Analysis method of polymyxin B sulfate related substances for injection

Abstract

The invention provides an analysis method of polymyxin B sulfate related substances, which comprises the following steps: dissolving polymyxin B sulfate for injection in water-acetonitrile (80: 20) to obtain a test solution; the method comprises the following steps: dissolving a polymyxin B standard substance in water-acetonitrile (80: 20), and uniformly shaking to obtain a standard substance solution; detecting the test solution and the standard solution by adopting a high performance liquid chromatography, wherein the detection adopts linear gradient elution; wherein the mobile phase A is a sodium sulfate solution; and the mobile phase B is acetonitrile. According to the technical scheme, a high performance liquid chromatography method is adopted, automatic operation can be achieved, repeatability is good, accuracy is high, sensitivity is high, component content and related substance inspection are combined into one method, the component content in a sample can be effectively detected, related substance inspection can also be considered, the detection time needed during quality control is shortened, and the analysis efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a method for analyzing related substances of polymyxin B sulfate for injection. Background Art

[0002] Polymyxins are a group of polypeptide antibiotics produced by Bacillus polymyxa, of which polymyxin B and E are used for medicinal purposes.

[0003] Polymyxin B Sulfate (Aerosporin) is a sterile lyophilized product mainly used for wounds, urinary tract infections, eye, ear, trachea and other infections caused by Pseudomonas aeruginosa and other Pseudomonas. In order to better control and improve the quality of drugs, polymyxin B sulfate for injection has been developed. However, the existing technology lacks analysis of polymyxin B sulfate for injection and its related substances, and there are problems such as long detection time and low analysis efficiency. Summary of the invention

[0004] The main purpose of the present invention is to provide an analysis method for polymyxin B sulfate for injection and related substances, aiming to solve the technical problem of low analysis efficiency of polymyxin B sulfate for injection and its related substances.

[0005] To achieve the above object, the present invention provides a method for analyzing related substances of polymyxin B sulfate for injection, comprising the following steps: Dissolve polymyxin B sulfate for injection in water-acetonitrile (80:20) to obtain the test solution; Dissolve the polymyxin B standard in water-acetonitrile (80:20) and shake well to obtain a standard solution; The test solution and the standard solution are tested by high performance liquid chromatography, wherein the test adopts linear gradient elution; Among them, mobile phase A is sodium sulfate solution; mobile phase B is acetonitrile.

[0006] Optionally, the elution conditions of the gradient elution are as follows: At 0 min, mobile phase A was 78% and mobile phase B was 22%; At 46 min, mobile phase A was 78% and mobile phase B was 22%; At 51 min, mobile phase A was 75% and mobile phase B was 25%; At 69 min, mobile phase A was 75% and mobile phase B was 25%; At 70 min, mobile phase A was 78% and mobile phase B was 22%; At 80 min, mobile phase A was 78% and mobile phase B was 22%.

[0007] Optionally, the chromatographic column for detection uses octadecylsilane bonded silica gel as a filler.

[0008] Optionally, the column temperature of the detection is 15~25°C.

[0009] Optionally, the detection wavelength of the detection is 200~230nm.

[0010] Optionally, the flow rate of the mobile phase for the detection is 0.8 mL / min.

[0011] Optionally, the injection volume of the detection is 20 μL.

[0012] Optionally, the mobile phase A is prepared by dissolving 4-5 g of anhydrous sodium sulfate in 900 mL of water, adjusting the pH value to 2-3 with dilute phosphoric acid, and then diluting the mixture to 1000 mL with water.

[0013] Optionally, the concentration of the test solution is 40~60g / L.

[0014] Optionally, the concentration of the standard solution is 40-60 g / L.

[0015] Optionally, the effective components of polymyxin B sulfate for injection include polymyxin B1, polymyxin B2, polymyxin B3, and polymyxin B1-I.

[0016] Optionally, the related substances detected by the analytical method include polymyxin B related substance E, polymyxin B related substance F, polymyxin B related substance G, polymyxin B related substance J, polymyxin B related substance H, polymyxin B related substance I, polymyxin B related substance M, and polymyxin B related substance P.

