Method for testing the microbial limit of estradiol dydrogesterone tablets

Abstract

The present application belongs to the technical field of drug detection, and particularly relates to a microbial limit test method for estradiol dydrogesterone tablets, which comprises the following steps: preparation of bacterial suspension, preparation of test solution, culture of different mixtures of bacterial suspension and test solution, and bacterial counting. The present application effectively neutralizes the bacteriostatic effect of estradiol and dydrogesterone steroid components by selecting 1% Tween 80-containing pH 7.0 sodium chloride-peptone buffer as a neutralization and dilution system, thereby solving the problem of matrix interference; a two-stage gradient test solution is designed to balance the detection sensitivity of control bacteria and the counting recovery rate; a three-stage bacterial separation process of Escherichia coli is matched with positive and negative controls to accurately detect the contamination control bacteria of tablets. The complete matching method has a wide application prospect, and the recovery rates of the six standard strains are stable and meet the requirements of the pharmacopoeia method verification, which can be directly used for the microbial quality detection of raw materials, finished products and factory microbial quality detection of enterprises.

Description

Technical Field

[0001] This invention belongs to the field of drug testing technology, specifically relating to a method for microbial limit testing of estradiol dydrogesterone tablets. Background Technology

[0002] Postmenopausal women experience a sustained decline in endogenous estrogen levels, leading to systemic metabolic and reproductive tract physiological changes. This not only causes typical menopausal symptoms such as sleep disturbances, mood swings, and osteoporosis, but also results in reproductive tract mucosal atrophy, reduced epithelial glycogen content, a decrease in the dominant vaginal lactobacillus flora, and a significant decline in the reproductive tract's immune barrier function. Estradiol-dydrogesterone tablets are an oral hormonal compound preparation, belonging to the steroid hormone class of drugs. Composed of two active ingredients: the estrogen estradiol and the progesterone dydrogesterone, it effectively treats postmenopausal estrogen deficiency symptoms, protects the endometrium, improves the reproductive tract microecological environment, and reduces the risk of pathogenic microorganism infection, demonstrating excellent clinical application value and safety.

[0003] According to the Chinese Pharmacopoeia's guidelines for microbial limit testing of non-sterile drugs and ICH quality risk management principles, oral preparations must strictly control the total number of aerobic bacteria, the total number of molds and yeasts, and the detection of controlled bacteria such as Escherichia coli. Microbial limit testing refers to the determination of the total number of microorganisms and pathogenic bacteria in medicinal materials or preparations to evaluate their degree of microbial contamination and safety. The steroidal antibacterial components in estradiol dydrogesterone tablets can affect the growth and detection of microorganisms. Therefore, even if a sample contains a large number of microorganisms, the interference of the steroidal antibacterial active ingredients may prevent their normal growth and detection, resulting in low microbial limit test results or false negatives.

[0004] To address the interference of antimicrobial components in medicinal materials or preparations on microbial limit testing, existing technologies mainly employ the following methods: membrane filtration, which uses a rinsing solution to wash the filter membrane, removing the antimicrobial active ingredient and thus reducing its antimicrobial effect; and plate counting, which uses a diluent or culture medium containing a neutralizing agent, which can bind to the antimicrobial component and reduce its antimicrobial effect.

[0005] Currently, there is no suitable microbial detection method for estradiol dydrogesterone tablets. There is an urgent need to solve the problem of steroid matrix interference with the antibacterial activity of estradiol dydrogesterone tablets, so as to achieve precise control of microbial contamination of tablets and improve the batch quality stability of products. Summary of the Invention

[0006] The purpose of this invention is to provide a method for microbial limit testing of estradiol dydrogesterone tablets, solving the problem of interference of the steroid matrix in estradiol dydrogesterone tablets with microbial limit testing, forming a complete, reproducible, and pharmacopoeia-compliant method applicability verification scheme, achieving precise control of tablet microbial contamination, reducing the risk of clinical infection, and improving the batch quality stability of the product.

