A method for detecting bacterial endotoxins in drugs containing quinoline ring structures

Abstract

The present invention provides a method for detecting bacterial endotoxins in drugs containing a quinoline ring structure, and relates to the technical field of bacterial endotoxin detection. Specifically comprising the following steps: first dissolving a drug containing a quinoline ring structure with a solvent to obtain a test sample stock solution to release bacterial endotoxins in the drug; then diluting the stock solution to reduce the interference of the drug's own properties on bacterial endotoxins; and finally detecting bacterial endotoxins by dynamic colorimetry. Wherein, the solvent is N, N-dimethylformamide with a volume fraction of 60-70%; the drug containing a quinoline ring structure includes imiquimod; the test sample stock solution concentration is 0.1-10 mg / ml, and the stock solution dilution multiple is 10-1000 times. The technical solution provided by the present invention solves the problem that drugs containing a quinoline ring structure in the prior art are insoluble in water, and effectively improves the accuracy of detecting the bacterial endotoxin content of drugs containing a quinoline ring structure. Moreover, the conventionally adopted gel method is avoided, thereby solving the problem of false negative results of taking the supernatant and detecting it by gel method.

Description

Technical Field

[0001] The invention belongs to the technical field of bacterial endotoxin detection, and in particular relates to a method for detecting bacterial endotoxin in a drug containing a quinoline ring structure. Background Art

[0002] Bacterial endotoxin is a unique structure on the cell wall of Gram-negative bacteria, and its main chemical substance is lipopolysaccharide. Endotoxin is an exogenous pyrogen, which can activate neutrophils and other substances, causing them to release an endogenous pyrogen, which acts on the body temperature regulation center to cause fever. In severe cases, it can lead to hypotension, toxic shock, disseminated intravascular coagulation, acute respiratory distress syndrome, multiple organ failure and even death. Therefore, biological products, injectable drugs, chemical drugs, radioactive drugs, antibiotics, vaccines and other drugs must pass the bacterial endotoxin test before they can be used.

[0003] At present, the methods for detecting bacterial endotoxins are: gel method and photometry. Among them, the gel method uses the principle of agglutination reaction between horseshoe crab reagent and bacterial endotoxin to qualitatively detect or semi-quantitatively detect endotoxin. The photometry method includes turbidity method and color matrix (colorimetric) method, which respectively uses the turbidity change during the reaction between horseshoe crab reagent and endotoxin and the amount of chromophore released by the generated coagulase to quantitatively determine endotoxin. The colorimetric method can be divided into endpoint colorimetric method and dynamic colorimetric method. This method has high sensitivity and precision. However, the two methods included in the pharmacopoeia only provide a basis for bacterial endotoxin detection, and are not specific to a certain drug. In actual application, some drugs cannot use the gel method or photometry method due to their own particularity, and can only use the rabbit pyrogen test method; some drugs cannot use the gel method due to their own particularity and the sensitivity of horseshoe crab reagent, but can use a highly sensitive and high-precision photometric quantitative test method.

[0004] Drugs containing quinoline ring structures are generally insoluble in water and have great interference with the detection of bacterial endotoxins. For example, imiquimod is a small molecule immunomodulator with a molecular formula of C 14 H 16 N 4 , CAS registration number 99011-02-6. At present, imiquimod preparations are mainly topical ointments, and bacterial endotoxin control is not required. With the continuous in-depth research on imiquimod, it is very likely that imiquimod injection will be made in the future, so the research on the detection method of imiquimod bacterial endotoxin is forward-looking. When the inventor tried to take imiquimod supernatant for gel test, he found that false negative results would appear, and the test results were deviated from the true value, which was not conducive to the accurate detection of bacterial endotoxins.

[0005] Therefore, designing a method that can accurately and truly detect drug bacterial endotoxins containing quinoline ring structures is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0006] The purpose of the present invention is to provide a method for detecting bacterial endotoxins in drugs containing a quinoline ring structure, which solves the problem in the prior art that drugs containing a quinoline ring structure are poorly soluble in water and easily interfere with and cause false negatives, and effectively improves the accuracy of detecting the bacterial endotoxin content of drugs containing a quinoline ring structure.