[0017] In the technical scheme of the present invention, high performance liquid phase method is adopted, which can be operated automatically, has good repeatability, high accuracy and high sensitivity, and the content of each component and the detection of related substances are combined into one method, which can effectively detect the content of each component in the sample, and can also take into account the inspection of related substances, reduce the detection time required during quality control, and improve the efficiency of analysis. Wherein the polymyxin B1, polymyxin B2, polymyxin B3, polymyxin B1-I components and polymyxin B related substances E, polymyxin B related substances F, polymyxin B related substances G, polymyxin B related substances J, polymyxin B related substances H, polymyxin B related substances I, polymyxin B related substances M, polymyxin B related substances P and other target substances in the component content can be effectively separated, and the impurities in the sample can be effectively detected in this method, which provides a reasonable basis for the improvement of the formulation quality standard. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0019] Figure 1 The present invention is a schematic flow chart of an embodiment of a method for analyzing related substances of polymyxin B sulfate for injection provided by the present invention.

[0020] Figure 2 is a chromatogram of a standard solution in an embodiment of an analytical method for related substances of polymyxin B sulfate for injection provided by the present invention; Figure 3 It is a chromatogram of the test solution in an embodiment of the method for analyzing related substances of polymyxin B sulfate for injection provided by the present invention.

[0021] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0022] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0023] It should be noted that, in the embodiments, those without specifying specific conditions are carried out according to normal conditions or conditions recommended by the manufacturer. Those without specifying the manufacturer of reagents or instruments used are conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, and "A and / or B" is taken as an example, including schemes A, B, or A and B that meet the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of the technical schemes is contradictory or cannot be achieved, it should be considered that the combination of such technical schemes does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work, all belong to the scope of protection of the present invention.

[0024] Polymyxin B sulfate for injection includes its effective ingredients polymyxin B1, polymyxin B2, polymyxin B3, etc., and also contains related substances as impurities, such as polymyxin B related substance E, polymyxin B related substance F, polymyxin B related substance G, polymyxin B related substance J, polymyxin B related substance H, polymyxin B related substance I, polymyxin B related substance M, polymyxin B related substance P. The prior art lacks an analytical method for polymyxin B sulfate for injection and its related substances, and there are problems such as long detection time and low analysis efficiency.

[0025] In view of this, the present invention proposes a method for analyzing related substances of polymyxin B sulfate for injection, combining Figure 1 Schematic diagram of the analytical method for related substances of polymyxin B sulfate for injection Figure 1 In one embodiment, the analysis method comprises the following steps.

[0026] Step S10, dissolving polymyxin B sulfate for injection in water-acetonitrile (80:20) to obtain a test solution.

[0027] In specific implementation, accurately weigh, take 40-60 mg of polymyxin B sulfate for injection of this product, put it in a 100 mL volumetric flask, add water-acetonitrile (water and acetonitrile are mixed in a volume ratio of 80:20) to dissolve and quantitatively dilute to the scale, and shake well.

[0028] Step S20, dissolve the polymyxin B standard in water-acetonitrile (80:20), and shake well to obtain a standard solution.

[0029] In the specific implementation, 40-60 mg of polymyxin B standard was accurately weighed and placed in a 100 mL volumetric flask, and water-acetonitrile (water and acetonitrile were mixed in a volume ratio of 80:20) was added to dissolve and quantitatively diluted to the scale, and shaken well.

[0030] Step S30: Analyze the obtained test solution and standard solution by high performance liquid chromatography.

[0031] In specific implementation, the chromatographic conditions are as follows: use a chromatographic column filled with octadecylsilane bonded silica gel (ThermoHypersil GOLDTM, 4.6mm×250mm, 5μm chromatographic column or a chromatographic column with equivalent performance) for gradient elution; the column temperature is 15~25°C, preferably 20°C; the detection wavelength is 200~230nm, preferably 215nm; the flow rate is 0.8mL per minute, and the injection volume is 20μL.

[0032] The mobile phase used for the gradient elution is: mobile phase A is a sodium sulfate solution, and its preparation process is: 4-5g (preferably 4.46g) of anhydrous sodium sulfate is dissolved in 900mL of water, and the pH value is adjusted to 2-3 (preferably 2.3) with dilute phosphoric acid, and then diluted to 1000mL with water, that is, the mass concentration of the sodium sulfate solution is 4-5g / L; mobile phase B is acetonitrile.

[0033] The elution conditions of the gradient elution are as follows: At 0 min, mobile phase A was 78% and mobile phase B was 22%; At 46 min, mobile phase A was 78% and mobile phase B was 22%; At 51 min, mobile phase A was 75% and mobile phase B was 25%; At 69 min, mobile phase A was 75% and mobile phase B was 25%; At 70 min, mobile phase A was 78% and mobile phase B was 22%; At 80 min, mobile phase A was 78% and mobile phase B was 22%.

