A method for detecting bacterial endotoxins in glacial acetic acid

Abstract

The present invention provides a method for detecting bacterial endotoxins in glacial acetic acid, which is detected by gel method, and the test solution is prepared by the method of redissolving after evaporation. The preparation method of the test solution is: first, an appropriate volume of glacial acetic acid solution is drawn into a pyrogen-free evaporating dish, heated at 15°C-90°C to evaporate it, and then redissolved with an appropriate amount of bacterial endotoxin test water, vortexed for 2-10 minutes to dissolve it, and finally a test solution of appropriate concentration is obtained. Compared with the prior art, the pretreatment method proposed in the present invention effectively solves the problem of false negative interference in endotoxin detection, is simple and reliable to operate, has strong reproducibility, and high sensitivity, which is of great significance to ensuring the safety of the preparation.

Description

Technical Field

[0001] The invention belongs to the field of microbial testing and relates to a method for detecting bacterial endotoxins in glacial acetic acid auxiliary materials. Technical Background

[0002] Glacial acetic acid, also known as anhydrous acetic acid, is one of the important organic acids. It is non-toxic and non-irritating, and has antifungal, antiseptic, disinfectant and corrosive effects. It is often used in injections, and its main functions include acidity regulator, acidifier, antimicrobial agent, etc.

[0003] In the development of injection products, in order to ensure that patients will not experience adverse reactions such as fever after drug injection, the bacterial endotoxin content in the raw materials must be strictly controlled to ensure product safety. Bacterial endotoxin is a component called lipopolysaccharide in the cell wall of many Gram-negative bacteria and is the main component of pyrogens. When a large amount of bacterial endotoxin enters the blood, it will trigger a thermal reaction, leading to fever, leukocyte reaction, endotoxin shock, and diffuse intravascular coagulation. When glacial acetic acid is used as an injection excipient, the control of its bacterial endotoxin limit is one of the key control items of the product.

[0004] The gel method is one of the bacterial endotoxin test methods included in the 2015 edition of the Chinese Pharmacopoeia. This method is easy to operate, accurate, sensitive, practical, and low cost. When using the gel method to test bacterial endotoxins, the maximum effective dilution (MVD) of the sample is first calculated based on the sample endotoxin control limit and the sensitivity of the selected horseshoe crab reagent. After the sample is diluted within the maximum effective dilution (MVD), the endotoxin limit test is performed. However, studies have found that when glacial acetic acid is diluted to the maximum dilution, there is still false negative interference, resulting in the inability of the positive control group of the test sample to agglutinate. The selection of horseshoe crab reagents with different sensitivities cannot solve the problem, so the direct dilution method cannot be used for endotoxin limit testing using the glacial acetic acid gel method.

[0005] CN10171870A discloses a method for detecting bacterial endotoxins. In the patent, the sample solution is prepared and diluted to 2-8 times, and about 10 drops of phosphate standard buffer are added to adjust the pH to about 6.8 to eliminate false negative interference. During the research process, it was found that when the glacial acetic acid test solution was adjusted to a pH of about 6-8 with different concentrations of sodium carbonate or other commercial regulators, false negative interference still existed in the positive control group of the test sample, indicating that adding regulators could not solve the interference problem.

[0006] Our research found that when using the Chinese Pharmacopoeia 2015 edition bacterial endotoxin test method-gel method to detect the bacterial endotoxin limit of glacial acetic acid excipients, the interference could not be eliminated after the sample was diluted to the maximum effective dilution multiple (MVD). Then, regulators such as sodium carbonate were added to further remove the interference. However, the results showed that the above methods were not suitable for the control of bacterial endotoxin limits in glacial acetic acid excipients. Therefore, new methods are needed to control the bacterial endotoxin limits in glacial acetic acid excipients. Summary of the invention

[0007] In order to overcome the technical problem of false negative interference in the prior art endotoxin detection method in glacial acetic acid excipient, the present invention provides a new detection method, which avoids the problem of false negative interference in conventional methods. The method is simple to operate, has high accuracy and good reproducibility, and is of great significance to ensuring the safety of injections.

