阅读说明：本技术 一种可快速判断头孢菌类抗生素中聚合物含量的方法 (Method capable of quickly judging content of polymer in cephalosporin antibiotics ) 是由 张超 高宏伟 刘美平 朱春丽 邵正飞 赵佳 于 2020-10-29 设计创作，主要内容包括：本发明属于化合物的检测技术领域,具体涉及一种可快速判断头孢菌类抗生素中聚合物含量的方法。本发明的方法为：通过薄层色谱法对待测头孢菌类抗生素进行检测,根据待测头孢菌类抗生素与标准品所形成的斑点的颜色深浅判断待测样品中聚合物的含量；检测的过程中以乙酸乙酯、醚类、二氯甲烷和有机酸的复合体系作为展开剂；所述醚类为小分子醚,所述有机酸为小分子酸。上述方法可通过薄层色谱颜色的深浅快速地判断待测抗生素中聚合物的含量,为药品研发提供及时的指导。(The invention belongs to the technical field of compound detection, and particularly relates to a method for rapidly judging the content of a polymer in cephalosporin antibiotics. The method comprises the following steps: detecting the cephalosporin antibiotics to be detected through a thin-layer chromatography, and judging the content of the polymer in the sample to be detected according to the color depth of spots formed by the cephalosporin antibiotics to be detected and the standard substance; in the detection process, a composite system of ethyl acetate, ethers, dichloromethane and organic acid is used as a developing agent; the ether is micromolecular ether, and the organic acid is micromolecular acid. The method can rapidly judge the content of the polymer in the antibiotic to be detected through the depth of the color of the thin-layer chromatography, and provides a timely guidance for drug research and development.)

1. A method capable of rapidly judging the content of polymers in cephalosporin antibiotics is characterized in that the cephalosporin antibiotics are detected by thin-layer chromatography, and the content of the polymers in a sample to be detected is judged according to the color depth of spots formed by the cephalosporin antibiotics to be detected and a standard product of the cephalosporin antibiotics to be detected;

in the detection process, a composite system of ethyl acetate, ethers, dichloromethane and organic acid is used as a developing agent; the ethers are small molecular ethers, and the organic acids are small molecular organic acids.

2. The method of claim 1, wherein the volume ratio of ethyl acetate, ether, dichloromethane and organic acid is (4-6): 3-5): 4-6): 5-7.

3. The method for rapidly determining the polymer content in cephalosporin antibiotics as claimed in claim 1, wherein the small molecular ether is methyl tert-butyl ether, ethyl ether or isopropyl ether.

4. The method for rapidly determining the polymer content in cephalosporin antibiotics in claim 1 or 3, wherein the small-molecule organic acid is formic acid or acetic acid.

5. The method as claimed in any one of claims 1 to 4, wherein the cephalosporin antibiotics are cefazolin sodium, cefepime, cefminox sodium or cefathiamidine.

6. The method for rapidly determining the polymer content in cephalosporin antibiotics according to claim 1 or 4, characterized in that a complex system of ethyl acetate, diethyl ether, dichloromethane and formic acid is used as a developing agent or a complex system of ethyl acetate, methyl tert-butyl ether, dichloromethane and formic acid is used as a developing agent.

7. The method for rapidly judging the content of the polymer in the cephalosporin antibiotics according to any one of claims 1 to 6, characterized in that a silica gel G plate is selected as a thin layer plate in the detection process by thin layer chromatography.

8. The method for rapidly determining the polymer content in the cephalosporin antibiotics according to claim 7, wherein the thickness of the silica gel G thin layer is in a range of 0.20-0.50 mm, preferably 0.20-0.25 mm.

9. The method for rapidly determining the polymer content in cephalosporin antibiotics as claimed in claim 1, characterized in that it comprises the following steps:

1) respectively spotting the cephalosporin antibiotics to be detected and the standard substances thereof on a thin-layer plate, and placing the thin-layer plate in a chromatographic cylinder for development;

2) and after the development is finished, taking out the thin layer, preserving heat, cooling, placing in an iodine saturation container for color development, and judging the content of the polymer in the sample to be detected according to the color depth of the spots formed by the sample to be detected and the standard product thereof.

