阅读说明：本技术 一种用于测定磷霉素钠杂质的高效液相色谱方法 (High performance liquid chromatography method for determining fosfomycin sodium impurities ) 是由 郑玉林 陈玉双 刘丽娟 于 2019-12-03 设计创作，主要内容包括：本发明涉及一种用于测定磷霉素钠杂质的高效液相色谱方法。所述高效液相色谱方法使用利用十八烷基硅烷键合硅胶为填充剂的色谱柱并采用磷酸水溶液-甲醇混合液为流动相,其中所述磷酸水溶液中磷酸的浓度质量分数为0.04％～0.06％,磷酸水溶液与甲醇的体积比为99:1 ～99:5。根据本发明的方法增强了对强极性杂质的保留,能够有效分离磷霉素钠复杂的杂质体系,具有较好的通用性和特定杂质的专属性,灵敏度高,操作方便,分析时间适中,为该化合物的质量控制提供了有效保障。(The invention relates to a high performance liquid chromatography method for determining fosfomycin sodium impurities. The high performance liquid chromatography method uses a chromatographic column which uses octadecylsilane chemically bonded silica as a filler and uses a phosphoric acid aqueous solution-methanol mixed solution as a mobile phase, wherein the concentration mass fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.04-0.06%, and the volume ratio of the phosphoric acid aqueous solution to methanol is 99: 1-99: 5. The method enhances the retention of strong polar impurities, can effectively separate a complex impurity system of fosfomycin sodium, has better universality and specificity of specific impurities, has high sensitivity, convenient operation and moderate analysis time, and provides effective guarantee for the quality control of the compound.)

1. The high performance liquid chromatography method for determining fosfomycin sodium impurities is characterized by using a chromatographic column using octadecylsilane chemically bonded silica as a filler and adopting a phosphoric acid aqueous solution-methanol mixed solution as a mobile phase, wherein the phosphoric acid concentration mass fraction in the phosphoric acid aqueous solution is 0.04-0.06%, and the volume ratio of the phosphoric acid aqueous solution to methanol is 99: 1-99: 5.

2. The high performance liquid chromatography method according to claim 1, wherein the concentration of phosphoric acid in the phosphoric acid aqueous solution is 0.05 mass%.

3. The high performance liquid chromatography method according to claim 1, wherein the column has a particle size of 5 μm, a column length of 250mm, and a column inner diameter of 4.6 mm.

4. The high performance liquid chromatography method of claim 1, wherein the column has a working temperature of 8 ℃ to 12 ℃.

5. The high performance liquid chromatography method according to claim 1, wherein the flow rate of the mobile phase is 0.5ml/min to 0.7ml/min, the detection wavelength is 205nm, and the injection volume is 10 μ l.

6. The high performance liquid chromatography method of claim 4, wherein the flow rate of the mobile phase is 0.6 ml/min.

7. The high performance liquid chromatography method according to claim 4, wherein the column temperature is 8 ℃, 9 ℃, 10 ℃, 11 ℃ or 12 ℃.

8. The high performance liquid chromatography method according to claim 1, wherein an ultraviolet detector is used as the detector.

9. The high performance liquid chromatography method according to claim 1, wherein a mixed solution comprising sodium allene phosphate, sodium cis-propenoate, phenylethylamine 2, 3-allylphosphate and sodium trans-propenoate is used as the system-compatible solution.

10. The high performance liquid chromatography method of claim 1, wherein the method detects at least the following four impurities present in fosfomycin sodium: sodium allene phosphate, sodium cis-propenyl phosphate, phenylethylamine 2, 3-allyl phosphate and sodium trans-propenyl phosphate.

Technical Field

The invention belongs to the technical field of drug analysis, and particularly relates to a high performance liquid chromatography method for determining fosfomycin sodium impurities.

Background

Fosfomycin is a novel broad-spectrum antibiotic developed jointly by Merck and CEPA in 1967, structurally belonging to a phosphoric acid derivative, having a unique chemical structure and a small molecular weight. Fosfomycin has a unique mechanism of action, can inhibit the early stage of cell wall synthesis, does not produce cross resistance to other antibiotics, and is mainly used for treating gram-negative and gram-positive bacterial infections.

At present, 4 varieties of fosfomycin are mainly used, namely fosfomycin sodium salt, fosfomycin calcium salt, fosfomycin trometamol salt and fosfomycin benzylamine salt. Sodium and calcium fosfomycin salts were marketed in europe in 1975, first in spain and then in italy and germany. The product of Mingmu corporation was marketed in Japan in 1980. Fosfomycin trometamol was marketed in europe in 1988; approved by the U.S. FDA in the united states for marketing in 1996 month 12. In terms of use conditions, sodium salts and calcium salts are used in europe, japan and southeast asia, and the japanese pharmacopoeia only contains two varieties of fosfomycin sodium salts and fosfomycin calcium salts. In the United states, fosfomycin trometamol is mainly used and is sold in a large amount.