[0008] The present invention discloses a method for detecting endotoxin in glacial acetic acid, which adopts a gel method for detection, and the test solution is prepared by a method of re-dissolving after evaporation.

[0009] In the method for detecting endotoxin in glacial acetic acid described in the present invention, all glassware used are dried to remove exogenous endotoxin interference, and the preferred drying condition is drying at 250° C. for more than 2 hours.

[0010] The present invention discloses a method for detecting endotoxin in glacial acetic acid. The method for preparing a test solution is as follows: aspirate an appropriate volume of glacial acetic acid solution into a pyrogen-free evaporating dish, heat it at 15°C-90°C to evaporate it, re-dissolve it with an appropriate amount of bacterial endotoxin test water, vortex it for 2min-10min to dissolve it, and obtain a test solution of appropriate concentration.

[0011] The present invention provides a preferred heating temperature of 30°C-90°C, more preferably 32°C-42°C.

[0012] In the method for detecting endotoxin in glacial acetic acid described in the present invention, the bacterial endotoxin limit of the glacial acetic acid auxiliary material is not higher than L=2.5EU / mL, and the maximum effective dilution factor (MVD)=L / λ. According to the labeled sensitivity of the horseshoe crab reagent (λ=0.0625EU / mL), the MVD is calculated to be no more than 40 times, that is, the maximum effective dilution factor of reconstitution with water is no more than 40 times.

[0013] In addition, the present invention also provides a method for detecting bacterial endotoxins in glacial acetic acid, comprising the following steps:

[0014] a) Take an appropriate amount of glacial acetic acid solution and evaporate it at 15℃-90℃, then take 5 times the volume of bacterial endotoxin test water to reconstitute it to prepare the test sample stock solution;

[0015] b) Verify the sensitivity of the horseshoe crab reagent using bacterial endotoxin standards at concentrations of 2λ, λ, 0.5λ, and 0.25λ, and record the results.

[0016] c) Mix the test sample stock solution in a) with the bacterial endotoxin standard in proportion; dilute with bacterial endotoxin test water to the final concentration of 2λ, λ, 0.5λ, and 0.25λ, vortex for 1 min, perform interference experiment, and record the results.

[0017] d) Dilute the test sample stock solution in a) to 40 times with water for bacterial endotoxin test to prepare the test solution. The test sample solution is reacted with the horseshoe crab reagent to perform bacterial endotoxin test on the test sample and record the results.

[0018] In the method for detecting endotoxin in glacial acetic acid of the present invention, the horseshoe crab reagent reaction is incubated in a test tube thermostat at 37° C. for 60 min±2 min. After the incubation, the test tube is gently taken out from the test tube thermostat to avoid vibration, and is slowly inverted 180° for observation.

[0019] The beneficial technical effect of the present invention is that the interference of glacial acetic acid on the endotoxin limit detection of gel bacteria is eliminated by the heating volatilization method, thereby avoiding the problem of false negative interference in the prior art. The detection result is accurate and reliable, the operation is simple, and the reproducibility is good, which is of great significance for ensuring the safety of the injection. DETAILED DESCRIPTION

[0020] In order to more fully understand the present invention, the following specific examples are given, but the present invention is not limited to the following examples.

[0021] 1. Experimental materials and instruments

[0022] Experimental Materials:

[0023] Horseshoe crab reagent: Zhanjiang Andus, specification: 0.1mL / tube, sensitivity: 0.0625EU / mL; bacterial endotoxin working standard: China Food and Drug and Biological Products Control Institute, potency: 80EU / tube, batch number: 150601-201886; water for bacterial endotoxin test: China Food and Drug and Biological Products Control Institute, batch number: 1803090.

[0024] Instruments: dry heat sterilizer, electric constant temperature water bath, water-proof constant temperature incubator, vortex mixer, etc.

