Sterility test method for levofloxacin injection
By using a neutralizing agent and appropriate rinsing methods in the sterility test of levofloxacin injection, the problem of microbial inhibition by levofloxacin injection was solved, achieving accuracy and reliability in sterility testing, meeting pharmacopoeia requirements, and ensuring drug safety.
Patent Information
- Application Number
- CN202511671794.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-14
- Publication Date
- 2026-01-06
Smart Images
Figure SMS_1 
Figure SMS_2 
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of drug testing, specifically relating to a method for sterility testing of levofloxacin injection. Background Technology
[0002] According to Chapter 1101, "Sterility Testing Methods," of the Chinese Pharmacopoeia, existing sterility testing methods may be affected by the inhibitory effect of the drug itself when handling certain drugs, especially those containing antibacterial components. This inhibition of microbial growth can affect the accuracy and reliability of sterility testing. Levofloxacin, as a broad-spectrum antibacterial drug, may significantly inhibit microorganisms during sterility testing of its injection solution, making existing sterility testing methods unable to accurately reflect its sterility status.
[0003] Levofloxacin injection is widely used in clinical treatment, primarily for various bacterial infections. Because it enters the bloodstream directly, sterility is a key indicator of its quality control. Inaccurate sterility testing methods can lead to infection risks during clinical use, seriously threatening patient safety. Therefore, developing a sterility testing method that effectively eliminates the microbial inhibitory effect of levofloxacin injection is of great significance for ensuring the safety of clinical medication.
[0004] Existing methods for sterility testing of levofloxacin lactate sodium chloride injection involve adding a neutralizing agent. However, levofloxacin lactate and levofloxacin have different chemical structures, resulting in different chemical properties, stability, and antibacterial activities in solution. The conditions for eliminating the inhibitory effect of the drug solution on microorganisms also differ significantly. Therefore, there is an urgent need to develop a sterility testing method for levofloxacin injection. Summary of the Invention
[0005] To address the shortcomings of existing technologies, this invention provides a method for aseptic testing of levofloxacin injection.
[0006] This invention provides a method for sterility testing of levofloxacin injection, comprising: (1) Preparation of test solution: Transfer the test sample into a 0.1% sterile peptone aqueous solution containing 1.5-2.5 mol / L neutralizing agent; (2) Rinsing: Use a 0.1% sterile peptone aqueous solution containing 0.46-0.54 mol / L neutralizing agent as the rinsing solution. Filter the test solution by membrane filtration. Rinse 6 times with a rinsing volume of 600 mL / membrane. Filter and collect the microorganisms trapped in the test sample. (3) Microbial culture: The filter membrane that has been filtered and retained from the test sample is mixed with culture medium and neutralizing agent and cultured.
[0007] Preferably, in step (1), the test sample is transferred into a 0.1% sterile peptone aqueous solution containing 1.5 to 2.5 mol / L neutralizing agent and then left to stand for 10 minutes.
[0008] Preferably, in step (1), the volume fraction of the test sample in the test solution is 80%.
[0009] Preferably, in step (2), during the final rinse, a neutralizing agent is added to the rinsing solution to make the neutralizing agent content 0.5-0.6 mol / L.
[0010] Preferably, in step (2), during the final rinse, a neutralizing agent is added to the rinsing solution and the solution is left to stand for 8-12 minutes.
[0011] Preferably, in step (3), the microbial culture is carried out in a microbial culture device, and the concentration of the neutralizing agent in each culture medium sleeve is 0.009-0.01 mol / L.
[0012] Preferably, in step (3), the culture medium used for microbial culture is selected from at least one of tryptic soy broth and thioglycolate fluid medium.
[0013] Preferably, in step (3), when the culture medium used for the microbial culture is tryptic soy liquid culture medium, the culture temperature is 20-25℃ and the culture time is ≥14 days; Alternatively, when the culture medium used for the microbial culture is thioglycolate fluid medium, the culture temperature is 30-35℃ and the culture time is ≥14 days.
[0014] Preferably, in step (3), the aseptic test is performed according to the General Chapter 1101 of the Chinese Pharmacopoeia, with Escherichia coli and Staphylococcus aureus as positive control bacteria.
