A kind of sterility detection method of benzathine penicillin
By adding appropriate amount of agar to the sterile detection method of benzine penicillin and expanding the volume of the culture medium bottle, the problems of low detection accuracy and long cycle in the existing methods are solved, and fast and accurate sterile detection results are achieved.
Patent Information
- Application Number
- CN202411938635.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-26
- Publication Date
- 2025-04-04
AI Technical Summary
The existing sterile detection methods of benzine penicillin have problems with low test accuracy, long cycle and increased consumables, especially because benzine penicillin is very easy to precipitate in some culture media and has a slow dissolution rate.
A sterile detection method including the following steps is adopted: the test solution is fully mixed with the penicillin enzyme solution to prepare the test solution; then the test solution is directly inoculated into the polysorbate 80 sulfhydryl glycolate fluid medium and the polysorbate 80 pancreatic soybean liquid culture medium, culture for no less than 14 days, and it is determined whether it is sterile by visual inspection. This method ensures that the test sample is completely dissolved within 5-7 days by adding an appropriate amount of agar to the culture medium and expanding the volume of the culture medium bottle.
This method can shorten the sterile detection cycle of benzyl penicillin, improve detection accuracy, reduce detection costs, avoid secondary contamination, and accurately perform qualitative detection.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of medicine production, and specifically relates to a sterility detection method for benzathine penicillin. Background Art
[0002] Benzathine Benzylpenicillin is an organic compound with the chemical formula C 48 H 56 N6O8S2 is an antibiotic that can resist a variety of bacterial infections. It is absorbed very slowly and has a low blood concentration. It is suitable for patients who need long-term penicillin prevention, such as patients with chronic rheumatic heart disease.
[0003] Benzathine penicillin is a white crystalline powder that is easily soluble in N,N-dimethylformamide or formamide, slightly soluble in ethanol, and very slightly soluble in water. Due to the fact that benzathine penicillin is extremely insoluble in water, the direct inoculation method should be used in accordance with the sterility test method specified in the pharmacopoeia. The current common sterility test method for benzathine penicillin is to inactivate benzathine penicillin with penicillinase, then inoculate it into the culture medium and culture it for no less than 14 days. After 14 days, transfer it to the same fresh culture medium and continue to culture it for no less than 4 days before judging the results. However, since benzathine penicillin is very easy to precipitate in trypticase soytone liquid culture medium and the dissolution rate is very slow, this method has the defects of low test accuracy and long cycle, and this method increases the consumables in the transfer process.
[0004] At present, my country has not issued specific standards for the sterility test method of benzathine penicillin, so it is very necessary to establish a sterility test method for benzathine penicillin. Summary of the invention
[0005] The purpose of the invention is to establish a sterility detection method for benzathine penicillin so as to shorten the sterility detection period of benzathine penicillin and improve its accuracy.
[0006] In order to achieve the above object, the present invention adopts the following technical solutions:
[0007] A method for detecting the sterility of benzathine penicillin comprises the following steps:
[0008] (1) Mix the benzathine penicillin test sample and penicillinase solution thoroughly to prepare a test solution containing ≥ 7500 units of penicillinase per 1 mg of benzathine penicillin;
[0009] (2) The test solution is directly inoculated into polysorbate 80 thioglycollate fluid culture medium and polysorbate 80 trypticase soytone liquid culture medium, respectively, and cultured for not less than 14 days; direct observation is performed to determine whether the culture is sterile; wherein the polysorbate 80 trypticase soytone liquid culture medium contains agar.
[0010] Preferably, each 100 ml of polysorbate 80 trypticase soytone liquid medium contains 0.06-0.08 g of agar.
[0011] Preferably, in step (2), the volume ratio of the test solution to the polysorbate 80 thioglycollate fluid culture medium and the polysorbate 80 trypticase soy liquid culture medium is 1:20-25.
[0012] More preferably, in step (2), the volume ratio of the test solution to the polysorbate 80 thioglycollate fluid culture medium and the polysorbate 80 trypticase soy liquid culture medium is 1:20.
[0013] Preferably, the ratio of the volume of the polysorbate 80 trypticase soytone liquid culture medium bottle to the volume of the polysorbate 80 trypticase soytone liquid culture medium therein is ≥2.5.
[0014] Preferably, 5 mL of the test solution is directly inoculated into 100 mL of polysorbate 80 thioglycollate fluid culture medium and 100 mL of polysorbate 80 tryptic soytone liquid culture medium, respectively, wherein the volume of the polysorbate 80 tryptic soytone liquid culture medium bottle is 250 mL, and each 100 mL of polysorbate 80 tryptic soytone liquid culture medium contains 0.07 g of agar.