[0034] According to the high performance liquid chromatography method (General Chapter 0512 of Part IV of the Chinese Pharmacopoeia 2020 Edition), the system suitability requirements and limits of the test solution and standard solution are as follows.

[0035] If there are impurity peaks in the chromatogram of the test solution, calculated by the area normalization method, the percentage of polymyxin B related substance E, polymyxin B related substance F, polymyxin B related substance G, polymyxin B related substance J, polymyxin B related substance H, polymyxin B related substance I, polymyxin B related substance M, and polymyxin B related substance P shall not exceed 3.0%. The percentage of a single impurity shall not exceed 3.0%. The total amount of impurities shall not exceed 17.0%. Peaks with an area less than 0.7 times the main peak of the standard solution (2) shall be ignored.

[0036] In the chromatogram of the standard solution, the relative retention times of polymyxin B2, polymyxin B3, polymyxin B1-I and polymyxin B1 are approximately 0.5min, 0.55min, 0.8min and 1.0min, respectively. The separation between the polymyxin B2 peak and the polymyxin B3 peak should be no less than 3.0. Calculated by the external standard method based on the corresponding peak area, the content of polymyxin B3 shall not exceed 6.0%, 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%.

[0037] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention. Example 1

[0038] 1. Materials Test solution: Take 50 mg of polymyxin B sulfate for injection (Fujian Fukang Pharmaceutical Co., Ltd.), accurately weigh it, put it in a 100 mL volumetric flask, add water-acetonitrile (80:20) to dissolve and quantitatively dilute to the scale, and shake well.

[0039] Standard solution: Accurately weigh 50 mg of polymyxin B (Fujian Fukang Pharmaceutical Co., Ltd.) standard, place in a 100 mL volumetric flask, add water-acetonitrile (80:20) to dissolve and quantitatively dilute to the scale, and shake well.

[0040] 2. Chromatographic conditions Instrument: ThermoFisher UltiMate 3000 Chromatographic column: octadecylsilane bonded silica as filler; Thermo Hypersil GOLDTM, 4.6mm×250mm, 5μm chromatographic column; Mobile phase: Sodium sulfate solution (dissolve 4.46 g of anhydrous sodium sulfate in 900 mL of water, adjust the pH value to 2.3 with dilute phosphoric acid, and then dilute with water to 1000 mL) as mobile phase A; acetonitrile as mobile phase B; perform gradient elution according to Table 1 below; Flow rate: 0.8 mL per minute; Column temperature: 20°C; Detection wavelength: 215nm; Injection volume: 20 μL.

[0041] Table 1 Gradient elution

[0042] Determined according to high performance liquid chromatography (General Rules 0512 of Part IV of the Chinese Pharmacopoeia 2020 Edition).

[0043] Accurately measure the test solution and standard solution, inject them into the liquid chromatograph, and record the chromatogram. Figure 2 , 3 , calculate the content of each component, the component content calculation formula is as follows: (1) Correction factor f = ; (2) Content of each component % = ; In the formula, A u : The peak area of ​​each component peak in the test solution; A S : Average peak area of ​​each component in the control solution; C u : Concentration of the test solution, in mg / mL; C s: The concentration of the control solution, in mg / mL; P: The content of each component of the reference substance. The calculation results are shown in the following table:

[0044] See also Figure 2 and 3 , the target substances such as polymyxin B1, polymyxin B2, polymyxin B3, polymyxin B1-I components and polymyxin B related substances E (also impurity E), polymyxin B related substances F (also impurity F), polymyxin B related substances G (also impurity G), polymyxin B related substances J (also impurity J), polymyxin B related substances H (also impurity H), polymyxin B related substances I (also impurity I), polymyxin B related substances M (also impurity M), polymyxin B related substances P (also impurity P) in the component content can be effectively separated, and the impurities in the sample can be effectively detected in this method. It can be seen that this detection method provides a reasonable basis for the improvement of the quality standards of preparations, and the detection of related substances can use the normalization method, without the need for impurity reference substances as external standards, saving detection costs.

[0045] Among them, the structural formula and nomenclature of the separated related substances (refer to the analysis report on related substances of polymyxin B by Guangzhou Aiqixi Pharmaceutical Technology Co., Ltd.) are as follows: The structure of the polymyxin B related substance E is: .