10. The method for rapidly judging the content of the polymer in the cephalosporin antibiotics according to claim 9, characterized in that the temperature of the heat preservation is 30-70 ℃ and the time is 5-30 min; preferably, the temperature for heat preservation is 45-55 ℃ and the time is 18-22 min.

Technical Field

The invention belongs to the technical field of compound detection, and particularly relates to a method for rapidly judging the content of a polymer in cephalosporin antibiotics.

Background

Cephalosporin antibiotics are widely used anti-infective drugs at present, the most serious adverse reaction is anaphylactic reaction, and a large number of clinical studies prove that the allergen which causes the anaphylactic reaction is not the antibiotic per se, but high-molecular polymer impurities in the antibiotic sometimes cause toxic reaction and seriously harm the life safety of patients, so the content control of the polymer is particularly important.

At present, the most strict control standard of the polymer content is antibiotics for injection, polymer impurity information (including polymeric impurities, dimers, trimers or multimers and the like with molecular weights lower than a main component and between the main component and the dimers) needs to be clarified, the chromatographic behavior of a polymer inspection impurity peak under relevant substance inspection conditions is inspected by adopting a two-dimensional chromatographic column switching method, and impurities (as a polymer inspection item or a relevant substance II) which are not controlled by the relevant substance inspection method are inspected and controlled by the polymer inspection method. The whole method is long in development time and complex in operation, and based on the situation, the method which is simple in operation and can quickly and preliminarily judge the polymer content of the cephalosporin antibiotics is urgently needed to be provided for quality judgment of products in the early stage in the research and development stage.

Disclosure of Invention

Aiming at the problems of complex operation and long time consumption in judging the content of the polymer in the antibiotics in the prior art, the invention provides a method which is simple and convenient to operate and can quickly judge the content of the polymer in the cephalosporin antibiotics, and the method comprises the following steps: detecting the cephalosporin antibiotics through thin-layer chromatography, and judging the content of the polymer in the sample to be detected according to the color depth of spots formed by the cephalosporin antibiotics to be detected and the standard substance of the cephalosporin antibiotics;

in the detection process, a composite system of ethyl acetate, ethers, dichloromethane and organic acid is used as a developing agent; the ethers are small molecular ethers, and the organic acids are small molecular organic acids.

Preferably, the volume ratio of the ethyl acetate to the ethers to the dichloromethane to the organic acid is (4-6) to (3-5) to (4-6) to (5-7).

Preferably, the ether is methyl tert-butyl ether, diethyl ether or isopropyl ether.

Preferably, the organic acid is formic acid or acetic acid.

Preferably, the cephalosporin antibiotics are cefazolin sodium, cefepime, cefminox sodium or cefathiamidine.

Preferably, a mixture of ethyl acetate, diethyl ether, dichloromethane and formic acid or a mixture of ethyl acetate, methyl tert-butyl ether, dichloromethane and formic acid is used as the developing agent.

Preferably, a silica gel G plate is selected as the thin layer plate during detection by thin layer chromatography.

Preferably, the thickness range of the silica gel G thin layer is 0.20-0.50 mm, and further preferably, the thickness range of the silica gel G thin layer is 0.20-0.25 mm.

As a preferred mode of operation, the method of the invention comprises the steps of:

1) respectively spotting the cephalosporin antibiotics to be detected and the standard substances thereof on a thin-layer plate, and placing the thin-layer plate in a chromatographic cylinder for development;

2) and after the development is finished, taking out the thin layer, preserving heat, cooling, placing in an iodine saturation container for color development, and judging the content of the polymer in the sample to be detected according to the color depth of the spots formed by the sample to be detected and the standard product thereof.

Preferably, the temperature for heat preservation is 30-70 ℃, and the time is 5-30 min; preferably, the temperature for heat preservation is 45-55 ℃ and the time is 18-22 min.