[0015] Preferably, the polysorbate 80 trypticase soy broth is shaken once a day.
[0016] Preferably, the polysorbate 80 content in the polysorbate 80 thioglycollate fluid culture medium is 0.5%; the polysorbate 80 content in the polysorbate 80 trypticase soytone liquid culture medium is 0.5%.
[0017] Preferably, the culture temperature of the polysorbate 80 thioglycollate fluid culture medium is 30°C to 35°C, and the culture temperature of the polysorbate 80 trypticase soy liquid culture medium is 20°C to 25°C.
[0018] The beneficial effects of the present invention are:
[0019] The present invention provides a method for detecting aseptic benzathine penicillin, which can judge the detection result by visual inspection, and is convenient and quick. The present invention can make the test sample completely dissolved within 5-7 days by adopting the operation method of adding an appropriate amount of agar to the culture medium and expanding the volume of the culture medium bottle, which not only saves the transfer step, shortens the inspection cycle, reduces the inspection cost, but also avoids secondary contamination and improves the accuracy of the inspection result. Methodological experiments show that the method of the present invention has high precision, good durability and high sensitivity, and can accurately perform qualitative detection on benzathine penicillin. DETAILED DESCRIPTION
[0020] The present invention will be further described below by way of examples, which are not intended to further limit the present invention. It should be understood by those skilled in the art that equivalent replacements or corresponding improvements made to the technical features of the present invention still fall within the scope of protection of the present invention.
[0021] Example 1 Establishment of Sterility Detection Method for Benzathine Penicillin
[0022] 1. Check the suitability of the culture medium
[0023] 1. The culture medium used in this embodiment includes polysorbate 80 thioglycollate fluid culture medium and polysorbate 80 tryptic soytone liquid culture medium (wherein, the polysorbate 80 tryptic soytone liquid culture medium bottle volume is 250ml, and the polysorbate 80 tryptic soytone liquid culture medium in each culture medium bottle is 100ml, and contains 0.07g agar). The culture medium used in the embodiment of the present invention is prepared and sterilized according to the Chinese Pharmacopoeia method. The experimental bacteria strains are all supervised by the China Pharmaceutical and Biological Products Inspection Institute.
[0024] 2. Culture medium sterility test results
[0025] Five bottles were randomly taken from the above culture medium, and each culture medium was placed at a specified temperature for 14 days. The results are shown in Table 1 below. As can be seen from the table, the above blank culture medium was sterile after 14 days of culture, which met the requirements of the sterility validation test.
[0026] Table 1
[0027]
[0028] 3. Preparation of bacterial solution
[0029] Inoculate fresh cultures of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Bacillus subtilis onto a tryptic soy agar medium, inoculate fresh cultures of Clostridium sporogenes onto a thioglycollate fluid medium, and culture at 30°C to 35°C for 18 to 24 hours; inoculate fresh cultures of Candida albicans onto a Sabouraud dextrose agar medium, and culture at 20°C to 25°C for 24 to 48 hours. Prepare bacterial suspensions containing no more than 100 cfu per ml of the above cultures using 0.9% sterile sodium chloride solution.
[0030] Inoculate fresh culture of Aspergillus niger onto Sabouraud glucose agar slant medium, culture at 20°C to 25°C for 5 to 7 days, add 5 ml of 0.9% sodium chloride solution containing 0.05% (ml / ml) polysorbate 80 to elute the spores, then aspirate the spore suspension into a sterile test tube, and use 0.9% sodium chloride solution containing 0.05% (ml / ml) polysorbate 80 to prepare a spore suspension containing no more than 100 cfu per 1 ml.
[0031] 4. Culture medium sensitivity test
[0032] Take 7 bottles of polysorbate 80 thioglycollate fluid culture medium, each with a filling volume of 100 ml, inoculate two bottles with no more than 100 cfu of Staphylococcus aureus, two bottles with no more than 100 cfu of Pseudomonas aeruginosa, two bottles with no more than 100 cfu of Clostridium sporogenes, and the other bottle is not inoculated with any bacterial solution as a blank control, and culture for 3 days.
[0033] Take 7 bottles (bottle volume 250 ml) of polysorbate 80 tryptic soytone liquid culture medium (containing 0.07 g agar / 100 ml culture medium) with a capacity of 100 ml per bottle, inoculate two bottles with no more than 100 cfu of Bacillus subtilis solution, two bottles with no more than 100 cfu of Candida albicans solution, two bottles with no more than 100 cfu of Aspergillus niger solution, and leave the other bottle uninoculated as a blank control. Culture for 5 days and observe the results.