[0046] Chemical formula: C 54 H 94 N 16 O 13 , relative molecular mass: 1175.45, chemical name: N -((2 S )-4-amino-1-((2 S , 3 R )-1-(((2 S )-4-amino-1-oxo-1-((3 S , 6 S , 9 S , 15 R , 18 S )-6,9,18-tris(2-aminoethyl)-15-benzyl-3-(( R )-1-hydroxyethyl)-12-isobutyl-2,5,8,11,14,17,20-heptaoxo-1,4,7,10,13,16,19-heptaazacyclotrisin-21-yl)amino)butan-2-yl)amino)-3-hydroxy-1-oxobutan-2-yl)amino)-1-oxobutan-2-yl)heptylamide.

[0047] The structure of the polymyxin B related substance F is: .

[0048] Chemical formula: C 56 H 98 N 16 O 14 , relative molecular mass: 1219.50, chemical name: N -(2 S )-4-amino-1-((2 S , 3 R )-1-((2 S )-4-amino-1-oxo-1-((3 S , 6 S , 9 S , 12 S , 15 R , 18 S )-6,9,18-tris(2-aminoethyl)-15-benzyl-3-(( R )-1-hydroxyethyl)-12-isobutyl-2,5,8,11,14,17,20-heptaoxo-1,4,7,10,13,16,19-heptaazacyclotrisin-21-yl)amino)butan-2-yl)amino)-3-hydroxy-1-oxobutan-2-yl)amino)-1-oxobutan-2-yl)-3-hydroxy-6-methyloctanamide.

[0049] The structure of the polymyxin B related substance G is: .

[0050] Chemical formula: C 55 H 96 N 16 O 13 , relative molecular mass: 1189.47, chemical name: N -(2 S )-4-amino-1-((2 S , 3 R )-1-((2 S )-4-amino-1-oxo-1-((3 S , 6 S , 9 S , 12 S , 15 R , 18 S )-6,9,18-tris(2-aminoethyl)-15-benzyl-12-( S )-sec-butyl)-3-(( R)-1-hydroxyethyl)-2,5,8,11,14,17,20-heptaoxo-1,4,7,10,13,16,19-heptaazacyclotrisin-21-yl)amino)butyl-2-yl)amino)-3-hydroxy-1-oxobutyl-2-yl)amino)-1-oxobutyl-2-yl)-6-methylheptylamide.

[0051] The structure of the polymyxin B related substance H is: .

[0052] Chemical formula: C 56 H 98 N 16 O 13 , relative molecular mass: 1203.50, chemical name: N -(2 S )-4-amino-1-((2 S , 3 R )-1-((2 S )-4-amino-1-oxo-1-((3 S , 6 S , 9 S , 12 S , 15 R , 18 S )-6,9,18-tris(2-aminoethyl)-15-benzyl-12-( S )-sec-butyl)-3-(( R )-1-hydroxyethyl)-2,5,8,11,14,17,20-heptaoxo-1,4,7,10,13,16,19-heptaazacyclotrisin-21-yl)amino)butyl-2-yl)amino)-3-hydroxy-1-oxobutyl-2-yl)amino)-1-oxobutyl-2-yl)-6-methylheptylamide.

[0053] The structure of polymyxin B related substance I is: .

[0054] Chemical formula: C 56 H 98 N 16 O 13 , relative molecular mass: 1203.50, chemical name: N -(2 S )-4-amino-1-((2 S , 3 R )-1-((2 S )-4-amino-1-oxo-1-((3 S , 6 S , 9 S , 12S , 15 R , 18 S )-6,9,18-tris(2-aminoethyl)-15-benzyl-3-( R )-1-hydroxyethyl)-12-isobutyl-2,5,8,11,14,17,20-heptaoxo-1,4,7,10,13,16,19-heptaazacyclotrisin-21-yl)amino)-3-hydroxy-1-oxobutan-2-yl)amino-1-oxobutan-2-yl)nonanamide.

[0055] The structure of polymyxin B related substance J is: .

[0056] Chemical formula: C 55 H 96 N 16 O 13 , relative molecular mass: 1189.47, chemical name: N -((2 S )-4-amino-1-(2 S )-1-((2 S )-4-amino-1-oxo-1-((3 S , 6 S , 9 S , 12 S , 15 R , 18 S )-6,9,18-tris(2-aminoethyl)-15-benzyl-3-(( R )-1-hydroxyethyl)-12-isobutyl-2,5,8,11,14,17,20-heptaoxo-1,4,7,10,13,16,19-heptaazacyclotrisin-21-yl)amino)butan-2-yl)amino)-3-hydroxy-1-oxopropyl-2-yl)amino)-1-oxobutan-2-yl)-6-methyloctanamide.

[0057] The structure of the polymyxin B related substance M is: .

