阅读说明：本技术 一种7β-甲氧基头孢美唑的分离纯化方法 (Separation and purification method of 7 beta-methoxy cefmetazole ) 是由 王辉 夏易能 于 2020-03-26 设计创作，主要内容包括：本发明属于医药技术领域,具体涉及一种7β-甲氧基头孢美唑的分离纯化方法。该分离纯化方法经过二次分离纯化,包括步骤如下：1)样品溶解；2)第一次分离纯化；3)第二次分离纯化；4)浓缩和冻干,得到目标产物7β-甲氧基头孢美唑,其中,两次分离纯化色谱柱均选用十八烷基硅烷键合硅胶填料,流动相为TFA水溶液和乙腈,当流动相平衡系统后,进行等度洗脱,分段收集目标组分。本发明方法成功地从头孢美唑异构体混合物中分离得到7β-甲氧基头孢美唑,纯度高达98%,重现性好,便于操作。该杂质对于头孢美唑的质量研究具有重要意义,为其杂质研究提供了优质的对照品,以便于更好的控制药物质量。(The invention belongs to the technical field of medicines, and particularly relates to a separation and purification method of 7 beta-methoxy cefmetazole. The separation and purification method comprises the following steps of performing secondary separation and purification: 1) dissolving a sample; 2) first separation and purification; 3) separating and purifying for the second time; 4) concentrating and freeze-drying to obtain a target product 7 beta-methoxy cefmetazole, wherein octadecylsilane chemically bonded silica filler is selected for chromatographic columns for two times of separation and purification, mobile phases are TFA aqueous solution and acetonitrile, isocratic elution is carried out after a mobile phase equilibrium system, and target components are collected in sections. The method successfully separates and obtains the 7 beta-methoxyl cefmetazole from the cefmetazole isomer mixture, the purity is as high as 98 percent, the reproducibility is good, and the operation is convenient. The impurities have important significance for the quality research of cefmetazole, and provide a high-quality reference substance for the impurity research so as to better control the quality of the medicine.)

1. A separation and purification method of 7 beta-methoxy cefmetazole is characterized in that: the separation and purification method comprises the following specific steps of secondary separation and purification:

1) sample dissolution: dissolving a mixture containing 7 beta-methoxy cefmetazole isomer in an acetonitrile solvent to obtain a mixed solution, controlling the concentration of the mixed solution, and injecting the mixed solution into a reversed-phase high-performance preparative liquid chromatography after filtration;

2) first separation and purification: the chromatographic column adopts octadecylsilane chemically bonded silica filler, mobile phases are TFA aqueous solution and acetonitrile, isocratic elution is carried out after a mobile phase balance system, and target components are collected in sections;

3) and (3) second separation and purification: selecting octadecylsilane chemically bonded silica filler as a chromatographic column, using TFA aqueous solution and acetonitrile as mobile phases, balancing the column by using the acetonitrile, loading the target component subjected to first separation and purification onto a separation column, performing isocratic elution, and collecting target peak fractions in a segmented manner;

4) concentration and freeze-drying: and (3) distilling the target component 7 beta-methoxy cefmetazole with qualified purity, which is collected by the second separation and purification, at low temperature under reduced pressure, removing most of organic solvent, and then putting the product into a freeze dryer for freeze drying to obtain the target product 7 beta-methoxy cefmetazole.

2. The method for separating and purifying 7 β -methoxy cefmetazole according to claim 1, wherein: the mass-volume ratio of the mixture containing the 7 beta-methoxy cefmetazole isomer and the acetonitrile solvent in the step 1) is (0.5 g-1 g): 10mL, and the concentration of the mixed solution is 5-100 mg/mL.

3. The method for separating and purifying 7 β -methoxy cefmetazole according to claim 1, wherein: in the step 2) and the step 3), the size of the chromatographic column is 20 multiplied by 250 mm, and the particle size of the octadecylsilane chemically bonded silica filler is 10 mu m.

4. The method for separating and purifying 7 β -methoxy cefmetazole according to claim 1, wherein: in step 2), the mobile phase was 0.1% TFA: acetonitrile = 75: 25, the flow rate is 20 mL/min, the sample volume is 2 mL, the temperature is room temperature, the pressure is 4 MPa, an ultraviolet detector is adopted, and the detection wavelength is 220 nm.

