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This study focuses on the microbial quality control of the Chinese herbal decoction pieces. In view of the shortcomings of traditional culture methods such as slow detection speed and inability to detect unculturable microorganisms, a new method based on ATP bioluminescence technology combined with statistical analysis methods was established to rapidly predict and quantitatively detect the total aerobic microbial count (TAMC) and total yeast and mold count (TYMC) contaminated Bupleurum chinense DC. decoction pieces. Based on the optimized ATP bioluminesence detection system, accurate detection of pure bacterial solution of Escherichia coli, Bacillus subtilis and Staphylococcus aureus can be achieved, with detection limits of 47.86, 89.13 and 1 862.09 CFU·mL-1, respectively. The detection time was 6.5 h, and the detection cost was as low as 2 yuan/time. The upper and lower warning limits of TAMC were determined by the misjudgment rates of 10% and 20%, respectively. And the warning limit of TYMC was determined by the misjudgment rate of 20%. The proposed crossing method could quickly predict the amount of microbial contamination in Bupleurum chinense DC. decoction pieces. The constructed partial least squares regression (PLSR) model could accurately quantify the quantity of microbial contamination in Bupleurum chinense DC. decoction pieces. The optimal PLSR prediction model for TAMC had a correction coefficient (R2) of 0.826, a root mean square error of correction set (RMSEE) of 0.468 and a root mean square error of cross-validation set (RMSECV) of 0.465. The R2, RMSEE and RMSECV in the prediction model of TYMC were 0.778, 0.543 and 0.541, respectively. The aim of this study is to establish a kind of rapid detection method and prediction models for the microbial limit of traditional Chinese medicine and Chinese herbal decoction pieces, and to provide a more convenient and sensitive detection technology for the microbial quality process control of traditional Chinese medicine products.
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ObjectiveTo establish microbial limit test methods for 44 pediatric drugs. MethodsAccording to the general guidelines in Chinese Pharmacopoeia （2015 and 2020 edition， volume Ⅳ），a suitability test of the methods for 44 drugs was carried out by pour-plate method， neutralization method or dilution method. ResultsTotal aerobic microbial count： chemical oral liquid samples can be tested by 1∶10 plate method；traditional Chinese medicine need to be neutralized firstly. Then oral liquids could be tested by 1∶10 plate method and 1∶100 plate method was used for granules. Total count of molds and yeasts： all the samples can be tested by the 1∶10 plate method. The recoveries of five test strains were between 0.5 and 2.0. The specified microorganisms were all detected in the test group， while not found in the negative control group. ConclusionThe microbial limit test methods for the 44 pediatric drugs are established and the results are reliable and can be used in the quality control．
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【Objective】 To establish a microbial limit test method for diatomite and pearlite, and verify its applicability. 【Methods】 According to the requirements of general rule 1105, Microbial Limit Test for Non Sterile Products of Pharmacopoeia of the People′s Republic of China (2020 Edition), the applicability test of microbial counting methods for three batches of perlite and diatomite was conducted before the microbial limit test of samples. The microbial growth of filter aid was analyzed and the recovery rate of each test bacterium was calculated. 【Results】 The ratio of the colony number of the test group minus the colony number of the test sample control group to the bacterial liquid control group was in the range of 0.5~2.0. 【Conclusion】 The method is accurate, reliable and can be used for microbial limit test of diatomite and perlite.
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Objective: To establish the quality standard for Fuzheng capsules. Methods: TLC was adopted to identify Astragali Radix and Glycyrrhiza uralensis. The method validation for Fuzheng capsules was conducted by microbial limit test as described in the appendixes of Chinese Pharmacopeia (2015 edition). The content of epimedii in Epimedium brevicornu was determined by HPLC. The chromatographic separation was carried out on an Agilent TC-C18(2) (250 mm×4. 6 mm, 5 μm) column. The mobile phase consis-ted of acetonitrile-water( 30: 70) with gradient elution at a flow rate of 1. 0 ml·min-1,and the injection volume was 5 μl. The detec-tion wavelength was 270 nm. Results: The spots in TLC were clear without any interference. The methods of plating and direct inocu-lation could be used for the microbial limit test. The linear range was 0.101-1.008 μg (r =0.999 7). The average recovery was 99. 36% with the RSD of 0. 81% (n=6). Conclusion: The method is simple with high specificity and good repeatability, which can be used as the quality control method for Fuzheng capsules.