[0034] The culture results are shown in Table 2.
[0035] Table 2
[0036]
[0037] Example 2 Method Applicability Verification
[0038] Test sample: benzathine penicillin, specification: 1g / vial, 7 vials in total, manufacturer: North China Pharmaceutical Group Xiantai Pharmaceutical Co., Ltd.
[0039] Mix the benzathine penicillin sample with the penicillinase solution (add not less than 7500 units of penicillinase per 1 mg of benzathine penicillin) to prepare the test solution.
[0040] a) Test group
[0041] 5 ml of the test solution was taken and directly inoculated into 100 ml of polysorbate 80 thioglycollate fluid culture medium and 100 ml of polysorbate 80 tryptic soytone liquid culture medium (polysorbate 80 tryptic soytone liquid culture medium bottle volume 250 ml, and containing 0.07 g agar / 100 ml culture medium), cultured for not less than 14 days, and observed. The results are shown in Table 3. Among them, the culture temperature of the polysorbate 80 thioglycollate fluid culture medium after inoculation was 30°C to 35°C, and the culture temperature of the polysorbate 80 tryptic soytone liquid culture medium after inoculation was 20°C to 25°C.
[0042] b) Experimental Group 1
[0043] Take 5 ml of the test solution and inoculate it into 6 bottles of 100 ml polysorbate 80 thioglycollate fluid culture medium in sequence. After each group of polysorbate 80 thioglycollate fluid culture medium and the test solution are evenly mixed, add Staphylococcus aureus liquid containing no more than 100 cfu to two bottles of polysorbate 80 thioglycollate fluid culture medium, add Escherichia coli standard liquid containing no more than 100 cfu to two bottles of polysorbate 80 thioglycollate fluid culture medium, and add Clostridium sporogenes standard liquid containing no more than 100 cfu to two bottles of polysorbate 80 thioglycollate fluid culture medium. The inoculated polysorbate 80 thioglycollate fluid culture medium is cultured at 30℃~35℃ for no more than 5 days and observed.
[0044] Take 5 ml of the test solution and inoculate it into 6 bottles of 100 ml polysorbate 80 tryptic soytone liquid medium (containing 0.06 g agar / 100 ml medium, medium bottle volume 250 ml) in sequence. After each group of polysorbate 80 tryptic soytone liquid medium and the test solution are evenly mixed, add a standard bacterial solution of Bacillus subtilis with a bacterial count of no more than 100 cfu to two bottles of polysorbate 80 tryptic soytone liquid medium, add a standard bacterial solution of Aspergillus niger with a bacterial count of no more than 100 cfu to two bottles of polysorbate 80 tryptic soytone liquid medium, and add a standard bacterial solution of Candida albicans with a bacterial count of no more than 100 cfu to two bottles of polysorbate 80 tryptic soytone liquid medium. The inoculated polysorbate 80 tryptic soytone liquid medium is cultured at 20°C to 25°C for no more than 5 days and observed.
[0045] The preparation of each standard bacterial solution is the same as "3. Preparation of bacterial solution" in Example 1.
[0046] c) Experimental Group 2
[0047] The remaining conditions were the same as those of test group 1, except that 0.08 g agar was contained in each 100 ml of polysorbate 80 tryptic soy broth.
[0048] d) Experimental Group 3
[0049] The remaining conditions were the same as those of test group 1, except that 0.10 g agar was contained in each 100 ml of polysorbate 80 tryptic soy broth.
[0050] The results are shown in Table 3.
[0051] Table 3
[0052]
[0053]
[0054] The results of the validation test showed that compared with the test sample group, the test bacteria in each bottle of test group 1 and test group 2 grew well, the test amount of the test sample had no antibacterial effect under the test conditions, the amount of penicillinase was sufficient to inactivate all the test samples, and the amount of agar had no antibacterial effect. In test group 3, due to excessive agar, the standard strain was inhibited, and Bacillus subtilis grew slowly.
[0055] The above results show that the amount of penicillinase and agar in test group 1 and test group 2 is suitable for the test of the test product. This sterility test method can fully verify that the sterility test of the test product meets the regulations, and the sterility test of the test product can be carried out according to this test method and test conditions.
[0056] Example 3 Comparative test of benzathine penicillin sterility detection method
[0057] Test plan: The test methods shown in 1-5 were used for sterility testing and comparison in the test group and control group.