The invention has the following beneficial effects:

the invention detects the material by thin-layer chromatography, and can judge whether the polymer in the product to be detected meets the specification or not by the color depth of the spots formed by the thin-layer chromatography and the standard substance, so as to guide the research and development. If the color of the formed spot is darker than that of the antibiotic for injection, the content of the polymer in the sample to be detected is higher than that of the polymer of the antibiotic for injection. The method can quickly judge the content of the polymer in the antibiotic to be detected, and is convenient for timely adjusting the preparation method of the drug in the drug research and development process.

Detailed Description

The invention is discussed in detail below with additional technical features.

The method of the invention comprises the following steps: detecting the cephalosporin antibiotics through thin-layer chromatography, and judging the content of the polymer in the sample to be detected according to the color depth of spots formed by the cephalosporin antibiotics to be detected and the standard substance of the cephalosporin antibiotics;

in the detection process, a composite system of ethyl acetate, ethers, dichloromethane and organic acid is used as a developing agent; the ethers are small molecular ethers, and the organic acids are small molecular organic acids.

Cephalosporin antibiotics can be prepared into various forms of medicines, such as oral preparations or injections, the injections have the highest requirements for safety, and if polymer impurity information (including polymer impurities, dimers, trimers or multimers and the like with molecular weights lower than the main component and between the main component and the dimers) needs to be determined, a two-dimensional chromatographic column switching method is usually adopted for research and determination of the molecular structure of the cephalosporin antibiotics, and reverse chromatographic conditions are developed to control the cephalosporin antibiotics. However, in the development stage of the medicine, the polymer in the medicine is not required to be subjected to the above-mentioned explicit analysis, and the content of the polymer is in compliance so as to provide effective guidance for the development.

The micromolecular ether is common ether with a short carbon chain, such as ether with 2-5 carbon atoms, and the micromolecular organic acid is organic acid with 1-3 carbon atoms.

According to some preferred embodiments, the volume ratio of the ethyl acetate, the ethers, the dichloromethane and the organic acid is (4-6): (3-5): (4-6): (5-7). The volume ratio of ethyl acetate, ether, methylene chloride and organic acid is in the above range, and the formed complex system has large solubility difference to the cephalosporin antibiotics and the polymer thereof, and can separate the polymer formed by the antibiotics and the antibiotics ideally.

According to some preferred embodiments, the ether is methyl tert-butyl ether, diethyl ether or isopropyl ether. The ethers can improve the separation effect of the polymer and other impurities. In the process of detecting cephalosporin antibiotics, besides cephalosporin compounds and polymers thereof, other impurities are usually present, and the occurrence of the impurities can also have certain influence on the separation of the cephalosporin compounds and the polymers thereof.

According to some preferred embodiments, the organic acid is formic acid or acetic acid. The acid can adjust pH of the developing agent, thereby improving the roundness of the spots.

According to some preferred embodiments, the cephalosporin antibiotic is cefazolin sodium, cefepime, cefminox sodium or cefathiamidine.

According to some preferred embodiments, a mixture of ethyl acetate, diethyl ether, dichloromethane and formic acid is used as the developing agent or a mixture of ethyl acetate, methyl tert-butyl ether, dichloromethane and formic acid is used as the developing agent.

As a preferred mode of operation, the volume ratio of ethyl acetate, diethyl ether, dichloromethane and formic acid is 5:4:5: 6;

as a preferred mode of operation, the volume ratio of ethyl acetate, methyl tert-butyl ether, methylene chloride and formic acid is 4:5:5: 6.

As a preferable operation mode, a mixed system with the volume ratio of ethyl acetate, diethyl ether, dichloromethane and formic acid being 5:4:5:6 is selected as a developing agent, or a mixed system with the volume ratio of ethyl acetate, methyl tert-butyl ether, dichloromethane and formic acid being 4:5:5:6 is selected as a developing agent, and the cephalosporin antibiotics are cefazolin sodium, cefepime or minox sodium.