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Objective:To investigate the microbial contamination status of Chinese herbal pieces from Guangxi regions and provide reference for the microbial limit standard. Methods:According to Chinese Pharmacopoeia (2015 edition), the herbal pieces were analyzed for the total number of aerobe microbial, yeasts, molds, thermophilic bacteria and bile-resistant gram-negative bacteria,and salmonella was also detected. Results:In 90 batches of Chinese herbal pieces,the lg average value of the total number of aerobe microbial was 3.7,and that of the total yeasts and molds was 1.7,that of thermophilic bacteria was 0.9 and that of bile-resistant gram-negative bacteria was 1.1,and Salmonella was not detected out. There were statistically significant differences (P < 0.05) in the microbial contamination levels of herbal pieces with different processing techniques and packaging materials. Conclusion:According to different processing methods and administration, microbial limit standard should be established for classification in order to guide the standardized production of Chinese herbal pieces and improve their quality gradually.
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Objective To establish a method for the microbial limit test of zinc oxide and talcum powder lotion in Chinese Pharmacopoeia 2015.Methods Microbial enumeration test and specified microorganisms test with instructions were conducted from Chinese Pharmacopoeia 2015,which involved 10 batches zinc oxide and talcum powder lotion and five species of bacteria in all.The samples were treated by centrifugation and membrane filtration.Microbial enumeration test:the total number of aerobic bacteria using TSA medium to examine,strains were Staphylococcus aureus,Pseudomonas aeruginosa,Bacillus subtilis,Candida albicans,and Aspergillus niger;The total number of molds and yeasts using SDA culture medium to examine,strains were Candida albicans and Aspergillus niger.Each strain was divided into two groups:Adding bacteria test group and bacteria control group.Samplecontrol group and negative control group of each culture medium was prepared respectively.Count the colonies and calculate the rate of recovery.Specified microorganisms test:Pseudomonas aeruginosa and Staphylococcus aureus were applicated and set the bacteria validation group,sample control group,and negative control group.The culture medium of each group was crossed on the corresponding medium plate,and Microflex LT mass spectrometer was used to identify the bacteria.Results The recoveries of all kinds of strains in microbial enumeration test,total yeast and mold count were 0.75-1.16 in all batches.In the validations of Staphylococcus aureus and Pseudomonas aeruginosa,all kinds of strains were respectively detected in bacteria validation group,and there were no bacterial growth in sample control group and negative control group.Conclusion The microbiological examination methods for zinc oxide and talcum powder lotion can meet the requirements of Chinese Pharmacopoeia 2015.
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Objective:To investigate the neutralization of lecithin and polysorbate-80 in antibacterial activity of oral solid tradition-al Chinese medicine preparations without Chinese medicinal herb powder. Methods:According to the requirements in Chinese Pharma-copeia (2015 edition), the applicability of microbial counting method was tested in the diluent containing lecithin and polysorbate-80 and the conventional diluent, respectively, and the results were compared. Results: The recovery results of applicability of microbial counting method using neutralizers (lecithin and polysorbate-80) conformed to the standard of Chinese pharmacopoeia (2015 edition). Conclusion:The combination of lecithin and polysorbate-80 has notable neutralization in antibacterial activity of oral solid traditional Chinese medicine preparations without Chinese medicinal herb powder.
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Objective: To establish a microbial limit test method for compound benzocaine gel.Methods: According to the general principles of Chinese Pharmacopoeia (2015 edition), method applicable experiments were performed respectively for the routine method, neutralization method and dilution & neutralization method.Using the recovery ratio of test bacteria as the index, the medium of dilution & neutralization method was adopted in the total aerobic microbial count and total combined yeasts and molds count, and the neutralization method was used for the control bacteria detection.The neutralizing agents were polysorbate 80 and lecithin.Results: The method of dilution & neutralization could eliminate the bacteriostasis of the drug, and the recovery ratio of each test bacteria was within the range of 0.5-2.0, which was in line with the requirements of Chinese Pharmacopoeia.Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus could be detected out in the positive control bacterial test,and bacteria were not detected out in the negative test.Conclusion: The method can be used for the microbial limit test for compound benzocaine gel.