[0015] Preferably, the neutralizing agent is selected from magnesium chloride or magnesium sulfate.
[0016] This invention provides a sterility testing method for levofloxacin injection. By screening the concentration of the neutralizing agent in the test solution and rinsing solution, the rinsing volume, and the number of rinsing cycles, the inhibitory effect of levofloxacin injection on microorganisms is eliminated. Under preferred conditions, this sterility testing method meets the applicability requirements of General Chapter 1101 of the Chinese Pharmacopoeia, improving the effectiveness and reliability of sterility testing for levofloxacin injection. It provides a basis for drug testing and antibiotic quality control, and has promising application prospects.
[0017] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.
[0018] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Detailed Implementation
[0019] Unless otherwise specified, the experimental methods used in the following examples and experimental cases are conventional methods. Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available products.
[0020] All instruments, equipment, or measuring instruments used in the following examples and experimental cases have been calibrated or verified and are within their validity period. See Table 1 below for details: Table 1 Detailed information on the strains involved in the following examples and experimental cases is shown in Table 2: Table 2 The preparation methods of the test bacterial solutions in the following examples and experimental cases are as follows: (1) Staphylococcus aureus, Escherichia coli and Bacillus subtilis were inoculated onto tryptic soy agar medium and cultured at 30-35°C for 18-24 hours. The cultures were then prepared into bacterial suspensions with a bacterial count of less than 100 CFU per 0.1 ml using sterile sodium chloride-peptone buffer or 0.9% sterile sodium chloride solution at pH 7.0.
[0021] (2) Inoculate Clostridium sporogenes into thioglycolate fluid medium and incubate at 30-35°C for 18-24 hours. Then, prepare a bacterial suspension with less than 100 CFU per 0.1 ml using sterile sodium chloride-peptone buffer or 0.9% sterile sodium chloride solution at pH 7.0 for later use.
[0022] (3) Inoculate Candida albicans onto Sabouraud dextrose agar medium and incubate at 20-25°C for 24-48 hours. Prepare a bacterial suspension with less than 100 CFU per 0.1 ml using sterile sodium chloride-peptone buffer or 0.9% sterile sodium chloride solution at pH 7.0 for later use.
[0023] (4) Inoculate Aspergillus niger on Sabouraud dextrose agar slant medium, culture at 20 - 25°C for 5 - 7 days, take the Aspergillus niger slant culture, add an appropriate amount (about 5 ml) of sterile sodium chloride - peptone buffer solution with a pH of 7.0 containing 0.05% (ml / ml) polysorbate 80 or 0.9% sterile sodium chloride solution, elute the spores, aspirate the spore suspension, and prepare a spore suspension containing less than 100 cfu of spores per 0.1 ml with sterile sodium chloride - peptone buffer solution with a pH of 7.0 containing 0.05% (ml / ml) polysorbate 80 or 0.9% sterile sodium chloride solution for standby.
[0024] Example 1 Sterility test method for levofloxacin injection This example provides a sterility test method for levofloxacin injection, and the specific steps are as follows: 1. Experimental grouping The experiment is divided into a negative control group, a positive control group, a sample group, a test group, and a neutralizer control group.
[0025] The purpose of setting the negative control group is to confirm the sterility of the rinsing solution and the culture medium; the purpose of setting the positive control group is to confirm the ability of the culture medium to promote the growth of microorganisms; the purpose of setting the sample group is to confirm whether the test sample is sterile; the purpose of setting the test group is to confirm whether the test sample has antibacterial properties under the experimental conditions; the purpose of setting the neutralizer control group is to confirm that the neutralizer is non - toxic to microorganisms.
[0026] 2. Sample quantity (1) According to the provisions of General Principles 1101 <Sterility test method> in Part IV of the Chinese Pharmacopoeia: When the output of each batch of products exceeds 500 pieces, the minimum number of test samples required for inoculating each type of culture medium is 20 pieces; When the volume (V) of the test sample is within the range of "1 ml < V ≤ 40 ml", half of each test sample is inoculated into each type of culture medium, but not less than 1 ml.