[0007] To achieve the above objectives, the present invention provides a method for microbial limit testing of estradiol dydrogesterone tablets, comprising the following steps: S1. Prepare bacterial suspension; S2. Preparation of test solution: Grind the diol dydrogesterone tablets and prepare the test solution with diluent; S3. Mix the bacterial suspension and test solution according to different combinations, and then inject them into the petri dish for incubation. S4. Counting of bacterial strains: Count the strains in the culture dishes after S3 culture.

[0008] Preferably, in step S1, the bacterial strains in the bacterial suspension include Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger, and Escherichia coli. Among them, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Aspergillus niger are aerobic bacteria; Candida albicans is a yeast; Aspergillus niger is a mold; and Escherichia coli is a control bacterium.

[0009] Preferably, in step S1, the cultures of Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans are prepared into bacterial suspensions with a concentration of 5000-10000 cfu / mL using buffer solution. After culturing, the mold culture was prepared into a spore suspension with a bacterial concentration of 5000-10000 cfu / mL using a buffer solution; The cultured Escherichia coli was prepared into a bacterial suspension with a concentration of 10–100 cfu / mL using buffer solution; The buffer solution was a pH 7.0 sterile sodium chloride-peptone buffer.

[0010] Preferably, in step S2, the test solution is divided into a 1:10 test solution and a 1:50 test solution; The ratio of estradiol dydrogesterone tablets to diluent in the 1:10 test solution is 10g:90ml. The 1:50 test solution is prepared by diluting the 1:10 test solution.

[0011] Preferably, the diluent in step S2 is a sterile sodium chloride-peptone buffer at pH 7.0 containing 1% (g / mL) Tween 80.

[0012] Preferably, in step S3, the bacterial suspension obtained in S1 and the test solution obtained in S2 are used to culture for the determination of the total number of aerobic bacteria, molds and yeasts: Escherichia coli is first enriched and then cultured by streak plating.

[0013] Preferably, in step S3, the aerobic bacteria assay uses tryptic soy agar medium; the mold and yeast assay uses Sabouraud dextrose agar medium; and the Escherichia coli enrichment culture uses tryptic soy liquid medium and MacConkey liquid medium sequentially for enrichment culture, followed by streak culture on MacConkey agar plates.

[0014] Preferably, in step S3, the culture set for determining the total number of aerobic bacteria, molds and yeasts includes an experimental group containing a 1:50 test solution and a bacterial suspension, a bacterial control group containing a dilution and a bacterial suspension, and a test sample control group containing only a 1:50 test solution. Three control groups were set up for the culture of Escherichia coli: a test sample control group containing 1:10 test solution, a positive control group containing 1:10 test solution and Escherichia coli suspension, and a negative control group containing only dilution.

[0015] Preferably, in step S3, when determining the total number of aerobic bacteria, the culture temperature is 30-35℃ and the culture time is 3-5 days; when determining the total number of molds and yeasts, the culture temperature is 20-25℃ and the culture time is 5-7 days. Tryptic soy peptone liquid medium, culture temperature 30-35℃, culture time 18-24 hours; MacConkey liquid medium, culture temperature 42-44℃, culture time 24-48 hours; MacConkey agar streak plate, culture temperature 30-35℃, culture time 18-72 hours.

[0016] The present invention also provides the application of the aforementioned examination method in detecting the number of microorganisms in estradiol dydrogesterone tablets.

[0017] The advantages and positive effects of the microbial limit test method for estradiol dydrogesterone tablets described in this invention are: 1. It effectively eliminates matrix antibacterial interference, improves the accuracy of microbial counting and control bacteria screening, and effectively reduces the rate of exogenous microbial contamination during drug production and storage.

[0018] 2. Significantly improved the product's microbiological safety and batch quality stability, making up for the shortcomings of existing technologies in quality control and clinical safety. Detailed Implementation

[0019] The technical solution of the present invention will be further described below through embodiments.

