Method for simultaneously determining contents of four B components and related substances in polymyxin sulfate B raw material and preparation thereof

Abstract

The invention relates to a method for simultaneously determining contents of four B components and related substances in a polymyxin B sulfate raw material and a preparation thereof, which adopts ultra-high performance liquid chromatography combined with an ultraviolet detector, and uses octadecylsilane chemically bonded silica as a chromatographic column. A trifluoroacetic acid aqueous solution with the volume fraction of 0.07% is used as a mobile phase A, and a trifluoroacetic acid acetonitrile solution with the volume fraction of 0.05% is used as a mobile phase B; compared with a traditional method, the method has the advantages that the mobile phase is simple to prepare, the compound retention time is stable, the analysis time is shortened by one time, the analysis cost is reduced, the sensitivity is high, the reproducibility is good, and four B components and related substances thereof can be completely separated; the contents of the four B components and related substances in the polymyxin B sulfate can be rapidly and accurately measured at the same time.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and in particular to a method for simultaneously determining the contents of four B components and related substances in a polymyxin B sulfate raw material and a preparation thereof. Background Art

[0002] Polymyxin B sulfate is a group of antibacterial polypeptide antibiotics isolated from Bacillus polymyxa. Its sulfate is used clinically to treat infections caused by Gram-negative bacilli - Pseudomonas aeruginosa, including urinary tract infections, meningitis, lung infections, sepsis, and skin, soft tissue, eye, ear, and joint infections. It also has a good therapeutic effect on infections caused by other negative bacteria such as Clostridium perfringens, Escherichia coli, Klebsiella pneumoniae, and Haemophilus influenzae. Polymyxin B sulfate is a multi-component mixture with similar structures, including B1, B2, B3 and B 1- I has four components, among which B1 and B2 are active ingredients, B3 and B 1- I is the main impurity.

[0003] At present, EP11.0 includes the detection methods of "four B components" and "related substances" in polymyxin B sulfate raw materials. The 2020 edition of the Chinese Pharmacopoeia and USP-NF2023 both include the detection methods of "four B components" and "related substances" in polymyxin B sulfate raw materials and their preparations. The methods for "four B components" and "related substances" included in domestic and foreign pharmacopoeias are the same, all using the isocratic elution method of reversed-phase high-performance liquid chromatography with a sodium sulfate-phosphoric acid-water-acetonitrile system. Under the inspection item of "four B components", it is required that the content of polymyxin B3 shall not exceed 6.0%, and the content of polymyxin B1-I shall not exceed 15.0%, and the total content of polymyxin B1, B2, B3 and B1-I shall not be less than 80%. At the same time, under the inspection item of "related substances", it is required that a single impurity shall not exceed 3.0%, and the total amount of impurities shall not exceed 17.0%.

[0004] Disadvantages of existing technologies: complex preparation of mobile phase, long single-needle analysis time (about 50 minutes), low sensitivity, and poor reproducibility. Causes: The traditional method uses a sodium sulfate-phosphoric acid-water-acetonitrile system, which requires tedious operations such as weighing buffer salts, adjusting pH, and mixing aqueous and organic phases. The mobile phase preparation process is complicated; the traditional method uses isocratic elution, and the single-needle analysis time is long (about 50 minutes). The diffusion of the polymyxin B component on the chromatographic column is slow, resulting in broadening of the four B component peaks, low column efficiency, and low sensitivity; at the same time, slight changes in the ratio of aqueous and organic phases in isocratic elution will cause rapid changes in retention time, resulting in poor reproducibility of the method; in the process of using this method, it was also found that after the injection of polymyxin B sulfate, as the number of injection needles continued to increase, the four B component peaks and related substance peaks would become constantly tailing, and the peaks would become flatter and flatter until no peaks appeared and could not be reproduced. Typical chromatograms with poor reproducibility are as follows: Figure 1 shown. Summary of the invention

[0005] The purpose of the present invention is to overcome the problems of long analysis time, low sensitivity, poor repeatability, high precision requirement for mixing ratio of mobile phase aqueous phase and organic phase, etc. when detecting four B components and related substances in polymyxin B sulfate by traditional methods, thereby providing a method for simultaneously determining the contents of four B components and related substances in polymyxin B sulfate raw materials and preparations thereof.