[0058] Experimental Group:
[0059] 5 ml of the test solution of Example 2 was taken and directly inoculated into 100 ml of polysorbate 80 thioglycollate fluid culture medium (the volume of the culture medium bottle was 250 ml) and 100 ml of polysorbate 80 tryptic soy liquid culture medium (containing 0.07 g agar / 100 ml culture medium, the volume of the culture medium bottle was 250 ml).
[0060] The inoculated polysorbate 80 thioglycollate fluid culture medium is cultured at 30°C to 35°C for not less than 14 days, and the inoculated polysorbate 80 trypticase soytone liquid culture medium is cultured at 20°C to 25°C for not less than 14 days.
[0061] Control group 1:
[0062] 5 ml of the test solution of Example 2 was taken and directly inoculated into 100 ml of polysorbate 80 thioglycollate fluid culture medium (the volume of the culture medium bottle was 120 ml) and 100 ml of polysorbate 80 tryptic soy liquid culture medium (the volume of the culture medium bottle was 120 ml and did not contain agar).
[0063] The inoculated polysorbate 80 thioglycollate fluid culture medium is cultured at 30°C to 35°C for not less than 14 days, and the inoculated polysorbate 80 trypticase soytone liquid culture medium is cultured at 20°C to 25°C for not less than 14 days.
[0064] Control group 2:
[0065] Benzathine penicillin and penicillinase solution were mixed (no less than 7500 units of penicillinase were added for every 1 mg of benzathine penicillin) to prepare a test solution; 5 ml of the test solution of Example 2 were directly inoculated into 100 ml of polysorbate 80 thioglycollate fluid culture medium (the volume of the culture medium bottle was 250 ml) and 100 ml of polysorbate 80 tryptic soy liquid culture medium (without agar, the volume of the culture medium bottle was 250 ml).
[0066] The inoculated polysorbate 80 thioglycollate fluid culture medium is cultured at 30°C to 35°C for not less than 14 days, and the inoculated polysorbate 80 trypticase soytone liquid culture medium is cultured at 20°C to 25°C for not less than 14 days.
[0067] Control group 3:
[0068] Benzathine penicillin and penicillinase solution were mixed (no less than 7500 units of penicillinase were added for every 1 mg of benzathine penicillin) to prepare a test solution; 5 ml of the test solution of Example 2 were directly inoculated into 100 ml of polysorbate 80 thioglycollate fluid culture medium (the volume of the culture medium bottle was 250 ml) and 100 ml of polysorbate 80 tryptic soy liquid culture medium (containing 0.04 g agar / 100 ml culture medium, the volume of the culture medium bottle was 250 ml).
[0069] The inoculated polysorbate 80 thioglycollate fluid culture medium is cultured at 30°C to 35°C for not less than 14 days, and the inoculated polysorbate 80 trypticase soytone liquid culture medium is cultured at 20°C to 25°C for not less than 14 days.
[0070] Control group 4:
[0071] Benzathine penicillin and penicillinase solution were mixed (no less than 7500 units of penicillinase were added for every 1 mg of benzathine penicillin) to prepare a test solution; 5 ml of the test solution of Example 2 were directly inoculated into 100 ml of polysorbate 80 thioglycollate fluid culture medium (the volume of the culture medium bottle was 250 ml) and 100 ml of polysorbate 80 tryptic soy liquid culture medium (containing 0.06 g agar / 100 ml culture medium, the volume of the culture medium bottle was 250 ml).
[0072] The inoculated polysorbate 80 thioglycollate fluid culture medium is cultured at 30°C to 35°C for not less than 14 days, and the inoculated polysorbate 80 trypticase soytone liquid culture medium is cultured at 20°C to 25°C for not less than 14 days.
[0073] Control group 5:
[0074] Benzathine penicillin and penicillinase solution were mixed (no less than 7500 units of penicillinase were added for every 1 mg of benzathine penicillin) to prepare a test solution; 5 ml of the test solution of Example 2 were directly inoculated into 100 ml of polysorbate 80 thioglycollate fluid culture medium (the volume of the culture medium bottle was 250 ml) and 100 ml of polysorbate 80 tryptic soy liquid culture medium (containing 0.08 g agar / 100 ml culture medium, the volume of the culture medium bottle was 250 ml).
[0075] The inoculated polysorbate 80 thioglycollate fluid culture medium is cultured at 30°C to 35°C for not less than 14 days, and the inoculated polysorbate 80 trypticase soytone liquid culture medium is cultured at 20°C to 25°C for not less than 14 days.
[0076] The test results of the experimental group and the control group 1-5 are shown in Table 4.