[0058] Chemical formula: C55H96N16O13, relative molecular mass: 1189.47, chemical name: N-(2S)-4-amino-1-((2S,3R)-1-(2S)-4-amino-1-oxo-1-(3S,6S,9S,12S,15R,18S)6,9,18-tris(2-aminoethyl)-15-benzyl-12-((S)-sec-butyl)-3-((R)-1-hydroxyethyl)-2,5,8,11,14,17,20-heptaoxo-1,4,7,10,13,16,19-heptaazacyclotris-21-yl)amino)butan-2-yl)amino)-3-hydroxy-1-oxobutan-2-yl)amino)-1-oxobutan-2-yl)octanamide.

[0059] The structure of the polymyxin B related substance N is: .

[0060] Chemical formula: C 55 H 96 N 16 O 14 , relative molecular mass: 1205.47, chemical name: N -(2 S )-4-amino-1-((2 S , 3 R )-1-(2 S )-4-amino-1-oxo-1-((3 S , 6 S , 9 S , 12 S , 15 R , 18 S )-6,9,18-tris(2-aminoethyl)-15-benzyl-3-(( R )-1-hydroxyethyl)-12-isobutyl-2,5,8,11,14,17,20-heptaoxo-1,4,7,10,13,16,19-heptaazacyclotrisin-21-yl)amino)butan-2-yl)amino)-3-hydroxy-1-oxobutan-2-yl)-1-oxobutan-2-yl)-2-hydroxyoctanamide.

[0061] The technical features of the above embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0062] The above embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.

Claims

1. A method for analyzing related substances of polymyxin B sulfate for injection, characterized in that: The following steps are involved: Dissolve polymyxin B sulfate for injection in water-acetonitrile solution and shake well to obtain the test solution; Dissolve the polymyxin B standard in water-acetonitrile solution and shake well to obtain a standard solution; The test solution and the standard solution are tested by high performance liquid chromatography, wherein the test adopts gradient elution; The volume ratio of water to acetonitrile in the water-acetonitrile solution is 80:20; the mobile phase A is a sodium sulfate solution with a mass concentration of 4-5 g / L, and the mobile phase B is acetonitrile.

2. The method for analyzing related substances of polymyxin B sulfate for injection according to claim 1, characterized in that: The effective components of polymyxin B sulfate for injection include polymyxin B1, polymyxin B2, polymyxin B3, and polymyxin B1-I.

3. The method for analyzing related substances of polymyxin B sulfate for injection according to claim 1, characterized in that: The related substances detected by the analytical method include polymyxin B related substance E, polymyxin B related substance F, polymyxin B related substance G, polymyxin B related substance J, polymyxin B related substance H, polymyxin B related substance I, polymyxin B related substance M, and polymyxin B related substance P.

4. The method for analyzing related substances of polymyxin B sulfate for injection according to claim 2 or 3, characterized in that: The elution conditions of the gradient elution are as follows: At 0 min, mobile phase A was 78% and mobile phase B was 22%; At 46 min, mobile phase A was 78% and mobile phase B was 22%; At 51 min, mobile phase A was 75% and mobile phase B was 25%; At 69 min, mobile phase A was 75% and mobile phase B was 25%; At 70 min, mobile phase A was 78% and mobile phase B was 22%; At 80 min, mobile phase A was 78% and mobile phase B was 22%.

5. The method for analyzing related substances of polymyxin B sulfate for injection according to claim 4, characterized in that: The chromatographic column used for the detection uses octadecylsilane bonded silica gel as a filler.

6. The method for analyzing related substances of polymyxin B sulfate for injection according to claim 4, characterized in that: The column temperature of the detection is 15~25°C.

7. The method for analyzing related substances of polymyxin B sulfate for injection according to claim 4, characterized in that: The detection wavelength of the detection is 200~230nm.

8. The method for analyzing related substances of polymyxin B sulfate for injection according to claim 4, characterized in that: The flow rate of the mobile phase for the detection was 0.8 mL / min; the injection volume for the detection was 20 μL.

9. The method for analyzing related substances of polymyxin B sulfate for injection according to claim 1, characterized in that: The preparation process of the mobile phase A is to dissolve 4-5 g of anhydrous sodium sulfate in 900 mL of water, adjust the pH value to 2-3 with dilute phosphoric acid, and then dilute with water to 1000 mL.

10. The method for analyzing related substances of polymyxin B sulfate for injection according to claim 1, characterized in that: The concentration of the test solution is 40-60 g / L; the concentration of the standard solution is 40-60 g / L.

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