[0006] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: a method for simultaneously determining the contents of four B components and related substances in polymyxin B sulfate raw materials and preparations thereof, comprising:

[0007] (1) Standard solution: Weigh 50 mg of polymyxin B standard into a 100 ml volumetric flask, add water-acetonitrile to dissolve and quantitatively dilute to scale, shake well to obtain a standard solution;

[0008] (2) Sensitivity solution: Measure 1 ml of the standard solution into a 100 ml volumetric flask, dilute to the mark with water-acetonitrile, and shake well;

[0009] (3) Test solution: Take 40-60 mg of the sample to be tested, place it in a 100 ml volumetric flask, add water-acetonitrile to dissolve and quantitatively dilute to the scale, and shake well;

[0010] Ultra-high performance liquid chromatography combined with an ultraviolet detector was used, and octadecylsilane bonded silica gel was used as a chromatographic column; a 0.07% trifluoroacetic acid aqueous solution by volume fraction was used as a mobile phase A, and a 0.05% trifluoroacetic acid acetonitrile solution by volume fraction was used as a mobile phase B, and gradient elution was performed according to the following procedure;

[0011]

[0012] The flow rate was 0.3 ml per minute; the column temperature was 30°C; the detection wavelength was 215 nm; and the injection volume was 2 μL.

[0013] The preferred technical solution is: the volume ratio of water to acetonitrile is 80:20.

[0014] The preferred technical solution is: the chromatographic column is specifically: Waters ACQUITY UPLC CSH C18 150*2.1mm, 1.7μm.

[0015] The preferred technical solution is: in the chromatogram of the standard solution, the relative retention times of polymyxin B2, polymyxin B3, polymyxin B1-I and polymyxin B1 are 0.58-82, 0.65-0.70, 0.80-0.85 and 0.95-1.05, respectively.

[0016] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0017] Compared with the traditional method, the method of the present invention has simple mobile phase preparation, stable compound retention time, shortened analysis time by 1 time, reduced analysis cost, high sensitivity, good reproducibility, can completely separate four B components and related substances, and can more quickly and accurately achieve simultaneous determination of the contents of four B components and related substances in polymyxin B sulfate. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A typical chromatogram of the prior art.

[0019] Figure 2 The following are typical chromatograms showing the contents of four B components.

[0020] Figure 3 It is a typical chromatogram of the present invention. DETAILED DESCRIPTION

[0021] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0022] See also Figure 2-3. It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings in this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention, so they have no substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the effects and purposes that can be achieved by the present invention. At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description, and are not used to limit the scope of the implementation of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the implementation of the present invention without substantially changing the technical content.

[0023] Polymyxin B sulfate is a mixture of multiple components with similar structures, including B 1、 B 2、 B3 and B1-I are four components, among which B1 and B2 are active ingredients, and B3 and B1-I are main impurities. The chemical structure is as follows:

[0024]

[0025] Unless otherwise specified, the reagents or materials described in the following examples are commercially available.

[0026] Example 1: A method for simultaneously determining the contents of four B components and related substances in polymyxin B sulfate raw materials and preparations thereof

[0027] The technical problem solved by the present invention is to overcome the problems of long analysis time, low sensitivity, poor repeatability, high requirements on the mixing accuracy of the mobile phase aqueous phase and the organic phase ratio when detecting the four B components and related substances in polymyxin B sulfate by traditional methods, thereby providing a detection method for simultaneously determining the contents of the four B components and related substances in polymyxin B sulfate.

[0028] Compared with the traditional method, the method of the present invention has simple mobile phase preparation, stable compound retention time, shortened analysis time by 1 time, reduced analysis cost, high sensitivity, good reproducibility, can completely separate the four B components and their related substances, and can more quickly and accurately achieve simultaneous determination of the contents of the four B components and related substances in polymyxin B sulfate. The typical chromatogram is shown in FIG. Figure 2 shown.

[0029] Ultra-high performance liquid chromatography combined with an ultraviolet detector was used, and octadecylsilane bonded silica gel was used as a chromatographic column such as Waters ACQUITY UPLC CSH C18 150*2.1 mm, 1.7 μm; a 0.07% trifluoroacetic acid aqueous solution by volume fraction was used as mobile phase A, and a 0.05% trifluoroacetic acid acetonitrile solution by volume fraction was used as mobile phase B, and gradient elution was performed according to the table below; the flow rate was 0.3 ml per minute; the column temperature was 30°C; the detection wavelength was 215 nm; and the injection volume was 2 μL.

[0030] Time (minutes) Mobile phase A (volume fraction %) Mobile phase B (volume fraction %) 0 81 19 15 78 22 25 68 32

[0031] The above method was used to determine the contents of related substances and component B in three batches of commercial samples of polymyxin B sulfate for injection. The specific implementation plan is as follows:

[0032] 1. Instrument:

[0033] Thermo Vanquish ultra-high performance liquid chromatograph (UV detector).