[0027] (2) The sample quantity in this example is 20 pieces (specification: 20 ml: 0.5 g).
[0028] 3. Preparation of the test solution Transfer the test sample (levofloxacin injection to be tested) into 100 ml of 0.1% sterile peptone aqueous solution containing 2.0 mol / L magnesium chloride, dilute the test sample, mix evenly, and let it stand for 10 minutes.
[0029] For the negative control group and the neutralizer control group, do not add the test solution, but only add 100 ml of 0.1% sterile peptone aqueous solution containing 2. mol / L magnesium chloride (neutralizer).
[0030] For the positive control group, do not add the test solution.
[0031] 4. Filtration and rinsing The process conditions for the inspection method in this embodiment are shown in Table 3.
[0032] Table 3 The test solution was filtered using a membrane filtration method, passing through the sleeve (containing the filter membrane) of a two-piece bacterial culture device. In both the sample and test groups, each sleeve filtered 10 levofloxacin injection samples, and one two-piece bacterial culture device filtered 20 samples.
[0033] A 0.1% sterile peptone aqueous solution containing 0.5 mol / L magnesium chloride was selected as the rinsing solution. Each membrane was rinsed with 600 ml of the solution, 100 ml per rinse, for a total of 6 rinses. During the final rinse, different procedures were performed according to the group composition. For the negative control group, during the final rinse, approximately 100 ml of rinsing solution was added, followed by 10 ml of 1.0 mol / L magnesium chloride solution. After mixing thoroughly, the solution was filtered. For the sample group, during the final rinse, approximately 100 ml of rinsing solution was added, followed by 10 ml of 1.0 mol / L magnesium chloride solution. After mixing thoroughly, the solution was allowed to stand for 10 minutes, and then the solution was filtered immediately. For the experimental group and the neutralizing agent control group, during the final rinse, 100 ml of rinsing solution was added, followed by 10 ml of 1.0 mol / L magnesium chloride solution. After mixing thoroughly and allowing to stand for 10 minutes, an appropriate amount of bacterial culture was added, and the solution was filtered immediately. Regarding the addition of appropriate bacterial solutions, add 0.1 mL of Aspergillus niger, Candida albicans, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, and Clostridium sporogenes bacterial solutions to the sleeve respectively. The concentration of each bacterial solution is less than 1000 CFU / mL. A total of 3 sets of dual-unit bacterial collection and culture devices are used.
[0034] The positive control group was not rinsed; bacterial solution was added directly. The types of microorganisms, the amount of bacterial solution added, and the concentration were the same as those in the experimental group.
[0035] 5. Microbial culture After rinsing, collect the microorganisms trapped in the test sample and add 100 ml of tryptic soy peptone liquid medium or thioglycolate fluid medium to each sleeve. Except for the positive control group, add 5 mL of 0.2 mol / L magnesium chloride solution to each culture medium sleeve (i.e., 5 mL / sleeve).
[0036] For both the positive control and experimental groups, tryptic soy broth was added to the sleeves corresponding to Aspergillus niger, Candida albicans, and Bacillus subtilis, while thioglycolate fluid broth was added to the sleeves corresponding to Escherichia coli, Staphylococcus aureus, and Clostridium sporogenes. The culture conditions are shown in Table 4 below. Table 4 6. Test Results If the negative control group is sterile, and all microorganisms in the positive control group, neutralizing agent control group, and test group grow normally, then if the sample group shows no bacterial growth, it proves that the levofloxacin injection to be tested is sterile; if the sample group shows bacterial growth, it proves that the levofloxacin injection to be tested is bacterial.
[0037] The technical solution of the present invention will be further explained through experiments below.
[0038] Experimental Example 1 This experimental example screened the conditions, such as the preparation of the test solution and the rinsing of the rinsing solution, and found the process conditions that could effectively eliminate the inhibitory effect of the test sample on the test strain.
[0039] 1. Method 1 The process conditions for Method 1 are shown in Table 5.
[0040] Table 5 (1) Preparation of test solution: The sample solution is directly filtered by membrane filtration without dilution of the test solution before filtration.
