阅读说明：本技术 一种用于利福平胶囊微生物限度控制菌检查的抗菌活性去除方法 (Antibacterial activity removing method for rifampicin capsule microorganism limit control bacteria inspection ) 是由 刘彩香 胡建航 何遵卫 刘静 于 2019-11-27 设计创作，主要内容包括：本发明公开一种用于利福平胶囊微生物限度控制菌检查的抗菌活性去除方法,具体包括S1、在45℃的含有浓度为5～15％的甲醇或者0.1mol/L硫代硫酸钠的pH5.8～6.5的磷酸盐缓冲液中加入供试品,使供试品浓度为0.1g/mL；S2、保温45℃,30分钟充分溶解取上清液,即得去除抗菌活性的供试液,使得去除活性后的利福平胶囊供试液能够很好地适用于微生物限度控制菌检查。步骤S2中还可以另行加入离心搅拌的过程,能够使去除利福平药物抑菌活性的效果更好。(The invention discloses an antibacterial activity removing method for rifampicin capsule microorganism limit control bacteria inspection, which specifically comprises the steps of S1, adding a test sample into a phosphate buffer solution with the concentration of 5-15% of methanol or 0.1mol/L sodium thiosulfate and the pH value of 5.8-6.5 at 45 ℃ to ensure that the concentration of the test sample is 0.1 g/mL; s2, keeping the temperature at 45 ℃, fully dissolving the mixture for 30 minutes and taking the supernatant to obtain the test solution without the antibacterial activity, so that the rifampicin capsule test solution without the activity can be well suitable for the microbial limit control bacteria examination. In step S2, a centrifugal stirring process may be added to improve the effect of removing the bacteriostatic activity of rifampicin.)

1. A method for removing antibacterial activity for the microbial limit control bacteria inspection of rifampicin capsules, comprising the following steps:

s1, adding a test sample into a phosphate buffer solution with the concentration of 5-15% of methanol or 0.1mol/L of sodium thiosulfate and the pH value of 5.8-6.5 to make the concentration of the test sample be 0.1 g/mL;

s2, preserving the temperature, fully dissolving and taking supernatant to obtain the test solution with the antibacterial activity removed.

2. The method for removing antibacterial activity for the microbial limit control bacteria test of rifampicin capsule as claimed in claim 1, wherein the concentration of methanol in step S1 is 10%.

3. The method for removing antibacterial activity for the microbial limit control bacteria test of rifampicin capsule as claimed in claim 1, wherein in step S1: the pH of the phosphate buffer was 6.0.

4. The method for removing antibacterial activity in a rifampicin capsule according to any one of claims 1 to 3, wherein step S2 further comprises centrifugation, and step S2 specifically comprises: preserving heat, fully dissolving, centrifuging for 4-8 min at a speed of 400-700 r/min, and taking supernatant to obtain the test solution with the antibacterial activity removed.

5. The method of claim 4, wherein the centrifugation is performed at 500r/min in step S2.

6. The method for removing antibacterial activity for the microbial limit control bacteria test of rifampicin capsule as claimed in claim 4, wherein in step S2, the time for centrifugation is 5 min.

Technical Field

The invention belongs to the field of microbial limit inspection, and particularly relates to an antibacterial activity removing method for the microbial limit control bacteria inspection of rifampicin capsules.

Background

The microbial limit test is a method for testing the degree of contamination of an unspecified sterile preparation, raw materials thereof, and auxiliary materials by microorganisms, and the test items include microbial count (bacterial count, fungal count, yeast count) and control bacterial test.

Rifampin is a broad spectrum antibiotic drug belonging to rifamycin family, generally used for oral administration in the form of capsule or tablet, and mainly used for treating tuberculosis, meningitis and staphylococcus aureus infection, and for external application for treating trachoma. Rifampicin, as a non-sterile product, requires a microbial limit test. Because rifampicin itself has antibacterial activity, when a test sample is prepared for detection of microbial limit control bacteria, the antibacterial activity needs to be removed or inactivated first, however, general rule 1106 of the existing "pharmacopoeia of the people's republic of china (2015 edition) (hereinafter referred to as chinese pharmacopoeia)" check the microbial limit of non-sterile products: the "control bacteria test method" only discloses the neutralizing agent and inactivation method for common interferents, but does not discuss how a specific drug is inactivated.