[0020] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0021] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0022] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.

[0023] All test strains were purchased from the National Institutes for Food and Drug Control, and the strain numbers are as follows: Staphylococcus aureus [CMCC(B)26003] Pseudomonas aeruginosa [CMCC(B)10104] Bacillus subtilis [CMCC(B)63501] Escherichia coli [CMCC(B)44102] Candida albicans [CMCC(F)98001] Aspergillus niger [CMCC(F)98003] The pH 7.0 sodium chloride-peptone buffer used in the experiment was purchased from Beijing Sanyao Technology Co., Ltd., batch number 11218; Tween 80 was purchased from Sinopharm Chemical Reagent Co., Ltd., batch number 30189828.

[0024] Example 1: Detection of total aerobic bacteria, molds, and yeast counts (1) Preparation of bacterial suspension: Preparation of buffer solution: Weigh 14.6g of pH 7.0 sodium chloride-peptone buffer powder, dissolve in water to 1000ml, autoclave at 121℃ for 15 min, and set aside.

[0025] ① Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli Fresh cultures of Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis, which have been cultured at 30–35°C for 18–24 hours, are prepared into bacterial suspensions with a concentration of 5,000–10,000 CFU / mL using buffer solution.

[0026] Escherichia coli: Take fresh Escherichia coli culture that has been cultured at 30-35℃ for 18-24 hours and prepare a bacterial suspension with a concentration of 10-100 cfu / mL using buffer solution.

[0027] ② Candida albicans Fresh cultures of Candida albicans that have been cultured at 20–25°C for 2–3 days are prepared into a bacterial suspension with a concentration of 5000–10000 cfu / mL using buffer solution.

[0028] ③ Aspergillus niger Take a fresh culture of Aspergillus niger that has been cultured at 20-25℃ for 5-7 days, add 3-5 ml of buffer solution, wash off the spores and shake well to prepare a spore suspension. Transfer the spore suspension into a sterile test tube and prepare a spore suspension with a concentration of 5000-10000 cfu / mL per spore using buffer solution.

[0029] (2) Preparation of test solution: The diluent was prepared as follows: Weigh 1 g of Tween 80 and 1.46 g of pH 7.0 sodium chloride peptone buffer powder, dissolve them in water to 100 ml, autoclave at 121℃ for 15 min, and set aside.

[0030] Weigh 10g of estradiol dydrogesterone tablets and grind them into a fine powder in a mortar. Place the powder in a sterile Erlenmeyer flask, add 90ml of diluent, and shake well to prepare a 1:10 test solution. Take 20ml of the 1:10 test solution and add it to 80ml of diluent to prepare a 1:50 test solution.

[0031] (3) Determination of total aerobic bacteria, molds and yeasts: ① Experimental group Aerobic bacteria determination: Take 9.9 ml of 1:50 test solution from each of the five samples and mix them with 0.1 ml of bacterial suspensions of Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Aspergillus niger. Take 1 ml of each suspension and inject it into a Petri dish of tryptic soy agar medium. Incubate the Petri dishes upside down at 30-35℃ for 3 days. The counting results are shown in Table 1. Mold and yeast determination: Take two 9.9 ml portions of 1:50 test solution and mix them with 0.1 ml of Candida albicans and Aspergillus niger suspensions respectively. Take 1 ml of each suspension and inject it into a Petri dish of Sabouraud dextrose agar medium. Incubate the Petri dishes upside down at 20-25℃ for 5 days. The counting results are shown in Table 2.

[0032] ② Bacterial suspension control group Aerobic bacteria determination: Take 9.9 ml of each of the five dilutions and mix them with 0.1 ml of bacterial suspensions of Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Aspergillus niger, respectively. Take 1 ml of each suspension and inject it into a Petri dish of tryptic soy agar medium. Incubate the Petri dishes upside down at 30-35℃ for 3 days. The counting results are shown in Table 1. Mold and yeast enumeration: Take 2 portions of the diluent, mix each with 0.1 ml of Candida albicans and Aspergillus niger bacterial suspensions respectively in sterile test tubes, take 2 ml of each mixture and inoculate onto the plates of Sabouraud Dextrose Agar medium, incubate the plates in an inverted position at 20-25°C for 5 days, and the counting results are shown in Table 2.