[0041] (2) Process control of rinsing: 400 ml of sterile peptone aqueous solution containing 0.2 mol / L magnesium chloride was used to rinse each membrane, 100 ml / time, for 4 times.
[0042] (3) Determination of the amount of substances added to the culture medium: After rinsing, add 5 ml of 0.2 mol / L magnesium chloride solution to each culture medium sleeve (i.e., 5 ml / sleeve).
[0043] (4) Test results: The test showed that Bacillus subtilis, Escherichia coli and Staphylococcus aureus in the test group could not grow normally due to the inhibition of the test sample, indicating that the above test method could not effectively eliminate the inhibitory effect of the test sample on the test strain.
[0044] 2. Method 2 The process conditions for Method 2 are shown in Table 6.
[0045] Table 6 (1) Preparation of test solution: Transfer the test solution into 100 ml of 0.1% sterile peptone aqueous solution containing 0.2 mol / L magnesium chloride to dilute the test sample before filtration, thereby reducing the concentration of the test solution and thus reducing the adsorption of the test sample by the filter membrane.
[0046] (2) Process control of rinsing: 800 ml of sterile peptone aqueous solution containing 0.2 mol / L magnesium chloride was used to rinse each membrane, 100 ml / time, for a total of 8 rinses.
[0047] (3) Determination of the amount of substances added to the culture medium: After rinsing, add 5 ml of 0.2 mol / L magnesium chloride solution to each culture medium sleeve (i.e., 5 ml / sleeve).
[0048] (4) Test results: The test showed that Bacillus subtilis grew weakly in the test group, and Escherichia coli and Staphylococcus aureus could not grow normally due to the inhibition of the test sample. This indicates that the above test method cannot effectively eliminate the inhibitory effect of the test sample on the test strain.
[0049] 3. Method 3 The process conditions for Method 3 are shown in Table 7.
[0050] Table 7 (1) Preparation of test solution: Increase the concentration of magnesium chloride, the neutralizing agent, during the preparation of the test solution. Transfer the test solution into 100 ml of 0.1% sterile peptone aqueous solution containing 0.5 mol / L magnesium chloride to dilute the test sample before filtration, thereby reducing the concentration of the test solution and thus reducing the adsorption of the test sample by the filter membrane.
[0051] (2) Process control of rinsing: Each membrane was rinsed with 800 ml of 0.1% sterile peptone aqueous solution containing 0.2 mol / L magnesium chloride, 100 ml / rinse, for a total of 8 rinses. During the last rinse, 10 ml of 1.0 mol / L magnesium chloride solution was added first, mixed evenly and left to stand for 5 minutes, and then an appropriate amount of bacterial solution was added and filtered immediately.
[0052] (3) Determination of the amount of substances added to the culture medium: After rinsing, add 5 ml of 0.2 mol / L magnesium chloride solution to each culture medium sleeve (i.e., 5 ml / sleeve).
[0053] (4) Test results: The test showed that Bacillus subtilis grew weakly in the test group, and Escherichia coli and Staphylococcus aureus could not grow normally due to the inhibition of the test sample. This indicates that the above test method cannot effectively eliminate the inhibitory effect of the test sample on the test strain.
[0054] 4. Method 4 The process conditions for Method 4 are shown in Table 8.
[0055] Table 8 (1) Preparation of test solution: Transfer the test solution into 100 ml of 0.1% sterile peptone aqueous solution containing 0.5 mol / L magnesium chloride, mix well, let stand for 10 minutes, dilute the test sample before filtration, reduce the concentration of the test solution, thereby reducing the adsorption of the test sample by the filter membrane and prolonging the neutralizing agent action time.
[0056] (2) Process control of rinsing: Increase the concentration of neutralizing agent in the rinsing solution. Use 0.1% sterile peptone aqueous solution containing 0.5 mol / L magnesium chloride to rinse each membrane with 800 ml, 100 ml / rinse, for 8 rinses. In the last rinse, first add 10 ml of 1.0 mol / L magnesium chloride solution, mix well and let stand for 10 minutes to increase the neutralizing agent standing time in the last rinse. Then add an appropriate amount of bacterial solution and filter immediately.