[0007] To achieve the above-mentioned purpose, the present invention provides a method for detecting bacterial endotoxins in drugs containing a quinoline ring structure, comprising the following steps: firstly dissolving the drug containing a quinoline ring structure with a solvent to obtain a test sample stock solution to release the bacterial endotoxins in the drug; then diluting the stock solution to reduce the interference of the drug's own properties on the bacterial endotoxins; and finally detecting the bacterial endotoxins by a dynamic colorimetric method.

[0008] In a preferred embodiment, the solvent is N,N-dimethylformamide with a volume fraction of 60-70%. When exploring methods to improve the accuracy of detecting the bacterial endotoxin content of drugs containing quinoline ring structures, the inventors of the present application tried a variety of reagents, including strong acids (such as hydrochloric acid, sulfuric acid), weak acids (such as glacial acetic acid, phosphoric acid), dimethyl sulfoxide, etc. However, these reagents destroy the inactivated bacterial endotoxins while dissolving drugs containing quinoline ring structures, resulting in false negative test results and the inability to accurately measure the bacterial endotoxin content in the drug. Through a large number of creative experimental studies, it was found that when using N,N-dimethylformamide with a volume fraction of 60%-70%, it can completely dissolve a certain amount of drugs without destroying the bacterial endotoxins in the inactivated drugs, and can completely release the bacterial endotoxins in the drugs, making the test results more real and accurate.

[0009] However, less than 60% N,N-dimethylformamide can only dissolve a limited amount of drugs, and the concentration of the prepared solution is low, lower than the minimum effective dilution concentration, making the test results invalid; for more than 70% N,N-dimethylformamide, dilution to the maximum effective dilution multiple cannot eliminate the interference of the solvent in the detection of endotoxins, resulting in an endotoxin recovery rate of less than 50%.

[0010] In a preferred embodiment, the drug containing a quinoline ring structure includes imiquimod.

[0011] In a preferred embodiment, the concentration of the test solution is 0.1-10 mg / ml, and the dilution factor of the solution is 10-1000 times. Preferably, the concentration of the test solution is 0.2 mg / ml, 0.5 mg / ml, 0.8 mg / ml, 1 mg / ml, 1.2 mg / ml, 1.5 mg / ml, 1.8 mg / ml, 2 mg / ml, 5 mg / ml, 6 mg / ml, 8 mg / ml, and the dilution factor of the solution is 20 times, 50 times, 80 times, 100 times, 200 times, 400 times, 500 times, 600 times, 800 times. More preferably, when detecting endotoxin of imiquimod, the concentration of the test solution is 1 mg / ml, and the dilution factor of the solution is 200 times.

[0012] In a preferred embodiment, the method comprises the following steps:

[0013] S1 Preparation of standard curve reliability test solution: re-dissolve the bacterial endotoxin working standard to prepare 0.005EU / ml, 0.05EU / ml, and 0.5EU / ml bacterial endotoxin standard solutions;

[0014] S2 diluted N,N-dimethylformamide as solvent;

[0015] S3: preparing a test solution: dissolving the drug containing a quinoline ring structure with the solvent obtained in step S2 to obtain a test solution with a concentration of 0.1-10 mg / ml, and diluting the solution to obtain a test solution;

[0016] S4: preparing a test sample positive solution: diluting the bacterial endotoxin standard solution with the test sample solution obtained in step S3 to prepare a test sample positive solution containing 0.05 EU / ml bacterial endotoxin at the midpoint concentration of the standard curve;

[0017] S5: preparing a negative control solution by diluting the solvent obtained in step S2;

[0018] S6 detects bacterial endotoxins by dynamic colorimetry: reconstitute the dynamic colorimetric horseshoe crab reagent; take a 96-well plate, add negative control solution, standard curve reliability test solution, test sample solution, test sample positive solution in sequence, and add equal volumes of horseshoe crab reagent solution to the corresponding wells; place the 96-well plate after sample addition in an endotoxin detector for color development reaction; read the content of drug bacterial endotoxins containing a quinoline ring structure through the endotoxin detector.

[0019] In a preferred embodiment, in step S2, the volume fraction of the prepared solvent is 60-70%;

[0020] In a preferred embodiment, in step S3, the dissolution condition is heating in a water bath at 60-80°C for 2-8 minutes. Below this temperature, the dissolution rate of the drug is slow, and the time is prolonged. If the time is too long, there is a risk of damaging and inactivating endotoxins. Above this temperature, the dissolution rate cannot be greatly increased, but energy consumption is increased, so the dissolution temperature is set to 60-80°C.