[0025] Comparative Example 1:

[0026] The operation steps are as follows: weigh 0.2g of sodium carbonate and dissolve it in 2mL of endotoxin test water to prepare a solution with a concentration of 100mg / mL as a pH adjuster. Take 0.5mL of glacial acetic acid solution and vortex mix it with 2mL of test water to prepare the test sample stock solution. Take 0.15mL of the test sample stock solution and mix it with 0.15mL of test water, add 0.3mL of sodium carbonate solution and let it stand for 2min, vortex for 6min to obtain the test sample solution. Take 0.15mL of the test sample stock solution and mix it with 0.15mL of endotoxins of different concentrations, add 0.3mL of sodium carbonate solution and let it stand for 2min, vortex for 6min, and obtain test sample solutions containing 2λ, λ, 0.5λ, and 0.25λ endotoxins. The prepared test solution and the test solution containing 2λ, λ, 0.5λ, and 0.25λ endotoxin were reacted with the horseshoe crab reagent, respectively, and incubated in a test tube thermostat at 37℃±2℃ for 60min±2min, and the results were recorded, as shown in Table 1. Horseshoe crab reagent (λ=0.125EU / mL): Batch No. 1702101, Andus.

[0027] Table 1

[0028]

[0029] The results in Table 1 above show that when 100 mg / mL sodium carbonate solution was used as the pH adjuster to investigate the glacial acetic acid bacterial endotoxin method, there was false negative interference in the interference experiment, indicating that this method is not suitable for the limit control of bacterial endotoxins in glacial acetic acid auxiliary materials.

[0030] Comparative Example 2:

[0031] The operation steps are as follows: weigh 0.25g of sodium carbonate and dissolve it in 4mL of endotoxin test water to prepare a solution with a concentration of 62.5mg / mL as a pH adjuster. Take 0.5mL of glacial acetic acid solution and vortex mix it with 2mL of test water to prepare the test sample stock solution. Take 0.1mL of the test sample stock solution and mix it with 0.1mL of test water, add 0.2mL of test water, mix it, then add 0.4mL of sodium carbonate solution and let it stand for 2min, vortex it for 6min to get the test sample solution. Take 0.1mL of the test sample stock solution and mix it with 0.1mL of endotoxins of different concentrations, add 0.2mL of test water, mix it, then add 0.4mL of sodium carbonate solution and let it stand for 2min, vortex it for 6min, and get the test sample solutions containing 2λ, λ, 0.5λ, and 0.25λ endotoxins. The prepared test solution and the test solution containing 2λ, λ, 0.5λ, and 0.25λ endotoxin were reacted with the horseshoe crab reagent, respectively, and incubated in a test tube thermostat at 37℃±2℃ for 60min±2min, and the results were recorded as shown in Table 2. Horseshoe crab reagent (λ=0.125EU / mL): batch number 1702101, Andus.

[0032] Table 2

[0033]

[0034] The results in Table 2 above show that when 62.5 mg / mL sodium carbonate solution was used as a pH regulator to investigate the glacial acetic acid bacterial endotoxin method, there was false negative interference in the interference experiment, indicating that the bacterial endotoxin limit control in the glacial acetic acid auxiliary material cannot be achieved by adjusting the dosage of sodium carbonate.

[0035] Embodiment 1:

[0036] The operation steps are as follows: draw 0.5mL of glacial acetic acid solution into a pyrogen-free evaporating dish, heat it at 85℃±5℃ to evaporate it, reconstitute it with 2.5mL of bacterial endotoxin test water, vortex it for 5min to dissolve it, and obtain the test sample stock solution. Take 0.1mL of this stock solution and mix it with 0.7mL of test water to obtain the test sample solution. Take 0.1mL of the test sample stock solution and mix it with 0.1mL of endotoxin standard solution with concentrations of 16λ and 8λ, then add 0.6mL of test water to mix, and obtain the test sample solution containing 2λ and λ endotoxins. The above-prepared test sample solution and the test sample solution containing 2λ and λ endotoxins are reacted with the horseshoe crab reagent respectively, and incubated in a test tube thermostat at 37℃±2℃ for 60min±2min, and the results are recorded, as shown in Table 3 below. Horseshoe crab reagent (λ=0.125EU / mL): batch number 1805071, Andus.