[0057] (3) Determination of the amount of substances added to the culture medium: After rinsing, add 5 ml of 0.2 mol / L magnesium chloride solution to each culture medium sleeve (i.e., 5 ml / sleeve).
[0058] (4) Test results: The test showed that Bacillus subtilis in the test group could grow normally, while Escherichia coli and Staphylococcus aureus grew weakly due to the inhibition of the test sample. This indicates that the above test method can weaken the antibacterial effect of the test sample to a certain extent, but it still cannot effectively eliminate the inhibitory effect of the test sample on the test strain.
[0059] 5. Method 5 The process conditions for Method 5 are shown in Table 9.
[0060] Table 9 (1) Preparation of test solution: Increase the concentration of magnesium chloride, the neutralizing agent, during the preparation of the test solution. Transfer the test solution into 100 ml of 0.1% sterile peptone aqueous solution containing 3.0 mol / L magnesium chloride to dilute the test sample before filtration.
[0061] (2) Process control of rinsing: Each membrane was rinsed with 800 ml of 0.1% sterile peptone aqueous solution containing 0.5 mol / L magnesium chloride, 100 ml / rinse, for a total of 8 rinses. During the last rinse, 10 ml of 1.0 mol / L magnesium chloride solution was added, mixed evenly, and left to stand for 10 minutes. Then, an appropriate amount of bacterial solution was added and the membrane was filtered immediately.
[0062] (3) Determination of the amount of substances added to the culture medium: After rinsing, add 5 ml of 0.2 mol / L magnesium chloride solution to each culture medium sleeve (i.e., 5 ml / sleeve).
[0063] (4) Test results: The test showed that Bacillus subtilis in the test group could grow normally, while Escherichia coli and Staphylococcus aureus grew weakly due to the inhibition of the test sample. This indicates that the above test method can weaken the antibacterial effect of the test sample to a certain extent, but it still cannot effectively eliminate the inhibitory effect of the test sample on the test strain.
[0064] 6. Method 6 The process conditions for Method 6 are shown in Table 10.
[0065] Table 10 (1) Preparation of test solution: Reduce the concentration of magnesium chloride, the neutralizing agent, during the preparation of the test solution, and transfer the test solution into 100 ml of 0.1% sterile peptone aqueous solution containing 2.0 mol / L magnesium chloride to dilute the test sample before filtration.
[0066] (2) Process control of rinsing: Each membrane was rinsed with 800 ml of 0.1% sterile peptone aqueous solution containing 0.5 mol / L magnesium chloride, 100 ml / rinse, for a total of 8 rinses. During the last rinse, 10 ml of 1.0 mol / L magnesium chloride solution was added, mixed evenly, and left to stand for 10 minutes. Then, an appropriate amount of bacterial solution was added and the membrane was filtered immediately.
[0067] (3) Determination of the amount of substances added to the culture medium: After rinsing, add 5 ml of 0.2 mol / L magnesium chloride solution to each culture medium sleeve (i.e., 5 ml / sleeve).
[0068] (4) Test results: According to the test, Bacillus subtilis in the test group can grow normally, while Escherichia coli and Staphylococcus aureus grow weakly due to the inhibitory effect of the test product. The inhibitory effect of the test product on the test strains was still not effectively eliminated.
[0069] 7. Method 7 The process conditions for Method 7 are shown in Table 11.
[0070] Table 11 (1) Preparation of test solution: Transfer the test solution into 100 ml of 0.1% sterile peptone aqueous solution containing 3.0 mol / L magnesium chloride to dilute the test sample before filtration.
[0071] (2) Process control of rinsing: Use 0.1% sterile peptone aqueous solution containing 0.5 mol / L magnesium chloride to rinse each membrane with 600 ml, 100 ml / rinse, for 6 rinses. In the last rinse, first add 10 ml of 1.0 mol / L magnesium chloride solution, mix well and let stand for 10 minutes, then add an appropriate amount of bacterial solution and filter immediately.
[0072] (3) Determination of the amount of substances added to the culture medium: After rinsing, add 5 ml of 0.2 mol / L magnesium chloride solution to each culture medium sleeve (i.e., 5 ml / sleeve).