[0021] In a preferred embodiment, the specific operation steps of step S5 are: placing the solvent obtained in step S2 in a water bath at 60-80°C for 2-8 minutes, letting it stand to room temperature, and then diluting it 100-200 times. The purpose of this step is: the negative solution is used as a blank control solution, and the preparation process of the test sample stock solution must be consistent in order to more truly detect the content of endotoxin in the drug.

[0022] In a preferred embodiment, in step S6, the sensitivity of the kinetic colorimetric horseshoe crab reagent is 0.005-50EU / ml. The sensitivity of the above horseshoe crab reagent is a commercially available specification, which can be routinely selected according to the test drug. More preferably, when detecting endotoxin of imiquimod, the sensitivity of the kinetic colorimetric horseshoe crab reagent is 0.005EU / ml.

[0023] In a preferred embodiment, in step S6, the color development reaction condition is 36-38°C for 30-60 min. More preferably, in step S6, the color development reaction condition is 37°C for 45-60 min.

[0024] In a preferred embodiment, in order to avoid the influence of the external environment on the test results, before step S1, all the utensils used can also be pre-treated to remove exogenous endotoxins.

[0025] In a preferred embodiment, in order to reduce the impact of the operating steps on the test results, step S1 is to re-dissolve the bacterial endotoxin working standard, step S2 is to dilute N,N-dimethylformamide, step S5 is to dilute the solvent obtained in step S2, and step S6 is to re-dissolve the dynamic colorimetric horseshoe crab reagent, all of which are re-dissolved or diluted with water for bacterial endotoxin testing.

[0026] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0027] The present invention fully dissolves the drug containing a quinoline ring structure by selecting a specific solvent and optimizing the reaction conditions, so that the bacterial endotoxin in the drug containing a quinoline ring structure which is poorly soluble in water is completely released, and then the content of bacterial endotoxin can be quantified by a dynamic colorimetric method.

[0028] The overall detection method is simple and convenient, has low requirements on reaction conditions and reaction equipment, and effectively solves the problem of poor solubility of drugs containing quinoline ring structure; it avoids the use of gel method, thereby solving the problem of false negative results of traditional supernatant detection by gel method. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in detail below in conjunction with specific implementation methods, but it should be understood that the protection scope of the present invention is not limited by the specific implementation methods.

[0030] The embodiment of the present invention solves the problem of poor solubility of drugs containing quinoline ring structures in the prior art and the problem of false negative results when the supernatant is used as a test solution and the test result is detected by gel method by providing a method for detecting bacterial endotoxins in drugs containing quinoline ring structures.

[0031] The instruments, reagents and methods used in the method of the present invention are described as follows:

[0032] 1. Main instruments

[0033] Endotoxin detector, model 357, manufacturer: Zhanjiang Andus Biological Co., Ltd., purification workbench: SW-CJ-IFD, manufacturer: Sujing Group Antai Company, thermal resistance furnace, model: SRJX, manufacturer: Shanghai Pudong Rongfeng Scientific Instrument Co., Ltd.

[0034] 2. Reagents

[0035] Bacterial endotoxin working standard, titer: 10EU / tube, Zhanjiang Andus Biological Co., Ltd.; Horseshoe crab reagent, specification: 0.005EU / ml, Zhanjiang Andus Biological Co., Ltd.; Water for bacterial endotoxin test, specification: 50ml / bottle, Zhanjiang Andus Biological Co., Ltd.

[0036] Other raw materials, reagents, instruments and equipment not specifically specified can be purchased from the market or prepared by existing methods.

[0037] The technical solution of the present application is described in detail below through specific embodiments:

[0038] Example 1 Detection of Imiquimod Bacterial Endotoxin Content

[0039] (1) Test on the effect of solvent on bacterial endotoxins:

[0040] Preparation of S1 standard curve reliability test solution: Take the bacterial endotoxin working standard, reconstitute it with bacterial endotoxin test water, vortex it in a vortex shaker for 15 minutes, and then prepare standard curve endotoxin solutions of 0.005EU / ml, 0.05EU / ml, and 0.5EU / ml.

[0041] S2 dilutes N,N-dimethylformamide with water for bacterial endotoxin test to obtain a 65% volume fraction N,N-dimethylformamide solution as a solvent.