5. The method for separating and purifying 7 β -methoxy cefmetazole according to claim 1, wherein: in step 3), the mobile phase was 0.1% TFA: acetonitrile = 68: 32, the flow rate is 20 mL/min, the sample volume is 2 mL, the temperature is room temperature, the pressure is 4 MPa, an ultraviolet detector is adopted, the detection wavelength is 220 nm, and the column is firstly balanced by a mobile phase with acetonitrile accounting for 10% for 10 min.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a separation and purification method of 7 beta-methoxy cefmetazole.

Background

Cefmetazole, the chemical name is (6R, 7S) -7- [ (cyanomethylthio) acetamido ] -7-methoxy-3- [ 1-methyl-1H-tetrazole-5-thiomethyl ] -8-oxidation-5-thia-1-aza- [4.2.0] oct-2-ene-2-carboxylic acid, is a second generation semi-synthetic cephalosporin antibacterial drug, has the functions of broad spectrum, high efficiency and low toxicity, and the chemical structural formula is shown in figure 1.

The action mechanism of cefmetazole is similar to that of cephalosporins, and the cefmetazole plays a role in sterilization mainly by interfering the synthesis of bacterial cell walls in the proliferation stage. Cefmetazole is widely applied clinically, but the compound has a complex structure, multiple process steps, high synthesis difficulty, poor stability of partial intermediates, easy generation of impurities and higher impurity separation and identification difficulty.

The research and development of new medicines must comply with the basic principle of 'safety, effectiveness and controllable quality'. The method has the advantages that various countries or organizations successively demonstrate a plurality of guiding principles on the limit of impurities, and the core is that the actual and potential impurities in the medicine can be well controlled and removed as much as possible, so that the medicine is safer. Therefore, the preparation and separation of impurities have far-reaching significance for the control of the quality of the medicine and the safety of the medicine for people.

Kunio Atsumi et al (Tetrahedron Letters, 23, 2977-. With the research on cefmetazole production and preparation process and product quality conducted by the applicant for many years, after the original synthesis route is changed, a crude product is detected by HPLC-MS to determine an isomer containing cefmetazole, and the cefmetazole is successfully separated and purified through a large amount of work and is subjected to product quality research¹H-NMR、¹³C-NMR identification is carried out, and the cefmetazole 7 beta-methoxyl isomer is determined, and the chemical structural formula of the cefmetazole is shown in figure 2. At present, the obtained isomer has important significance for the medicine quality research and quality control of cefmetazole. No other literature about the separation and purification method of the impurity of the 7 beta-methoxy cefmetazole is available at present.

Disclosure of Invention

The invention aims to provide a method for separating and purifying 7 beta-methoxyl cefmetazole serving as a novel cefmetazole impurity, which is used for separating and purifying the novel impurity by using a preparation liquid.

The purpose of the invention is realized by the following technical scheme:

a separation and purification method of 7 beta-methoxy cefmetazole comprises the following steps of secondary separation and purification:

1) sample dissolution: dissolving a mixture containing 7 beta-methoxy cefmetazole isomer in an acetonitrile solvent to obtain a mixed solution, controlling the concentration of the mixed solution, and injecting the mixed solution into a reversed-phase high-performance preparative liquid chromatography after filtration;

2) first separation and purification: the chromatographic column adopts octadecylsilane chemically bonded silica filler, mobile phases are TFA aqueous solution and acetonitrile, isocratic elution is carried out after a mobile phase balance system, and target components are collected in sections;

3) and (3) second separation and purification: selecting octadecylsilane chemically bonded silica filler as a chromatographic column, using TFA aqueous solution and acetonitrile as mobile phases, balancing the column by using the acetonitrile, loading the target component subjected to first separation and purification onto a separation column, performing isocratic elution, and collecting target peak fractions in a segmented manner;

4) concentration and freeze-drying: and (3) distilling the target component 7 beta-methoxy cefmetazole with qualified purity, which is collected by the second separation and purification, at low temperature under reduced pressure, removing most of organic solvent, and then putting the product into a freeze dryer for freeze drying to obtain the target product 7 beta-methoxy cefmetazole.

Preferably, the mass-to-volume ratio of the mixture containing 7 beta-methoxy cefmetazole isomer and the acetonitrile solvent in the step 1) is (0.5 g-1 g): 10mL, and the concentration of the mixed solution is 5-100 mg/mL.

Preferably, in the step 2) and the step 3), the size of the chromatographic column is 20X 250 mm, and the particle size of the octadecylsilane chemically bonded silica filler is 10 μm.