Various impurities may be generated in the preparation and storage processes of the fosfomycin sodium, and the types and the contents of the impurities in the product are directly related to the quality and the medication safety of the medicine, so that the contents of the impurities related to the fosfomycin sodium need to be accurately determined before the medicine is used.

"high performance liquid chromatography for determining allene phosphate and cis-propenephosphate, key intermediates to fosfomycin by Liuhui et al" (Liuhui, Bright Red et al, analytical laboratories, 2014, 33(3):300) discloses a method for determining substances related to fosfomycin sodium. According to the method, the content determination of 2 intermediate impurities, namely allene phosphate and cis-propenyl phosphate, is researched by adopting an Agilent ZORBAX amino column as a fixed phase, a phosphate solution and acetonitrile as a mobile phase and an amino column as a chromatographic column. From the results, the method is suitable for measuring the content of allene phosphate and cis-propenylphosphoric acid in the fosfomycin synthesis reaction. However, the above method has many disadvantages, such as the adopted amino column is fragile, and the fosfomycin sodium series impurities have large polarity and fast peak, so that the complex impurity mixed system with large polarity cannot be effectively retained and separated.

Therefore, in order to effectively analyze and detect the quality of the medicine and ensure the safety of the medicine, an analysis method for conveniently and effectively detecting the fosfomycin sodium and the complex impurities thereof still needs to be developed.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a high performance liquid chromatography method for efficiently separating and determining fosfomycin sodium ((-) - (1R, 2S) -1, 2-epoxypropylphosphonic acid disodium salt, the structural formula is shown as (1)) impurities, thereby realizing accurate monitoring of the impurity spectrum and strict control of the quality.

The above object is achieved by the following scheme:

in one aspect, the invention provides a high performance liquid chromatography method for determining fosfomycin sodium impurities, which uses a chromatographic column using octadecylsilane chemically bonded silica as a filler and uses a phosphoric acid aqueous solution-methanol mixed solution as a mobile phase, wherein the phosphoric acid aqueous solution has a phosphoric acid concentration of 0.04-0.06% by mass and a volume ratio of the phosphoric acid aqueous solution to methanol of 99: 1-99: 5. In a preferred embodiment, the concentration of phosphoric acid in the phosphoric acid aqueous solution is 0.05% by mass.

Aiming at the characteristics of small molecular weight and large polarity of fosfomycin sodium impurities, the method adopts a chromatographic column (such as a Warters Atlantis T3 chromatographic column) taking octadecylsilane chemically bonded silica as a filler for detection, and the chromatographic column adopts a trifunctional C18 alkyl bonded phase, so that the bonding density is ensured, the retention of polar compounds is improved, and 100% aqueous phase mobile phase compatibility can be realized, thereby improving the separation effect.

Further preferably, the particle size of the chromatographic column is 5 μm, the length of the chromatographic column is 250mm, and the inner diameter of the chromatographic column is 4.6 mm.

By using the chromatographic column with the particle size of 5 mu m, the separation is faster and the column efficiency is higher. Meanwhile, the use of a longer length of column (e.g., 250mm) can improve the degree of separation, thereby improving the level of separation.

Further preferably, in the method, the operating column temperature of the chromatography column is 8 ℃ to 12 ℃. In a preferred embodiment, the column temperature is 8 ℃, 9 ℃, 10 ℃, 11 ℃ or 12 ℃. Further, in the method, the flow rate of the mobile phase is 0.5ml/min to 0.7ml/min, the detection wavelength is 205nm, and the injection volume is 10 mu l. In a preferred embodiment, the flow rate of the mobile phase is 0.6 ml/min.

In the scheme, the invention adopts low temperature (about 10 ℃) to finely distinguish isomers, ensures the separation of isomer (including cis-trans isomer) impurities with similar structures, and improves the separation effect. If the temperature is higher than 12 ℃ or lower than 8 ℃, the separation effect is poor, for example, impurity peaks are combined, and the like.

Further preferably, in the method, an ultraviolet detector is used as the detector.

In the scheme, the ultraviolet detector has the advantages of high sensitivity, low noise, non-destructiveness, wide linear range, insensitivity to flow rate and temperature change and the like.

Further preferably, in the method, a mixed solution containing sodium allene phosphate, sodium cis-propenoate, phenethylamine 2, 3-allylphosphate and sodium trans-propenoate is used as the system adaptability solution.

In the technical scheme, the mixed solution of the substances is used as a system adaptability solution, the separation effect among impurity peaks is observed, a chromatographic system can be tested and adjusted, and comprehensive characteristics in the aspects of electric signals, analysis operation, samples and the like are comprehensively detected.