Rifampin capsules belong to a film agent test article, and in the existing documents, for example, the validation of rifampin microbial limit detection method for eye use (royal bud, enemy proid, 2009; 26) and the establishment of rifampin microbial limit detection method for eye drop use (zhao snow wei, royal hongran, 2013, 06) disclose the rifampin microbial limit detection method for eye drop use, but the method for removing or inactivating rifampin antibacterial activity is not provided in the documents, and the requirements for removing or inactivating the rifampin antibacterial activity of the rifampin capsule test solution cannot be met by adopting the chinese pharmacopoeia and other published prior arts, thereby affecting the detection accuracy of the microbial limit. Therefore, there is a need to specifically study an inactivation method capable of meeting the requirements for rifampicin drugs.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an antibacterial activity removing method for the microbe limit control bacteria inspection of a rifampicin capsule, and the specific phosphate buffer solution is selected to effectively realize the bacteriostasis and inactivation of the rifampicin capsule. Specifically, the following technique is used.

A method for removing antibacterial activity for the microbial limit control bacteria inspection of rifampicin capsules comprises the following steps:

s1, adding a test sample into a phosphate buffer solution with the concentration of 5-15% of methanol or 0.1mol/L of sodium thiosulfate and the pH value of 5.8-6.5 to make the concentration of the test sample be 0.1 g/mL;

s2, preserving the temperature, fully dissolving and taking supernatant to obtain the test solution with the antibacterial activity removed.

The test solution prepared by the method is inoculated with Escherichia coli (namely escherichia coli) by adopting a membrane filtration method, then a filter membrane is transferred to a culture medium for culture, and finally, when a phosphate buffer solution containing 5-15% of methanol and having a pH value of 5.8-6.5 is used, the bacteria grow in the culture medium for 18-24 h, and when a phosphate buffer solution containing 0.1mol/L of sodium thiosulfate and having a pH value of 5.8-6.5 is used, the bacteria grow in the culture medium for 48 h. Finally, the identification confirms that the growing bacteria are the Escherichia coli added during inoculation. The concentration of added methanol or sodium thiosulfate, as well as the pH of the phosphate buffer, had a significant effect on the length of time over which bacterial growth was detected.

Preferably, in step S1, the concentration of methanol is 10%. The concentration is the optimum value.

Preferably, in step S1: the pH of the phosphate buffer was 6.0. The pH is the optimum value for a phosphate buffer.

When the concentration of methanol is 10% and the pH value of the phosphate buffer is 6.0, the growth of bacteria can be observed only in 18 hours by adopting the method, and the inoculated Escherichia coli can be identified.

Preferably, step S2 further includes a centrifugation process, and step S2 specifically is: preserving heat, fully dissolving, centrifuging for 4-8 min at a speed of 400-700 r/min, and taking supernatant to obtain the test solution with the antibacterial activity removed. Residual antibacterial active ingredients are better removed by utilizing the centrifugal effect, the purity of the test solution can be higher, and the time spent on detecting the growth of bacteria is only 16 hours.

Preferably, the liquid temperature in steps S1, S2 is 45 ℃. Preferably, in step S2, the time for sufficient dissolution is 30 min.

More preferably, in step S2, the rotation speed of the centrifuge is 500 r/min.

More preferably, in step S2, the time for centrifugation is 5 min.

Compared with the prior art, the invention has the advantages that:

1. aiming at the medicine rifampicin capsule, a method capable of effectively removing and inactivating antibacterial activity is provided, and the inactivation effectiveness and the non-toxicity to microorganisms are confirmed through verification;

2. provides direct guidance for a method for checking microbe limit control bacteria for rifampicin capsules, facilitates the understanding of detection personnel on the basis of the existing Chinese pharmacopoeia (2015 edition), and provides an effective reference scheme.

Detailed Description

The technical solutions of the present invention will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.