[0034] 2. Drug testing:

[0035] Polymyxin B standard (Source: USP; content of each component: Polymyxin B1: 74.1%; Polymyxin B2: 8.3%; Polymyxin B3: 2.1%; Polymyxin B1-I: 7.3%;)

[0036] 3. Chromatographic conditions:

[0037] Ultra-high performance liquid chromatography combined with an ultraviolet detector was used, with octadecylsilane bonded silica gel as the chromatographic column Waters ACQUITY UPLC CSH C18 150*2.1mm, 1.7μm; 0.07% trifluoroacetic acid aqueous solution was used as mobile phase A, and 0.05% trifluoroacetic acid acetonitrile solution was used as mobile phase B, and gradient elution was performed according to the table below; the flow rate was 0.3 ml per minute; the column temperature was 30°C; the detection wavelength was 215nm; and the injection volume was 2μL.

[0038] Time (minutes) Mobile phase A (volume fraction %) Mobile phase B (volume fraction %) 0 81 19 15 78 22 25 68 32

[0039] 4. Sample preparation process:

[0040] (1) Standard solution: Accurately weigh 50 mg of polymyxin B standard and place it in a 100 ml volumetric flask. Add water-acetonitrile (volume ratio 80:20) to dissolve and quantitatively dilute to the scale. Shake well.

[0041] (2) Sensitivity solution: Accurately measure 1 ml of the standard solution and place it in a 100 ml volumetric flask. Dilute to the mark with water-acetonitrile (volume ratio 80:20) and shake well.

[0042] (3) Test solution: Take about 50 mg of the product, accurately weigh it, place it in a 100 ml volumetric flask, add water-acetonitrile (volume ratio 80:20) to dissolve it and quantitatively dilute it to the scale, and shake well.

[0043] (4) System suitability requirements: In the chromatogram of the standard solution, the relative retention times of polymyxin B2, polymyxin B3, polymyxin B1-I and polymyxin B1 are approximately 0.60, 0.67, 0.83 and 1.0, respectively. The resolution between the polymyxin B2 peak and the polymyxin B3 peak should be no less than 3.0.

[0044] 5. Measurement results

[0045] (1) The system suitability results are shown in the following table and Figure 3 As shown:

[0046] name Relative retention time Separation <![CDATA[Polymyxin B2]]> 0.60 4.5 <![CDATA[Colistin B3]]> 0.67 11.4 <![CDATA[Polymyxin B1-I]]> 0.83 7.2 <![CDATA[Polymyxin B1]]> 1.00 -

[0047] It can be seen from the results in the above table that the peak shapes of the components of polymyxin B in this method are all good, and the separation between the components is greater than 1.5, which can meet the system suitability requirements.

[0048] (2) Sample test results

[0049]

[0050] This method was used to test three batches of commercial samples of a certain product. The results showed that this method has accurate detection results and good reproducibility, and is suitable for the determination of related substances and B component content in polymyxin B sulfate raw materials or polymyxin B sulfate preparations for injection.

[0051] The above description is only used to explain the preferred embodiments of the present invention, and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

Claims

1. A method for simultaneously determining the contents of four B components and related substances in polymyxin B sulfate raw materials and preparations thereof, characterized in that: include: (1) Standard solution: Weigh 50 mg of polymyxin B standard into a 100 ml volumetric flask, add water-acetonitrile to dissolve and quantitatively dilute to scale, shake well to obtain a standard solution; (2) Sensitivity solution: Measure 1 ml of the standard solution into a 100 ml volumetric flask, dilute to the mark with water-acetonitrile, and shake well; (3) Test solution: Take 40-60 mg of the sample to be tested, place it in a 100 ml volumetric flask, add water-acetonitrile to dissolve and quantitatively dilute to the scale, and shake well; Ultra-high performance liquid chromatography combined with an ultraviolet detector was used, and octadecylsilane bonded silica gel was used as a chromatographic column; a 0.07% trifluoroacetic acid aqueous solution by volume fraction was used as a mobile phase A, and a 0.05% trifluoroacetic acid acetonitrile solution by volume fraction was used as a mobile phase B, and gradient elution was performed according to the following procedure; The flow rate was 0.3 ml per minute; the column temperature was 30°C; the detection wavelength was 215 nm; and the injection volume was 2 μL.

2. The method for simultaneously determining the contents of four B components and related substances in polymyxin B sulfate raw materials and preparations thereof according to claim 1, characterized in that: The volume ratio of water to acetonitrile was 80:

20.

3. The method for simultaneously determining the contents of four B components and related substances in polymyxin B sulfate raw materials and preparations thereof according to claim 1, characterized in that: The specific chromatographic column is: Waters ACQUITY UPLC CSH C18150*2.1mm, 1.7μm.

4. The method for simultaneously determining the contents of four B components and related substances in polymyxin B sulfate raw materials and preparations thereof according to claim 1, characterized in that: In the chromatogram of the standard solution, the relative retention times of polymyxin B2, polymyxin B3, polymyxin B1-I and polymyxin B1 were 0.58-82, 0.65-0.70, 0.80-0.85 and 0.95-1.05, respectively.

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