Further preferably, the method is capable of detecting at least the following four impurities present in fosfomycin sodium: sodium allene phosphate, sodium cis-propenyl phosphate, phenylethylamine 2, 3-allyl phosphate and sodium trans-propenyl phosphate.

As mentioned above, the method of the invention adopts the chromatographic column which uses octadecylsilane chemically bonded silica as the filler and works at a lower column temperature of 8-12 ℃, so that the separation and detection levels of fosfomycin sodium impurities are greatly improved, and the method of the invention can effectively reserve and separate a complex impurity mixed system with larger polarity.

In summary, according to the technical scheme, the invention can achieve the following excellent technical effects:

(1) the method adopts a chromatographic column (for example, a Warters atlantis T3 chromatographic column) which uses octadecylsilane chemically bonded silica as a filler for detection, and the chromatographic column adopts a trifunctional C18 alkyl bonded phase, so that the bonding density is ensured, the retention of a polar compound is improved, and 100% aqueous phase mobile phase compatibility can be realized, thereby improving the separation effect;

(2) the invention adopts low temperature (about 10 ℃) to finely distinguish isomers, ensures the separation of isomer (including cis-trans isomer) impurities with similar structures, thereby improving the separation effect;

(3) the method enhances the retention of strong polar impurities, can effectively separate a complex impurity system of fosfomycin sodium, has better universality and specificity of specific impurities, has high sensitivity, convenient operation and moderate analysis time, and provides effective guarantee for the quality control of the compound;

(4) the method can effectively separate and determine the related impurities of the fosfomycin sodium (especially a complex impurity mixed system with larger polarity), and therefore, the method can be used as an important component part for controlling the quality of the fosfomycin sodium.

Drawings

Some preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Those of ordinary skill in the art will understand that these drawings are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

FIG. 1 is a detection profile of a system suitability solution according to one embodiment of the invention;

FIG. 2 is a linear dependence of cis-propenophosphoric acid (peak area-concentration);

FIG. 3 is a linear relationship (peak area-concentration) of allene phosphate;

FIG. 4 is a linear relationship (peak area-concentration) of reverse propenophosphoric acid;

FIG. 5 is a linear plot of 2, 3-allylphosphoric acid (peak area-concentration).

Detailed Description

Some embodiments of the invention will be described below with reference to the accompanying drawings and examples. Those of ordinary skill in the art will appreciate that these examples are provided merely to illustrate how the invention can be practiced and are not intended to limit the scope of the invention in any way.

Phosphomycin is a novel broad-spectrum antibiotic with wide application. However, in the process of preparing specific various derivatives thereof (such as fosfomycin sodium), various impurities are inevitably introduced, and the impurities are not easy to detect and remove, thereby causing a number of obstacles to the application thereof. In order to solve this problem, the present inventors have conducted extensive studies on the synthesis process and forced degradation of fosfomycin (particularly, fosfomycin sodium), and as a result, found that process impurities including 4 kinds or more of raw materials, intermediates, by-products, and the like, such as sodium allene phosphate, sodium cis-propenoate, 2, 3-allylphenethylphosphate, sodium trans-propenoate, and the like, may be present in the prepared fosfomycin sodium. The presence of these impurities hinders the practical effectiveness of fosfomycin sodium.

Based on this finding, the present inventors have conducted extensive studies on a complex impurity mixed system in which the above at least four impurities are present, and have been attempting to find a method capable of separating and detecting the above at least four impurities at a time. As a result, the present inventors have found that the above-mentioned at least four main impurities can be separated and detected at one time by detection using a column (for example, a Warters Atlantis T3 column) using octadecylsilane bonded silica gel as a packing, thereby successfully solving the technical problems to be solved by the present invention. In addition, the separation effect can be further enhanced by using a lower column temperature (for example, 8 to 12 ℃). The present invention has been made based on the above findings.

Specifically, the invention provides a high performance liquid chromatography method for measuring fosfomycin sodium, which uses a chromatographic column using octadecylsilane chemically bonded silica as a filler and uses a phosphoric acid aqueous solution-methanol mixed solution as a mobile phase, wherein the phosphoric acid concentration mass fraction in the phosphoric acid aqueous solution is 0.04-0.06%, and the volume ratio of the phosphoric acid aqueous solution to methanol is 99: 1-99: 5. The chromatographic column adopts a trifunctional C18 alkyl bonding phase, ensures the bonding density, improves the retention of polar compounds, and can realize 100% aqueous phase mobile phase compatibility, thereby improving the separation effect.

The process according to the invention will be described and illustrated in more detail below with reference to examples. Those of ordinary skill in the art will appreciate that these examples are provided for illustrative purposes only, and that those of ordinary skill in the art will better understand how to implement the present invention and will not set forth any limitations on the scope of the present invention.