[0073] (4) Test results: According to the test, Bacillus subtilis in the test group can grow normally, while Escherichia coli and Staphylococcus aureus grow weakly due to the inhibitory effect of the test product. The inhibitory effect of the test product on the test strains was still not effectively eliminated.
[0074] 8. Method 8 The process conditions for Method 8 are shown in Table 12.
[0075] Table 12 (1) Preparation of test solution: Transfer the test solution into 100 ml of 0.1% sterile peptone aqueous solution containing 3.0 mol / L magnesium chloride to dilute the test sample before filtration.
[0076] (2) Process control of rinsing: Use 0.1% sterile peptone aqueous solution containing 0.5 mol / L magnesium chloride to rinse each membrane with 400 ml, 100 ml / rinse, for 4 rinses. In the last rinse, first add 10 ml of 1.0 mol / L magnesium chloride solution, mix well and let stand for 10 minutes, then add an appropriate amount of bacterial solution and filter immediately.
[0077] (3) Determination of the amount of substances added to the culture medium: After rinsing, add 5 ml of 0.2 mol / L magnesium chloride solution to each culture medium sleeve (i.e., 5 ml / sleeve).
[0078] (4) Test results: The test showed that the growth of Bacillus subtilis, Escherichia coli and Staphylococcus aureus in the test group was weak due to the inhibitory effect of the test product, and the inhibitory effect of the test product on the test strains was still not effectively eliminated.
[0079] 9. Method 9 The process conditions for Method 9 are shown in Table 13.
[0080] Table 13 (1) Preparation of test solution: To reduce the inhibitory effect of test sample on microorganisms during the preparation of test solution, the test sample solution is transferred into 100 ml of 0.1% sterile peptone aqueous solution containing 2.0 mol / L magnesium chloride. The test sample is diluted before filtration to reduce the concentration of test solution and thus reduce the adsorption of test sample on filter membrane.
[0081] (2) Process control of rinsing: Use 0.1% sterile peptone aqueous solution containing 0.5 mol / L magnesium chloride to rinse each membrane with 600 ml, 100 ml / rinse, for 6 rinses. In the last rinse, first add 10 ml of 1.0 mol / L magnesium chloride solution, mix well and let stand for 10 minutes, then add an appropriate amount of bacterial solution and filter immediately.
[0082] (3) Determination of the amount of substances added to the culture medium: After rinsing, add 5 ml of 0.2 mol / L magnesium chloride solution to each culture medium sleeve (i.e., 5 ml / sleeve).
[0083] (4) Test results: The test showed that all the test bacteria in the test group could grow normally (including Bacillus subtilis, Staphylococcus aureus and Escherichia coli), indicating that the above test method can effectively eliminate the inhibitory effect of the test sample on the test strain.
[0084] The comparison and summary of the methods and conditions in methods 1-9 above are shown in Table 14 below.
[0085] Table 14 The table above shows that the preparation of the test solution, the concentration of the neutralizing agent in the rinsing solution, the rinsing volume, and the number of rinsing cycles have a significant impact on eliminating the inhibitory effect of the test sample on the test strain. A comparison of methods 1-4 shows that increasing the concentration of the neutralizing agent in the test solution cannot eliminate the inhibition of Bacillus subtilis. Only by increasing the mixing time of the test solution with the neutralizing agent during preparation, increasing the concentration of the neutralizing agent in the rinsing solution, and increasing the mixing time of the neutralizing agent in the final rinsing, can normal growth of Bacillus subtilis be achieved. A comparison of methods 4-9 shows that increasing the concentration of the neutralizing agent in the test solution cannot eliminate the inhibition of Escherichia coli and Staphylococcus aureus. Only by appropriately increasing the concentration of the neutralizing agent in the test solution and appropriately reducing the rinsing volume can normal growth of Escherichia coli and Staphylococcus aureus be achieved. In summary, the preferred sterility testing conditions for levofloxacin injection solution are aseptic conditions of 2 mol / L in the test solution, 0.5 mol / L in the rinsing solution, 600 ml / membrane rinsing volume, and 6 rinsing times. Under these preferred conditions, all test bacteria can grow normally, eliminating the inhibitory effect of the test sample on the test bacteria.