[0042] S3: prepare the test solution: dissolve imiquimod in the solvent obtained in step S2 and place it in a water bath at 70° C. for 5 minutes until the sample is completely dissolved, to prepare a 1 mg / ml test solution; dilute the test solution 200 times with water for bacterial endotoxin test to obtain the test solution;

[0043] S4: preparing a test sample positive solution: diluting the bacterial endotoxin standard solution obtained in step S1 with the test sample solution obtained in step S3 to prepare a test sample positive solution containing 0.05 EU / ml bacterial endotoxin at the midpoint concentration of the standard curve;

[0044] S5: prepare a negative control solution: take the solvent obtained in step S2, place it in a 70°C water bath for 5 minutes, let it stand until it reaches room temperature, and then dilute it 200 times with water for bacterial endotoxin testing to serve as a negative control solution;

[0045] S6 detects bacterial endotoxins by dynamic colorimetry: reconstitute the dynamic colorimetric horseshoe crab reagent (sensitivity is 0.005EU / ml); take a 96-well plate, add negative control solution, standard curve reliability test solution, test sample solution, test sample positive solution in sequence, and add equal volumes of horseshoe crab reagent solution to the corresponding wells; place the 96-well plate after sample addition in an endotoxin detector, and perform color development reaction at 37.0℃ for 60 minutes; read the content of drug bacterial endotoxin containing quinoline ring structure through the endotoxin detector.

[0046] Three sets of parallel experiments were performed, and the results are shown in Table 1:

[0047] Table 1

[0048]

[0049] It can be seen from Table 1 that the correlation coefficients of the three batches of samples, the reaction time and recovery rate of the negative control can all meet the requirements, so the test values ​​are valid and true.

[0050] Comparative Example 1

[0051] The same three batches of imiquimod were used. The bacterial endotoxin test was conducted with water. The supernatant was used as the test solution. The sensitivity of the horseshoe crab reagent was 0.25EU / ML, 0.125EU / ML, 0.06EU / ML, and 0.03EU / ML, respectively. The bacterial endotoxin was detected using the gel method.

[0052] The specific steps of the gel method are as follows:

[0053] Minimum effective concentration (λ is the sensitivity of the horseshoe crab test, L is the bacterial endotoxin limit, and the bacterial endotoxin limit of imiquimod is 1.0EU / mg)

[0054] 1. Preparation of test sample: Weigh 50 mg of imiquimod and add 50 ml of bacterial endotoxin test water, shake well for 5 minutes, let stand and take the supernatant as the test sample stock solution, the concentration of the test solution is 1 mg / ml. Dilute the test sample stock solution to 0.25 mg / ml, 0.125 mg / ml, 0.06 mg / ml, and 0.03 mg / ml respectively as the test solution at different sensitivities.

[0055] 2. Preparation of positive control solution of bacterial endotoxin working standard: Take bacterial endotoxin working standard, reconstitute it with bacterial endotoxin test water, vortex it in a vortex shaker for 15 minutes, and then prepare 2λ (0.5EU / ML, 0.25EU / ML, 0.12EU / ML, 0.06EU / ML) standard positive control solutions with bacterial endotoxin test water respectively.

[0056] 3. Preparation of test sample positive solution: Take bacterial endotoxin working standard, reconstitute it with bacterial endotoxin test water, vortex it in a vortex shaker for 15 minutes, and then use the test sample solution to prepare 2λ (0.5EU / ML, 0.25EU / ML, 0.12EU / ML, 0.06EU / ML) test sample positive control solutions.

[0057] 4. Negative control solution: Take the water used for bacterial endotoxin test as the negative control.

[0058] 5. Sample addition: Take 8 tubes of horseshoe crab reagent (0.1ml), add 0.1ml of bacterial endotoxin test water to each tube to reconstitute it, add 0.1ml of the test sample solution diluted at a ratio not exceeding the maximum effective dilution multiple or the minimum effective dilution concentration to 2 tubes as test sample tubes, add 0.1ml of 2.0λ concentration endotoxin solution made from bacterial endotoxin working standard with bacterial endotoxin test water to 2 tubes as positive control tubes, add 0.1ml of bacterial endotoxin test water to 2 tubes as negative control tubes, add 0.1ml of 2.0λ concentration endotoxin solution made from the same bacterial endotoxin working standard with the test sample solution to 2 tubes as test sample positive control tubes, gently mix the solutions in the test tubes, seal the tube mouths with sealing film, and place them vertically in a constant temperature incubator at 37±1℃ and keep warm for 60±2 minutes.

