solution is sucked into an evaporation dish for removing pyrogen, the mixture is heated to volatilize under the condition of the heating temperature of 15-90 ℃, then a proper amount of water for bacterial endotoxin detection is used for redissolving, and the mixture is swirled for 2-10min to be dissolved, and finally a test solution with proper concentration is obtained.
4. The detection method according to claim 3, characterized in that: the heating temperature is selected from 30-90 ℃.
5. The detection method according to claim 4, characterized in that: the heating temperature is selected from 32-42 ℃.
6. Detection method according to one of claims 3 to 5, characterized in that: the maximum effective dilution multiple of the water redissolution is not more than 40 times.
7. A method for detecting bacterial endotoxin in glacial acetic acid comprises the following steps:
a) taking a proper amount of glacial acetic acid solution, volatilizing at 15-90 ℃, and re-dissolving 5 times of volume of water for bacterial endotoxin detection to prepare a test sample stock solution;
b) rechecking the sensitivity of limulus reagent by using bacterial endotoxin standard substances with the concentrations of 2 lambda, 0.5 lambda and 0.25 lambda, and recording the result;
c) respectively and uniformly mixing the stock solution of the test sample of a) with the bacterial endotoxin standard substance according to a proportion; diluting with bacterial endotoxin detection water to final concentrations of 2 lambda, 0.5 lambda and 0.25 lambda, vortexing for 1min, performing interference experiment, and recording the result;
d) diluting the stock solution of the test sample of a) to 40 times with water for bacterial endotoxin detection to obtain test sample solution, reacting the test sample solution with limulus reagent respectively, detecting the bacterial endotoxin of the test sample, and recording the result.
8. The detection method according to claim 7, characterized in that: the method for respectively reacting the test solution with the limulus reagent comprises the steps of culturing in a 37 ℃ test tube thermostat for 60min +/-2 min, gently taking out from the test tube thermostat after the culture is finished, avoiding vibration, slowly reversing the test solution for 180 degrees for observation, and recording results.
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