[0086] Experimental Example 2: Suitability Test of Aseptic Method for Levofloxacin Injection The suitability test of levofloxacin injection was conducted according to the method in Example 1, with three independent experiments performed. First, the test bacterial suspension was confirmed, and the results are shown in Table 15.
[0087] Table 15 The results of three independent aseptic tests are shown in Table 16-18 below.
[0088] Table 16 Table 17 Table 18 Three independent sterility tests were conducted on levofloxacin injection (batch number: S12191101). The results confirmed that levofloxacin injection had no antibacterial activity under the current testing volume and experimental conditions. Staphylococcus aureus and Escherichia coli can be selected as positive control bacteria for the sterility testing of levofloxacin injection.
[0089] As can be seen from the above embodiments and experimental examples, the present invention provides a method for sterility testing of levofloxacin injection. By screening the concentration of the neutralizing agent in the test solution and rinsing solution, the rinsing volume, and the number of rinsing times, the inhibitory effect of levofloxacin injection on microorganisms is eliminated. Under preferred conditions, this sterility testing method meets the applicability requirements of General Chapter 1101 of the Chinese Pharmacopoeia, improving the effectiveness and reliability of sterility testing of levofloxacin injection, providing a basis for drug testing and antibiotic quality control, and showing good application prospects.
Claims
1. A method for sterility testing of levofloxacin injection, characterized by, It comprises: (1) Preparation of test solution: the test product is transferred into 0.1% sterile aqueous protein peptone solution containing 1.5-2.5 mol / L neutralizing agent; (2) Washing: 0.1% sterile aqueous protein peptone solution containing 0.46-0.54 mol / L neutralizing agent is used as washing liquid, the test solution is filtered by membrane filtration method, the washing liquid is washed for 6 times with a washing amount of 600 mL / membrane, and the filtrate is collected to intercept microorganisms in the test product; (3) Microbial culture: the filter membrane containing the microorganisms intercepted from the test product after filtration is mixed with culture medium and neutralizing agent for culture.
2. The method of the aseptic test according to claim 1, characterized in that: In step (1), after the test product is transferred into 0.1% sterile aqueous protein peptone solution containing 1.5-2.5 mol / L neutralizing agent, it is allowed to stand for 10 minutes.
3. The method of the aseptic test according to claim 1, characterized in that: In step (1), the volume fraction of the test product in the test solution is 80%.
4. The method of the aseptic test according to claim 1, characterized in that: In step (2), the neutralizing agent is added to the washing liquid to make the content of the neutralizing agent 0.5-0.6 mol / L in the last washing.
5. The method of the aseptic test according to claim 4, characterized in that: In step (2), after the neutralizing agent is added to the washing liquid in the last washing, it is allowed to stand for 8-12 minutes.
6. The method of the aseptic test according to claim 1, characterized in that: In step (3), the microbial culture is carried out in a bacteria collector, and the concentration of the neutralizing agent in each culture medium sleeve is 0.009-0.01 mol / L.
7. The method of the aseptic test according to claim 1, characterized in that: In step (3), the culture medium used for the microbial culture is selected from at least one of tryptone soy peptone liquid medium and thioglycolate fluid medium.
8. The method of the aseptic test according to claim 7, characterized in that: In step (3), when the culture medium used for the microbial culture is tryptone soy peptone liquid medium, the culture temperature is 20-25℃, and the culture time is ≥14 days; Or, when the culture medium used for the microbial culture is thioglycolate fluid medium, the culture temperature is 30-35℃, and the culture time is ≥14 days.
9. The method of the sterility check according to claim 1, characterized in that: In step (3), the inspection is carried out according to the method for sterility test in Chinese Pharmacopoeia (Volume 4 General Rules 1101), and Escherichia coli and Staphylococcus aureus are used as positive control bacteria.
10. The method of the sterility check according to claim 1, characterized in that: The neutralizing agent is selected from magnesium chloride or magnesium sulfate.