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OBJECTIVE:To establish a method of microbial limit test for liquid preparation of Iron sucrose injection before filtration and sterilization.METHODS:According to the microbial limit test in the 1005 and general rules 1006 of 2015 edition of Chinese Pharmacopoeia (vol.Ⅴ),plate method and membrane filtration method were used to measure total number of aerobic bacteria (Staphylococcus aureus,Pseudomonas aeruginosa,Bacillus subtilis,Candida albicans,Aspergillus niger) and total number of molds and yeasts (C.albicans,A.niger).The optimal test method was obtained by comparing the bacterial recoveries.RESULTS:By plate method,the recoveries of P aeruginosa and B.subtilis were 2％ and 5％.The test sample was diluted 10 times with pH 7.0 sodium chloride-peptone buffer solution,and the bacterial recoveries were in the range of 88％ to 96％;but he medium was dark in color.By membrane filtration method,without rinse solution,the bacterial recoveries in the range of 88％ to 95％.Add rinse solution,the bacterial recoveries were in the range of 91％ to 103％.After validated,the recoveries of menbrane filtration method with tlushing fluid ranged 50％-200 ％,which was in line with the requnements.CONCLUSIONS:The membrane filtration method established in this experiment has higher bacterial recovery rate than the plate method.The bacterial recoveries rate were higher after adding rinse solution,and no dark substance in the surface of filter membrane affect the accotmt.It can be used as the microbial limit test method for preparation liquid of Iron sucrose injection before filtration and sterilization.
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Objective:To eliminate the interference of matrix in urea cream on the microbial limit test. Methods: A certain a-mount of magnesium sulfate was added to the test solution followed by stirring and placing sometime to make the cream layering, and the water layer was used for the microbial limit detection. Results:After magnesium sulfate was added to urea cream, microbial limit test was not affected by the matrix. The average recovery of Escherichia oli, Staphylococcus aureus, Bacillus subtilis, Candida albicans and Aspergillus niger was 96. 00%, 97. 00%, 100. 00%, 99. 00% and 101. 00%, respectively. The negative and positive results of Psudomonas aeruginosa and Staphylococcus auress were reliable. Conclusion:In the detection of microbial limit for urea cream, mag-nesium sulfate can be used as a good auxiliary agent to make the detection method more convenient and reliable.
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Objective: To provide a method of microbial limit test for Changyanning tablets and carry out the verification of the mothod.Methods: A plate method was used in the total aerobic microbial count and the concentration of the test liquid was 1∶100.A plate method was used in the total yeast and mold count while the concentration of the test liquid was 1∶10.The direct inoculation method was used for the detection of Escherichia coli,bile-tolerant gram-negative bacteria and Salmonella enterica subsp.Results: The recoveries of the five strains were from 0.5 to 2.0.Conclusion: The method is suitable for the microbial limit examination of Changyanning tablets.
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Objective:To establish the microbial limit test methods for three preparations containing chlorhexidine acetate. Meth-ods:The validation of microbiological limit test for three preparations including chlorhexidine acetate solution, chlorhexidine acetate ointment and chlorhexidine acetate cream was carried out respectively using the conventional method,dilution method,membrane filtra-tion method and neutralization method. Results:The recovery rates of five strains in the verification of counting method by membirane filtration and nearailzation method were more than 50%. Every positive test micrcorganism could be detected while negative control bacteria could not grow in the examination of control bacteria.Conclusion:The membrane filtration method can be used for the deter-mination of control bacteria for chlorhexidine acetate solution and chlorhexidine acetate ointment,and the neutralization method can be applied in the determination of control bacteria for chlorhexidine acetate cream.
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Objective To establish the methods of microbial limit test for three kinds of preparations with heavy metals, such asDingxian Pills,Pizhi Lotion andJiawei Huangqin Ointment.Methods According to Chinese Pharmacopoeia 2010, the recovery rates of bacteria, fungus and yeast treated by three preparations were detected. And the methods of testing control bacteria were also validated.Results Culture medium dilution method was proved to be applicable forDingxian Pills. Culture medium dilution method combined with pre-filtration method was proper for Pizhi Lotion. And the extraction method was adopted forJiawei Huangqin Ointment. The recovery rates of these five validation strains reached 70% by appropriate methods. And the same methods were used for validation of the control bacteria.Conclusion The methods of microbial limit test for these three different preparations were established through this study.
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Decoction pieces of Chinese herbal medicine (CHM) was processed by Chinese medicinal materials, and generally the sources of medicinal materials were from animals, botanical, mineral drugs, etc., with itself microorganisms which could be introduced in the production process too. The microbial growth and reproduction in pieces will affect the quality of the pieces, even some of the harmful microorganisms affect the health and safety of patients through the oral administration. Therefore, it is important to sterilize the pieces under the controlled microbial limits. This article summarizes the microbial control of CHM research status and commonly sterilization methods in the production process, considers and discusses the problems on sterilization issues of CHM, and puts forward the suggestion on the thoughts of a special focus on "prototype pieces" sterilization, the establishment of its microbial limit, which could improve the quality standard of CHM so as to ensure the safely and effecacy of CHM pieces.