[0077] Table 4
[0078]
[0079] The test sample of the test group was completely dissolved at 5-7 days of culture, and no transfer was required after 14 days of culture. During observation and judgment, the thioglycollate fluid culture medium and the trypticase soytone liquid culture medium solution showed an obvious negative reaction, and there was no turbidity or other phenomena that interfered with the judgment results.
[0080] In control group 1, due to the small volume of the culture medium bottle and the absence of agar, the test sample precipitated at the bottom of the culture medium, which was not conducive to the full contact between the test sample and the enzyme. When observed for 14 days, the solution was still turbid and could not be judged. It was necessary to continue inoculating and culturing for 4 days before it could be judged as negative.
[0081] Although the volume of the culture medium bottle of control group 2 was increased, the test sample still precipitated at the bottom of the culture medium due to the absence of agar. The contact area with penicillinase was extremely small and the dissolution rate was slow. When observed for 14 days, the solution was still turbid and could not be judged. It was necessary to continue inoculating and culturing for 4 days before judging it as negative.
[0082] Although there is agar in control group 3, due to its low content, the test sample will still precipitate at the bottom of the culture medium, with a very small contact area with penicillinase, and the dissolution rate is slow. When observed for 14 days, the solution is still turbid and cannot be judged. It needs to be inoculated and cultured for another 4 days before being judged as negative.
[0083] The agar content of control group 4 and control group 5 can make the test sample suspended in the culture medium and fully contact with the penicillinase in the culture medium. When observed for 14 days, the test sample is completely dissolved and no transfer is required.
[0084] The above results show that the treatment process of control groups 1 to 3 interferes with the result judgment. The agar content in control group 4, control group 5 and the experimental group can make the test sample suspended in the culture medium and fully contact with the penicillinase in the culture medium. When observed and judged after 14 days of culture, the culture medium solution showed an obvious negative reaction, and there was no turbidity or other phenomena that interfered with the judgment result.
[0085] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.
Claims
1. A method for detecting the sterility of benzathine penicillin, characterized in that: The following steps are involved: (1) Mix the benzathine penicillin test sample and penicillinase solution thoroughly to prepare a test solution containing ≥ 7500 units of penicillinase per 1 mg of benzathine penicillin. (2) Take the test solution and directly inoculate it into polysorbate 80 thioglycollate fluid culture medium and polysorbate 80 trypticase soytone liquid culture medium respectively, and culture for not less than 14 days; directly observe and determine whether it is sterile; wherein the polysorbate 80 trypticase soytone liquid culture medium contains agar.
2. The method for sterility detection of benzathine penicillin according to claim 1, characterized in that: Each 100 ml of polysorbate 80 tryptic soy broth contains 0.06-0.08 g agar.
3. The method for sterility detection of benzathine penicillin according to claim 1, characterized in that: In step (2), the volume ratio of the test solution to the polysorbate 80 thioglycollate fluid culture medium and the polysorbate 80 trypticase soy liquid culture medium is 1:20-25.
4. The method for sterility detection of benzathine penicillin according to claim 3, characterized in that: In step (2), the volume ratio of the test solution to the polysorbate 80 thioglycollate fluid culture medium and the polysorbate 80 trypticase soy liquid culture medium is 1:
20.
5. The method for sterility detection of benzathine penicillin according to claim 1, characterized in that: The ratio of the volume of the polysorbate 80 trypticase soytone liquid culture medium bottle to the volume of the polysorbate 80 trypticase soytone liquid culture medium therein is ≥2.
5.
6. The method for sterility detection of benzathine penicillin according to claim 1, characterized in that: Take 5 mL of the test solution and inoculate it directly into 100 mL of polysorbate 80 thioglycollate fluid culture medium and 100 mL of polysorbate 80 tryptic soytone liquid culture medium, respectively. The volume of the polysorbate 80 tryptic soytone liquid culture medium bottle is 250 mL, and each 100 mL of polysorbate 80 tryptic soytone liquid culture medium contains 0.07 g of agar.
7. The method for sterility detection of benzathine penicillin according to claim 1, characterized in that: The polysorbate 80 trypticase soy broth was shaken once daily.
8. The method for sterility detection of benzathine penicillin according to claim 1, characterized in that: The polysorbate 80 thioglycollate fluid culture medium contains 0.5% polysorbate 80; the polysorbate 80 trypticase soytone liquid culture medium contains 0.5% polysorbate 80.
9. The method for sterility detection of benzathine penicillin according to claim 1, characterized in that: The culture temperature of the polysorbate 80 thioglycollate fluid culture medium is 30°C to 35°C, and the culture temperature of the polysorbate 80 trypticase soytone liquid culture medium is 20°C to 25°C.