More preferably, in step 2), the mobile phase is 0.1% TFA: acetonitrile = 75: 25, the flow rate is 20 mL/min, the sample volume is 2 mL, the temperature is room temperature, the pressure is 4 MPa, an ultraviolet detector is adopted, and the detection wavelength is 220 nm.

More preferably, in step 3), the mobile phase is 0.1% TFA: acetonitrile = 68: 32, the flow rate is 20 mL/min, the sample volume is 2 mL, the temperature is room temperature, the pressure is 4 MPa, an ultraviolet detector is adopted, the detection wavelength is 220 nm, and the column is firstly balanced by a mobile phase with acetonitrile accounting for 10% for 10 min.

The technical advantages of the invention are as follows:

most of isomers with similar structures are difficult to be resolved, and the method successfully separates 7 beta-methoxy cefmetazole from a cefmetazole isomer mixture, has the purity of 98 percent, good reproducibility and convenient operation. The impurities have important significance for the quality research of cefmetazole, and provide a high-quality reference substance for the impurity research so as to better control the quality of the medicine.

Drawings

Fig. 1 is a chemical structural formula of cefmetazole.

FIG. 2 shows the chemical structural formula of 7 beta-methoxy cefmetazole.

FIG. 3 is an HPLC analysis chart of preparative fractions collected in stages in comparative example 1.

FIG. 4 is a HPLC analysis chart of preparative fractions collected in stages in comparative example 2.

FIG. 5 is a HPLC analysis chart of preparative fractions collected in the first separation and purification stage in example 3.

FIG. 6 is a HPLC analysis chart of the preparative fractions collected in the second separation and purification stage in example 3.

FIG. 7 shows the target impurity 7 beta-methoxy cefmetazole obtained in example 3¹H-NMR spectrum.

FIG. 8 shows the target impurity 7 β -methoxy cefmetazole obtained in example 3¹³C-NMR spectrum.

Detailed Description

The technical solution of the present invention will be further described with reference to the following specific examples, but the present invention is not limited to these examples.

The invention relates to a separation and purification method of 7 beta-methoxyl cefmetazole, wherein a reverse high-efficiency preparative liquid chromatography is adopted to separate and purify a cefmetazole isomer mixture; the conditions of the reversed phase high performance preparative liquid chromatography are as follows:

the preparation method comprises the steps of preparing a C18 liquid phase column, preparing an ultraviolet detector, detecting the wavelength of 190-400 nm, the flow rate of 1-30 mL/min, and the temperature of room temperature, wherein the first separation mobile phase is 0.1% TFA-acetonitrile, and the acetonitrile accounts for 20-30%.

The cefmetazole isomer mixture is dissolved by acetonitrile, the concentration is 1-100 mg/mL, the cefmetazole isomer mixture is filtered, and then the cefmetazole isomer mixture is injected into a reversed-phase high-efficiency preparation liquid phase for separation and purification.

After the primary separation, the impurities after the primary peak are large, and the secondary purification and separation are needed, the prepared liquid phase column is a C18 liquid phase column, the detector is an ultraviolet detector, the detection wavelength is 190-400 nm, the flow rate is 1-30 mL/min, the temperature is room temperature, the pressure is 4 MPa, the secondary separation mobile phase is 0.1% TFA-acetonitrile, and the acetonitrile accounts for 30-35%. And during the second separation and purification, the column is balanced by 10% acetonitrile for 10 min, the concentrated preparation component is loaded onto the separation column by a pump, elution is carried out, and the target component is collected in sections.

The preparation components separated by the reversed-phase high-performance preparative liquid chromatography are distilled at low temperature under reduced pressure and are freeze-dried by a vacuum freeze dryer to obtain refined purified substances.

A preferred preparative liquid chromatography column according to the invention is HT-ODS-P (10 μm, 20X 250 mm).

Example 1

The separation and purification method of the mixture containing the 7 beta-methoxy cefmetazole isomer comprises the following steps:

1. sample dissolution: dissolving 1 g of cefmetazole isomer mixture in 10mL of acetonitrile solvent to obtain a mixed solution, wherein the mass concentration of the mixed solution is 100 mg/mL, and injecting the mixed solution into a reverse phase high performance preparative liquid chromatography after filtration.

2. First separation and purification: the size of the chromatographic column is 20X 250 mm, the column is filled with octadecylsilane bonded silica gel filler, the particle size of the filler is 10 μm, and the mobile phase is 0.1% TFA: acetonitrile = 80: 20, the flow rate is 20 mL/min, the sample volume is 2 mL, the temperature is room temperature, the pressure is 4 MPa, an ultraviolet detector is adopted, the detection wavelength is 220 nm, isocratic elution is carried out after a mobile phase equilibrium system, and the target components are collected in sections.