[0037] Table 3

[0038]

[0039] The results in Table 3 above indicate that when the glacial acetic acid bacterial endotoxin method was investigated by heating at 85°C ± 5°C, there was no interference in the interference experiment, indicating that this method is suitable for the limit control of bacterial endotoxins in glacial acetic acid auxiliary materials.

[0040] Embodiment 2:

[0041] The operation steps are as follows: draw 0.5mL of glacial acetic acid solution into a pyrogen-free evaporating dish, heat at 35℃±5℃ to evaporate it, reconstitute it with 2.5mL of bacterial endotoxin test water, vortex it for 5min to dissolve it, and obtain the test sample stock solution. Take 0.1mL of this stock solution and mix it with 0.7mL of test water to obtain the test sample solution. Take 0.1mL of the test sample stock solution and mix it with 0.1mL of endotoxin standard solution with concentrations of 16λ and 8λ, then add 0.6mL of test water to mix, and obtain the test sample solution containing 2λ and λ endotoxins. The above-prepared test sample solution and the test sample solution containing 2λ and λ endotoxins are reacted with the horseshoe crab reagent respectively, and incubated in a test tube thermostat at 37℃±2℃ for 60min±2min, and the results are recorded, as shown in Table 4 below. Horseshoe crab reagent (λ=0.125EU / mL): batch number 1805071, Andus.

[0042] Table 4

[0043]

[0044]

[0045] The results in Table 4 above illustrate that when the glacial acetic acid bacterial endotoxin method was investigated by heating and evaporating at 35°C±5°C, the interference experiment passed, indicating that this method is suitable for the limit control of bacterial endotoxins in glacial acetic acid auxiliary materials.

[0046] In addition, the experimental steps of Example 2 were repeated, and the solution was heated at 20°C ± 5°C or 60°C ± 5°C to evaporate. The experimental results obtained were the same as those of Example 2, thus proving that the analytical method of the present invention has high accuracy and good reproducibility.

Claims

1. A method for detecting bacterial endotoxins in glacial acetic acid, characterized in that: The gel method was used for detection, and the test solution was prepared by evaporating and then re-dissolving. All glassware used in the gel method test was dried at 250°C for more than 2 hours to remove exogenous endotoxin interference; The test solution is prepared by evaporating and then re-dissolving, and the method is as follows: First, take an appropriate amount of glacial acetic acid solution into a pyrogen-free evaporating dish, heat it at a temperature of 15℃-90℃ to evaporate it, then re-dissolve it with an appropriate amount of bacterial endotoxin test water, vortex for 2min-10min to dissolve it, and finally obtain a test solution of appropriate concentration; The detection method specifically includes the following steps: a) Take an appropriate amount of glacial acetic acid solution and evaporate it at 15℃-90℃, then take 5 times the volume of bacterial endotoxin test water to reconstitute it to prepare the test sample stock solution; b) Review the sensitivity of the horseshoe crab test kit using bacterial endotoxin standards at concentrations of 2λ, λ, 0.5λ, and 0.25λ, and record the results; c) Mix the test sample stock solution in a) with the bacterial endotoxin standard in proportion; dilute with bacterial endotoxin test water to a final concentration of 2λ, λ, 0.5λ, and 0.25λ, vortex for 1 min, perform interference experiments, and record the results; d) Dilute the sample stock solution in a) to 40 times with water for bacterial endotoxin test to prepare the sample solution. The sample solution is reacted with horseshoe crab reagent to perform bacterial endotoxin test on the sample and the results are recorded.

2. The detection method according to claim 1, characterized in that: The heating temperature is selected from 30-90°C.

3. The detection method according to claim 1, characterized in that: The heating temperature is selected from 32-42°C.

4. The detection method according to claim 1, characterized in that: The method for reacting the test solution with the horseshoe crab reagent is to culture it in a test tube thermostat at 37°C for 60min±2min, and after the culture is completed, gently take it out of the test tube thermostat to avoid vibration, and slowly invert it 180° for observation and record the results.

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