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Objective:To establish a method for the microbial limit test of Jingzhi Guanxin tablets. Methods: According to the methods in the 2010 edition and 2015 edition of Chinese Pharmacopoeia, the test was performed respectively. Results:Jingzhi Guanxin tablets showed obvious inhibitory effect on Bacillus subtilis. The bacteria count could be carried out by the culture medium diluting methods in the 2010 edition. The total amount of aerobe could be detected by the membrane filtration method in the 2015 edition. The total combined molds and yeasts count could be performed by the plate count method and the specified microorganism could be tested with the routine method. Conclusion:The above methods can be used for the microbial limit test of Jingzhi Guanxin tablets.
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Objective To establish a method of microbial limit tests for compound Hongjingtian oral liquid and to per-form the validation of the method.Methods The validation on the microbial limit test method was conducted according to the counting method of bacteria,mycetes and yeasts and the control bacteria test method stated in the appendix of China pharmaco-poeia (2010 edition).Results The recoveries were over 70% when culture diluting method was used for staphylococcus aureus and routine test method was used for Escherichiacoli,Bacillus subtilis,Candida albicans and Aspergillus niger.In the test of positive control bacteria,the growth was detected,while the negative control was no growth detected.Conclusion The micro-bial limit test for compound Hongjingtian oral liquid is validated in this study.The amount of staphylococcus aureus can be de-termined using diluted culture method ;while the amounts of Escherichiacoli,Bacillus subtilis,Candida albicans,Aspergillus niger can be determined by conventional method.
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OBJECTIVE:To establish a method of microbial limit test for Ofloxacin gel. METHODS:Test sample solutions were prepared by MnSO4,CaCl2 and MgCl2 gelout with different concentrations,the numbers of bacteria and control bacteria were detected by membrane filtration method,and the numbers of molds and yeasts were determined by conventional method. RE-SULTS:The conventional method showed the recoveries of Candida albicaus and Aspergillus niger were more than 70% after not treated by gel breaker;the membrane filtration method showed the recoveries of Escherichia coli,Staphylococcus aurus and Baci-lus subtilis were more than 70% after treated by 5% MnSO4,3% CaCl2 and 1% MgCl2;the membrane filtration method could de-tect the control bacteria after treated by gel breaker. CONCLUSIONS:Gel breaker with appropriate concentrations can effectively eliminate the antibacterial activity of Ofloxacin gel. The established method is suitable for its microbial limit test.
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Objective To establish a microbial limit test for levofloxacin hydrochloride gel. Methods Ca2+ was added to neutralize the antibacterial activity of quinolone,and to form the insoluble precipitate by binding with carbomer, therefore destroying the stability of macromolecules in the gel. The microbial limit test for levofloxacin hydrochloride gel was confirmed combining with membrane filtration and plate methods. Results The normal plate counting was used in detecting the mold and yeast colony in the gel, the recovery was more than 70%.Simultaneously neutralization centrifugation and membrane filtration were used in bacterial count,with recovery less than 70%,and controlling bacteria test. Conclusion The method is accurate and practical for microbial limit test of levofloxacin hydrochloride gel, but is risky for bacterial counting and needs further risk assessment.
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Objective:To establish the method validation of microbial limit tests for hospital paste preparations, including com-pound salicylic acid paste, zinc oxide paste and compound pine tar paste. Methods:According to the microbial limit test described in China pharmacopoeia 2010 edition, the method validation of count of bacteria, fungi and yeasts and tests for specified microorganisms was established. Results:Medium dilution method could be used in bacteria, fungi and yeasts count and specified microorganisms ex-amination for compound salicylic acid paste and zinc oxide paste. For compound pine tar paste, bacteria, fungi and yeasts count and the pseudomonas aeruginosa examination could use medium dilution method, while the staphylococcus aureus examination should employ membrane filtration method. Conclusion:The methods of microbial limit tests for the three hospital paste preparations are established, which can be used to control the quality of hospital preparations effectively.
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OBJECTIVE: To carry out standardization research on pre-filtration method for microbial test solutions of drugs and establish a new double-membrane filtration method in microbial tests. METHODS: New filters with double-membranes of different materials or pore-sizes were designed and used as pre-filters for microbial tests. The effects of test solution pre-filtration on several wild-type strains were evaluated and compared with the methods described by Chinese Pharmacopoeia. RESULTS: No statistical difference was found between the double-membrane filtration method and the pharmacopoeia method. However, filter-clogging was significantly reduced by using the double-membrane filtration method, and the double-membrane filtration method could also eliminate the antibacterial activity of the samples, thus reducing the damage or loss of microorganisms in special samples. CONCLUSION: The double-membrane filtration method meets the requirements of the Chinese Pharmacopoeia; the test results are valid and the method can be used for sterility test and microbial limit test for drug products.