3. And (3) purity detection: the preparative fractions collected in fractions were subjected to HPLC purity detection.

4. And (3) second separation and purification: the size of the chromatographic column is 20X 250 mm, the column is filled with octadecylsilane bonded silica gel filler, the particle size of the filler is 10 μm, and the mobile phase is 0.1% TFA: acetonitrile = 70:30, the flow rate is 20 mL/min, the sample volume is 2 mL, the temperature is room temperature, the pressure is 4 MPa, an ultraviolet detector is adopted, and the detection wavelength is 220 nm. The column is balanced by 10% acetonitrile for 10 min, and then the first purified and separated component is concentrated and loaded on the separation column, and then isocratic elution is carried out, and the target peak is collected by sections.

5. And (3) purity detection: HPLC purity detection is carried out on the preparation components collected by sections, and the purity of the 7 beta-methoxy cefmetazole is 93%.

6. Concentration and freeze-drying: and (3) distilling the collected target component 7 beta-methoxy cefmetazole at low temperature under reduced pressure to remove most of organic solvent, and then putting the obtained product into a vacuum freeze dryer for freeze drying to obtain the target impurity 7 beta-methoxy cefmetazole.

Example 2

The separation and purification method of the mixture containing the 7 beta-methoxy cefmetazole isomer comprises the following steps:

1. sample dissolution: dissolving 1 g of cefmetazole isomer mixture in 10mL of acetonitrile solvent to obtain a mixed solution, wherein the mass concentration of the mixed solution is 100 mg/mL, and injecting the mixed solution into a reverse phase high performance preparative liquid chromatography after filtration.

2. First separation and purification: the size of the chromatographic column is 20X 250 mm, the column is filled with octadecylsilane bonded silica gel filler, the particle size of the filler is 10 μm, and the mobile phase is 0.1% TFA: acetonitrile = 70:30, the flow rate is 20 mL/min, the sample volume is 2 mL, the temperature is room temperature, the pressure is 4 MPa, an ultraviolet detector is adopted, the detection wavelength is 220 nm, isocratic elution is carried out after a mobile phase equilibrium system, and the target components are collected in sections.

3. And (3) purity detection: the preparative fractions collected in fractions were subjected to HPLC purity detection.

4. And (3) second separation and purification: the size of the chromatographic column is 20X 250 mm, the column is filled with octadecylsilane bonded silica gel filler, the particle size of the filler is 10 μm, and the mobile phase is 0.1% TFA: acetonitrile = 65:35, the flow rate is 20 mL/min, the sample volume is 2 mL, the temperature is room temperature, the pressure is 4 MPa, an ultraviolet detector is adopted, and the detection wavelength is 220 nm. The column is balanced by 10% acetonitrile for 10 min, and then the first purified and separated component is concentrated and loaded on the separation column, and then isocratic elution is carried out, and the target peak is collected by sections.

5. And (3) purity detection: HPLC purity detection is carried out on the preparation components collected by stages, and the purity of the 7 beta-methoxy cefmetazole is 95%.

6. Concentration and freeze-drying: and (3) distilling the collected target component 7 beta-methoxy cefmetazole at low temperature under reduced pressure to remove most of organic solvent, and then putting the obtained product into a vacuum freeze dryer for freeze drying to obtain the target impurity 7 beta-methoxy cefmetazole.

Example 3

The separation and purification method of the mixture containing the 7 beta-methoxy cefmetazole isomer comprises the following steps:

1. sample dissolution: dissolving 1 g of cefmetazole isomer mixture in 10mL of acetonitrile solvent to obtain a mixed solution, wherein the mass concentration of the mixed solution is 100 mg/mL, and injecting the mixed solution into a reverse phase high performance preparative liquid chromatography after filtration.

2. First separation and purification: the size of the chromatographic column is 20X 250 mm, the column is filled with octadecylsilane bonded silica gel filler, the particle size of the filler is 10 μm, and the mobile phase is 0.1% TFA: acetonitrile = 75: 25, the flow rate is 20 mL/min, the sample volume is 2 mL, the temperature is room temperature, the pressure is 4 MPa, an ultraviolet detector is adopted, the detection wavelength is 220 nm, isocratic elution is carried out after a mobile phase equilibrium system, and the target components are collected in sections.