[0033] ③ Test article control group Aerobic bacteria enumeration: Take 1 ml of the 1:50 test solution and inoculate it onto the plate of Tryptic Soy Agar medium, prepare 5 parallel experiments, incubate the plates in an inverted position at 30-35°C for 5 days, perform combined counting, and the counting results are shown in Table 1; Mold and yeast enumeration: Take 1 ml of the 1:50 test solution and inoculate it onto the plate of Sabouraud Dextrose Agar medium, prepare 5 parallel experiments, incubate the plates in an inverted position at 20-25°C for 7 days, perform combined counting, and the counting results are shown in Table 2.

[0034] ④ Negative control group Aerobic bacteria enumeration: Take 1 ml of the diluent and inoculate it onto the plate of Tryptic Soy Agar medium, prepare 3 parallel experiments, incubate the plates in an inverted position at 30-35°C for 5 days, perform combined counting, and the counting results are shown in Table 1; Mold and yeast enumeration: Take 1 ml of the diluent and inoculate it onto the plate of Sabouraud Dextrose Agar medium, prepare 3 parallel experiments, incubate the plates in an inverted position at 20-25°C for 7 days, perform combined counting, and the counting results are shown in Table 2.

[0035] After incubation, perform enumeration of the total number of aerobic bacteria, molds and yeasts: The ratio of the value obtained by subtracting the colony count of the test article group from the colony count of the test group to the colony count of the bacterial suspension group is within the range of 0.5 to 2.0, which is qualified; the measurement results are shown in Table 1 and Table 2. The ratios of aerobic bacteria, molds and yeasts are all within the range of 0.5 to 2.0, and the tested test article is qualified.

[0036] Table 1 Aerobic bacteria enumeration results

[0037] Table 2 Mold and yeast enumeration results

[0038] The results in Tables 1 and 2 show that the total aerobic bacteria count and the recovery rates of molds and yeasts in this experiment both met the acceptable range specified in the pharmacopoeia. This indicates that the selected sterile sodium chloride-peptone buffer solution containing 1% (g / mL) Tween 80 pH 7.0 effectively neutralized the inherent antibacterial active substances in the test sample, relieving the inhibitory effect of the test sample matrix on the indicator bacteria. The antibacterial components inherent in the drug itself can damage or inhibit the recovery and growth of contaminating microorganisms during microbial detection. If an effective neutralization and mitigation system is not used, the inoculated strains may not proliferate normally, resulting in a low recovery rate and, consequently, false negatives in the microbial limit test of the sample, failing to accurately reflect the actual microbial contamination level of the product.

[0039] This method, by adding a sterile sodium chloride-peptone buffer solution containing 1% (g / mL) Tween 80 at pH 7.0, neutralizes the antibacterial activity in the test sample, eliminates growth interference from the matrix, and allows the added test strains to resume normal growth. The recovery rates of aerobic bacteria, molds, and yeasts meet the standards, proving the applicability of this microbial limit method. Microbial limit testing of samples conducted under these conditions can effectively detect potential bacterial and fungal contamination in samples, and the test results are reliable and can objectively evaluate the microbial safety and quality of the drug. This also indirectly demonstrates that the type and volume of the buffer solution are well-matched with the experimental dilution system, achieving neutralization of the antibacterial components without toxicly inhibiting the indicator bacteria themselves. The effectiveness of the neutralization system is verified, providing a reliable experimental basis for the microbial limit testing of this product.