[0059] 6. Result judgment: Gently take the test tube out of the thermostat and slowly turn it upside down 180°. If gel is formed in the tube and the gel does not deform or slide off the tube wall, it is positive and recorded (+). If gel is not formed or the formed gel is not solid, deforms and slides off the tube wall, it is negative and recorded (-). The positive control tube is (+), the test sample positive tube is (+), and the negative control tube is (-). The test is valid.

[0060] If both test tubes are (-), the test sample is judged to be in compliance with the regulations; if both are (+), it is judged not to be in compliance with the regulations; if one of the two tubes is (+) and the other is (-), the test sample is retested in 4 parallel tubes according to the above method. If all the parallel tubes are negative, the test sample is judged to be in compliance with the regulations; otherwise, the test sample is judged not to be in compliance with the regulations.

[0061] The test results are shown in Table 2:

[0062] Table 2

[0063]

[0064] As can be seen from Table 2, the results of the three batches of samples tested by the gel method using the supernatant are unstable, the positive control of the test sample is not established, and there is a false negative phenomenon. Compared with the gel method, the technical solution of the present invention has stable results, solves the problem of imiquimod dissolution (neither destroying bacterial endotoxins in the sample nor introducing exogenous endotoxins), and the problem of interference of the test sample with the test, can accurately check the content of bacterial endotoxins, and overcome the false negative phenomenon of the gel method.

[0065] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A method for detecting bacterial endotoxins in drugs containing a quinoline ring structure, It is characterized in that The following steps are involved: First, the drug containing quinoline ring structure is dissolved in a solvent to obtain a test solution to release bacterial endotoxins in the drug; then the solution is diluted to reduce the interference of the drug's own properties on bacterial endotoxins; finally, the bacterial endotoxins are detected by dynamic colorimetry; The solvent is N,N-dimethylformamide with a volume fraction of 60-70%; The specific steps include: S1 Preparation of standard curve reliability test solution: re-dissolve the bacterial endotoxin working standard to prepare 0.005EU / ml, 0.05EU / ml, and 0.5EU / ml bacterial endotoxin standard solutions; S2 diluted N,N-dimethylformamide as solvent; S3: preparing a test solution: dissolving the drug containing a quinoline ring structure with the solvent obtained in step S2 to obtain a test solution with a concentration of 0.1-10 mg / ml, and diluting the solution to obtain a test solution; S4: preparing a test sample positive solution: diluting the bacterial endotoxin standard solution with the test sample solution obtained in step S3 to prepare a test sample positive solution containing 0.05 EU / ml bacterial endotoxin at the midpoint concentration of the standard curve; S5: preparing a negative control solution: diluting the solvent obtained in step S2; S6 Detect bacterial endotoxins by dynamic colorimetry: Redissolve the dynamic colorimetric horseshoe crab reagent; take a 96-well plate, add negative control solution, standard curve reliability test solution, test sample solution, test sample positive solution in sequence, and add equal volumes of horseshoe crab reagent solution to the corresponding wells; place the 96-well plate after sample addition in an endotoxin detector for color development reaction; read the content of drug bacterial endotoxins containing a quinoline ring structure through the endotoxin detector; Wherein, in step S3, the dissolution condition is heating in a water bath at 60-80°C for 2-8min; In step S6, the color development reaction condition is 36-38° C. for 30-60 min.

2. The method for detecting bacterial endotoxins in a drug containing a quinoline ring structure as claimed in claim 1, It is characterized in that The drug containing a quinoline ring structure includes imiquimod.

3. The method for detecting bacterial endotoxins in drugs containing a quinoline ring structure as claimed in claim 1, It is characterized in that The concentration of the test sample stock solution is 0.1-10 mg / ml, and the dilution multiple of the stock solution is 10-1000 times.

4. The method for detecting bacterial endotoxin in a drug containing a quinoline ring structure as claimed in claim 1, It is characterized in that In step S6, the sensitivity of the dynamic colorimetric horseshoe crab reagent is 0.005-50EU / ml.

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