3. And (3) purity detection: preparative fractions collected in fractions were subjected to HPLC purity testing as shown in fig. 5.

4. And (3) second separation and purification: the size of the chromatographic column is 20X 250 mm, the column is filled with octadecylsilane bonded silica gel filler, the particle size of the filler is 10 μm, and the mobile phase is 0.1% TFA: acetonitrile = 68: 32, the flow rate is 20 mL/min, the sample volume is 2 mL, the temperature is room temperature, the pressure is 4 MPa, an ultraviolet detector is adopted, and the detection wavelength is 220 nm. The column is balanced by 10% acetonitrile for 10 min, and then the first purified and separated component is concentrated and loaded on the separation column, and then isocratic elution is carried out, and the target peak is collected by sections.

5. And (3) purity detection: preparative fractions collected in fractions were subjected to HPLC purity testing as shown in fig. 6. As can be seen from FIG. 6, the purity of the target component 7 beta-methoxy cefmetazole reaches 98%.

6. Concentration and freeze-drying: and (3) distilling the collected target component 7 beta-methoxy cefmetazole with qualified purity at low temperature under reduced pressure to remove most of organic solvent, and then putting the target component into a vacuum freeze dryer for freeze drying to obtain the target impurity 7 beta-methoxy cefmetazole.

7. And (3) structure confirmation: 1H-NMR and 13C-NMR showed in FIGS. 7 and 8.

1H-NMR（400MHz, DMSO）：δ9.92 (s, 1H, -OH), 5.13 (s, 1H, -CH-), 4.35-4.20 (dd, 2H, -CH ₂-), 3.93(s, 3H, -CH ₃), 4.35-4.21 (dd, 2H, -CH ₂-), 3.78 (s, 2H, -CH ₂-), 3.76-3.47 (dd, 2H, -CH ₂-), 3.38(s, 3H, -CH ₃)。

13C-NMR（400MHz, DMSO）：δ169.5, 163.0, 160.4, 153.4, 126.6, 126.1, 118.2, 92.7, 63.5, 52.9, 35.9, 34.8, 34.2, 27.4, 17.2。

ESI-MS（m/z）：494.04[M+Na]+。

The applicant tried to perform a separation and purification once, as in comparative examples 1 and 2, but the impurities after the main peak after the separation once were large, and the separation and purification effect was poor.

Comparative example 1

The separation and purification method of the cefmetazole isomer mixture comprises the following steps:

1. sample dissolution: 50 mg of cefmetazole isomer mixture was dissolved in 10mL of 0.1% TFA: acetonitrile = 65:35, 5 mg/mL, filtered and injected into a reverse phase hplc.

2. Separation and purification: the size of the chromatographic column is 4.6X 250 mm, the column is filled with octadecylsilane bonded silica filler, the filler particle size is 5 μm, and the mobile phase is 0.1% TFA: acetonitrile = 65:35, the flow rate is 1 mL/min, the sample volume is 5 muL, the temperature is room temperature, the pressure is 2200 psi, an ultraviolet detector is adopted, the detection wavelength is 254 nm, isocratic elution is carried out after a mobile phase equilibrium system, and the target components are collected in sections.

3. And (3) purity detection: HPLC purity detection is carried out on the preparation components collected in sections, and the target component 7 beta-methoxy cefmetazole cannot be effectively separated, as shown in figure 3.

Comparative example 2

The separation and purification method of the cefmetazole isomer mixture comprises the following steps:

1. sample dissolution: 50 mg of cefmetazole isomer mixture was dissolved in 10mL of 0.1% TFA: acetonitrile = 70:30, 5 mg/mL, filtered and injected into a reverse phase hplc.

2. Separation and purification: the size of the chromatographic column is 4.6X 250 mm, the column is filled with octadecylsilane bonded silica filler, the filler particle size is 50 μm, and the mobile phase is 0.1% TFA: acetonitrile = 70:30, flow rate is 1 mL/min, sample volume is 5 muL, temperature is room temperature, pressure is 2200 psi, an ultraviolet detector is adopted, detection wavelength is 254 nm, isocratic elution is carried out after a mobile phase equilibrium system, and target components are collected in sections.

3. And (3) purity detection: HPLC purity detection is carried out on the preparation components collected in sections, and the target component 7 beta-methoxy cefmetazole cannot be effectively separated, as shown in figure 4.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the inventive concept of the present invention, and these changes and modifications are all within the scope of the present invention.