As a preferable operation mode, in the process of detecting the cefazolin sodium, a mixed system of ethyl acetate, diethyl ether, dichloromethane and formic acid with the volume ratio of 5:4:5:6 is used as a developing agent. The developing agent can realize the separation of the cefazolin sodium and the polymer thereof in the cefazolin sodium, and the separation distance is long, so that the existence of impurities can not influence spots.

In a preferred operation mode, a mixed system of ethyl acetate, methyl tert-butyl ether, dichloromethane and formic acid in a volume ratio of 4:5:5:6 is used as a developing agent in the detection process of cefepime. The developing agent can realize the separation of the cefazolin sodium and the polymer thereof in the cefazolin sodium, and the separation distance is long, so that the existence of impurities can not influence spots.

According to some preferred embodiments, a silica gel G plate is selected as the thin layer plate during detection by thin layer chromatography. The silica gel G plate does not contain fluorescent agent, thereby being beneficial to the subsequent color comparison.

Preferably, the thickness range of the silica gel G thin layer is 0.20-0.50 mm;

further preferably, the thickness of the silica gel G thin layer ranges from 0.20 mm to 0.25 mm. The thickness range can control the sample loading amount within a certain range, thereby ensuring that the shape and the size of the spot are in a better state.

According to some preferred embodiments, the method of the invention comprises the steps of:

1) respectively spotting the cephalosporin antibiotics to be detected and the standard substances thereof on a thin-layer plate, and placing the thin-layer plate in a chromatographic cylinder for development;

2) and after the development is finished, taking out the thin layer, preserving heat, cooling, placing in an iodine saturation container for color development, and judging the content of the polymer in the sample to be detected according to the color depth of the spots formed by the sample to be detected and the standard product thereof.

According to some preferred embodiments, the temperature of the heat preservation is 30-70 ℃ and the time is 5-30 min;

according to some preferred embodiments, the temperature of the heat preservation is 45-55 ℃ and the time is 18-22 min. The reproducibility of the retention time of the spots can be ensured by controlling the temperature and time of the heat preservation.

According to some preferred embodiments, the developing agent is saturated in the chromatographic cylinder for 1.5-2 hours before sample application, and the thin-layer plate is saturated in the chromatographic cylinder for 0.4-0.6 hours after sample application.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

This example relates to the detection of polymer in cefazolin sodium, comprising the following steps:

1) taking a proper amount of cefazolin sodium, adding water to prepare a solution of 1mg/ml, and destroying the solution in a water bath at 80 ℃ for 6 hours to serve as a polymer reference substance.

2) The content of the polymer in the sample was determined by two-dimensional column switching method, which is shown in the following table

The polymer impurity level was found to be about 1.5%.

3) Samples 1 and 2 to be detected are taken, water is added to prepare a solution with the concentration of 1mg/ml, and the solution is used as a test solution. Taking 10 μ l of each of the polymer control solution and the sample solution, dropping on a silica gel G thin layer, respectively, using ethyl acetate-diethyl ether-dichloromethane-formic acid (5:4:5:6) as developing agent, air drying, keeping the temperature (50 deg.C) for 20min, cooling, and placing in an iodine saturated container for color development.

The result shows that the polymer spot of the sample 1 to be detected is shallower than the polymer spot of the reference substance, the polymer spot of the sample 2 to be detected is deeper than the polymer spot of the reference substance, and then the two-dimensional column switching method which is the same as the step 2) is adopted to detect the sample 1 to be detected and the sample 2 to be detected, so that the polymer content in the sample 1 to be detected is 0.9%, and the polymer content in the sample 2 to be detected is 2.0%. From the above, it can be seen that the content of the polymer in the cephalosporin antibiotics can be rapidly and accurately determined by the thin layer chromatography described herein.

Example 2

This example relates to the detection of polymer in cefepime, comprising the following steps:

1) taking a proper amount of cefepime, adding water to prepare a solution of 1mg/ml, and standing at room temperature for 1 week to serve as a polymer reference substance.