[0040] Example 2: Examination of control bacteria Test for Escherichia coli: Test sample group: Take 10 ml of 1:10 test solution and place it in 400 ml of tryptic soy liquid medium, mix well, and incubate at 30-35℃ for 18-24 hours; take 1 ml of the above culture and inoculate it into 100 ml of MacConkey liquid medium, and incubate at 42-44℃ for 24-48 hours; take MacConkey liquid culture and streak it onto MacConkey agar plates, and incubate at 30-35℃ for 18-72 hours. The test results are shown in Table 3.

[0041] Positive control group: Take 10 ml of the 1:10 test sample and place it in 400 ml of tryptic soy liquid medium; add 1 ml of Escherichia coli suspension and mix well, and incubate at 30-35℃ for 18-24 hours; take 1 ml of the above culture and inoculate it into 100 ml of MacConkey liquid medium, and incubate at 42-44℃ for 24-48 hours; take MacConkey liquid culture and streak it onto MacConkey agar plates, and incubate at 30-35℃ for 18-72 hours. The test results are shown in Table 3.

[0042] Negative control group: Take 10 ml of sterile sodium chloride-peptone buffer (pH 7.0) and place it in 400 ml of tryptic soy broth. Mix well and incubate at 30–35°C for 18–24 hours. Take 1 ml of the above culture and inoculate it into 100 ml of MacConkey broth. Incubate at 42–44°C for 24–48 hours. Streak the MacConkey broth onto MacConkey agar plates and incubate at 30–35°C for 18–72 hours. The test results are shown in Table 3.

[0043] Table 3. Results of Escherichia coli test

[0044] Escherichia coli, as a representative indicator bacterium for the control microbial testing of non-sterile drugs, mainly originates from the intestines of humans and animals. It is an important marker reflecting whether a drug has been contaminated by feces, and also indicates the potential risk of Gram-negative pathogenic bacteria contamination in the sample. A passing result for the Escherichia coli test in the microbial limit control microbial test indicates that the target control bacterium was not detected in the test sample under the given test conditions, suggesting that the drug has not been contaminated by intestinal-derived microorganisms.

[0045] As shown in Table 3, the test sample contained antibacterial active substances, and the test background was neutralized using a sterile sodium chloride-peptone buffer solution containing 1% (g / mL) Tween 80 pH 7.0. The control bacteria test results were satisfactory, indicating that the neutralized test system would not cause the target bacteria to be suppressed or missed due to interference from the sample's antibacterial components. The test method can effectively detect Escherichia coli that may be present in the test sample. This result corroborates the applicability results of the aerobic bacteria and mold / yeast counting methods, further proving the applicability of the entire microbial limit test system. It can eliminate the risk of false negatives in control bacteria due to the sample's antibacterial effect, and can accurately reflect the microbial status of the product, providing experimental basis at the control bacteria level for the microbial safety evaluation of this drug.

[0046] Therefore, this invention employs a microbial limit test method for estradiol-dydrogesterone tablets with the aforementioned structure. A pH 7.0 sodium chloride-peptone buffer containing 1% Tween 80 is selected as the neutralization and dilution system, effectively neutralizing the antibacterial effects of estradiol and dydrogesterone steroidal components and solving the problem of matrix interference. A two-stage gradient test solution is designed (1:10 for control bacteria and 1:50 for colony counting), balancing the detection sensitivity and recovery rate of control bacteria. Differentiated culture media, culture temperatures, culture times, and dish volumes for aerobic bacteria, molds, and yeasts significantly reduce counting errors. A standardized three-stage enrichment and isolation process for Escherichia coli, combined with positive and negative controls, eliminates false negatives / false positives, accurately detecting control bacteria contaminating the tablets. This complete and comprehensive method applicability validation system shows stable and compliant recovery rates for six standard strains, meeting pharmacopoeia method validation requirements. The entire method is specifically developed for estradiol-dydrogesterone tablets and can be directly used for microbial quality testing of raw materials and finished products, demonstrating broad application prospects.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for microbial limit testing of estradiol dydrogesterone tablets, characterized in that, Includes the following steps: S1. Prepare bacterial suspension; S2. Preparation of test solution: Grind estradiol dydrogesterone tablets and prepare test solution with diluent; S3. Mix the bacterial suspension and test solution in different combinations and then inject them into a petri dish for incubation. S4. Counting of bacterial strains: Count the strains in the culture dishes after S3 culture.