2) The content of the polymer in the sample was determined by two-dimensional column switching method, which is shown in the following table

The polymer impurity level was found to be about 1.5%.

3) Samples 1 and 2 to be detected are taken, water is added to prepare a solution with the concentration of 1mg/ml, and the solution is used as a test solution.

Detecting by thin layer chromatography, sucking polymer control solution and sample solution 10 μ l each, dropping on silica gel G thin layer, respectively, taking ethyl acetate-methyl tert-butyl ether-dichloromethane-formic acid (4:5:5:6) as developing agent, air drying, keeping temperature (50 deg.C) for 20min, cooling, and placing in iodine saturated container for color development.

As a result, the polymer spots of the sample 1 were found to be lighter than those of the control, and the polymer spots of the sample 2 were found to be darker than those of the control. And then detecting the sample 1 to be detected and the sample 2 to be detected by adopting the two-dimensional column switching method which is the same as the step 2), wherein the polymer content in the sample 1 to be detected is 1.2%, and the polymer content in the sample 2 to be detected is 1.8%. From the above, it can be seen that the content of the polymer in the cephalosporin antibiotics can be rapidly and accurately determined by the thin layer chromatography described herein.

Example 3

Compared with example 1, the difference is that ethyl acetate-methyl tert-butyl ether-dichloromethane-formic acid (4:5:5:6) is used as developing agent.

As a result, it was found that the polymer spots and the main component spots were closer to each other but could be separated from each other and the spots were distinct as compared with example 1, and the results were the same as example 1, and the results were also used to determine the depth of the spots between the polymer of the sample to be measured and the polymer of the control substance.

Example 4

Compared with example 2, the difference is that ethyl acetate-diethyl ether-dichloromethane-formic acid (5:4:5:6) is used as developing agent.

As a result, it was found that the polymer spots and the main component spots were closer to each other but could be separated from each other and the spots were distinct as compared with example 1, and the results were the same as example 2, and the results were used to determine the depth of the spots between the polymer of the sample to be measured and the polymer of the control substance.

Example 5

This example relates to the detection of polymers in cefminox sodium, comprising the following steps:

1) taking a proper amount of cefminox sodium, adding water to prepare a solution of 1mg/ml, and standing at room temperature for 1 week to serve as a polymer reference substance.

2) Through the determination of molecular exclusion chromatography, spherical hydrophilic modified silica GEL (the molecular weight application range is polymer 500-15000) is used as a filler (TSK-GEL G2000SWXL, chromatographic column with 7.8mm multiplied by 300mm, 5 μm or equivalent efficiency); taking phosphate buffer solution [0.005mol/L disodium hydrogen phosphate solution-0.005 mol/L sodium dihydrogen phosphate solution (61:39) ] -acetonitrile (95:5) as a mobile phase, taking a polymer reference substance for HPLC sample analysis, and obtaining the polymer impurity content of about 1.4%.

3) Samples 1 and 2 to be detected are taken, water is added to prepare a solution with the concentration of 1mg/ml, and the solution is used as a test solution. Taking 10 μ l of each of the polymer control solution and the sample solution, dropping on a silica gel G thin layer, respectively, using ethyl acetate-diethyl ether-dichloromethane-formic acid (5:4:5:6) as developing agent, air drying, keeping the temperature (50 deg.C) for 20min, cooling, and placing in an iodine saturated container for color development.

The result shows that the polymer spots of the sample 1 to be detected are shallower than the polymer spots of the reference substance, the polymer spots of the sample 2 to be detected are deeper than the polymer spots of the reference substance, and then the molecular exclusion chromatography method which is the same as the step 2) is adopted to detect the sample 1 to be detected and the sample 2 to be detected, wherein the polymer content in the sample 1 to be detected is 0.4%, and the polymer content in the sample 2 to be detected is 1.9%. From the above, it can be seen that the content of the polymer in the cephalosporin antibiotics can be rapidly and accurately determined by the thin layer chromatography described herein.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.