2. The method for microbial limit testing of estradiol dydrogesterone tablets according to claim 1, characterized in that, In step S1, the bacterial strains used to prepare the bacterial suspension include Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger, and Escherichia coli. Among them, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Aspergillus niger are aerobic bacteria; Candida albicans is a yeast; Aspergillus niger is a mold; and Escherichia coli is a control bacterium.

3. The method for microbial limit testing of estradiol dydrogesterone tablets according to claim 2, characterized in that, In step S1, the cultures of Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans are prepared into bacterial suspensions with a concentration of 5000-10000 cfu / mL using buffer solution. After culturing, the mold culture was prepared into a spore suspension with a bacterial concentration of 5000-10000 cfu / mL using a buffer solution; The cultured Escherichia coli was prepared into a bacterial suspension with a concentration of 10–100 cfu / mL using buffer solution; The buffer solution was a pH 7.0 sterile sodium chloride-peptone buffer.

4. The method for microbial limit testing of estradiol dydrogesterone tablets according to claim 1, characterized in that: In step S2, the test solution is divided into a 1:10 test solution and a 1:50 test solution; The ratio of estradiol dydrogesterone tablets to diluent in the 1:10 test solution is 10g:90ml. The 1:50 test solution is prepared by diluting the 1:10 test solution.

5. The method for microbial limit testing of estradiol dydrogesterone tablets according to claim 4, characterized in that: In step S2, the diluent is a sterile sodium chloride-peptone buffer at pH 7.0 containing 1% (g / mL) Tween 80.

6. The method for microbial limit testing of estradiol dydrogesterone tablets according to claim 1, characterized in that: In step S3, the bacterial suspension obtained in S1 and the test solution obtained in S2 are used to culture aerobic bacteria, molds and yeasts for total count determination: Escherichia coli is first enriched and then cultured on a plate.

7. The method for microbial limit testing of estradiol dydrogesterone tablets according to claim 6, characterized in that: In step S3, tryptic soy agar medium was used for aerobic bacteria determination; Sabouraud dextrose agar medium was used for mold and yeast determination; and Escherichia coli enrichment culture was carried out sequentially using tryptic soy liquid medium and MacConkey liquid medium, followed by streak culture on MacConkey agar plates.

8. The method for microbial limit testing of estradiol dydrogesterone tablets according to claim 6, characterized in that: In step S3, the culture set for determining the total number of aerobic bacteria, molds and yeasts includes an experimental group containing 1:50 test solution and bacterial suspension, a bacterial control group containing dilution and bacterial suspension, and a test sample control group containing only 1:50 test solution. Three control groups were set up for the culture of Escherichia coli: a test sample control group containing 1:10 test solution, a positive control group containing 1:10 test solution and Escherichia coli suspension, and a negative control group containing only dilution.

9. The method for microbial limit testing of estradiol dydrogesterone tablets according to claim 7, characterized in that: In step S3, when determining the total number of aerobic bacteria, the culture temperature is 30-35℃ and the culture time is 3-5 days; when determining the total number of molds and yeasts, the culture temperature is 20-25℃ and the culture time is 5-7 days. Tryptic soy peptone liquid medium, culture temperature 30-35℃, culture time 18-24 hours; MacConkey liquid medium, culture temperature 42-44℃, culture time 24-48 hours; MacConkey agar streak plate, culture temperature 30-35℃, culture time 18-72 hours.

10. The application of the examination method according to any one of claims 1-9 in detecting the number of microorganisms in estradiol dydrogesterone tablets.

Images