Method for separating polymyxin sulfate B1, B2, B3 and B1-I and impurities

Abstract

The invention provides a method for separating polymyxin sulfate B1, B2, B3 and / or B1-I. The method is characterized by comprising the following steps: enabling polymyxin sulfate to pass through a preparative high performance liquid chromatograph and an ultraviolet detector so as to obtain the polymyxin sulfate B1, B2, B3 and / or B1-I. A mobile phase A is acetonitrile, a mobile phase B is a trifluoroacetic acid aqueous solution, and a mobile phase B is a mobile phase B; the volume percentage of trifluoroacetic acid in the trifluoroacetic acid aqueous solution is 0.01%-1.0%, and the volume ratio of the mobile phase A to the mobile phase B is 5: 95-40: 60; the method has the advantages that the sample loading amount is large, the purity and the yield of the separated polymyxin sulfate B1, B2, B3 and / or B1-I are high, the interference of impurities M and J can be eliminated, the operation is simple, the cost is low, and the like.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for separating polymyxin sulfate B1, B2, B3, and B1-I from polymyxin sulfate, and also to a method for separating impurities M and J (also referred to as compounds M and J) therefrom. Technical Background

[0002] Polymyxin B is a basic polypeptide antibiotic produced by Paenibacillus polymyxa and composed of multiple amino acids and fatty acids. Polymyxin B is a mixture containing multiple main components with similar structures, called polymyxin sulfate polymyxin B1, B2, B3, B1-I, and their structures are as follows:

[0003]

[0004]

[0005] Polymyxin B has a strong bactericidal effect on Gram-negative bacteria. Since it has the advantage of being able to quickly kill bacteria without causing them to develop drug resistance, it has become one of the treatment methods for preventing and treating drug-resistant Gram-negative bacterial strains. In clinical practice, polymyxin B is usually in the form of sulfate, which is mainly used to treat a variety of diseases caused by Gram-negative bacteria, including dysentery, meningitis, etc., and can also be used to prevent Gram-negative bacterial infections without developing drug resistance. However, polymyxin B sulfate also has toxicity and neuromuscular blocking effects, so its application in the medical field is greatly limited.

[0006] In order to more clearly and deeply study the mechanism of action of polymyxin B sulfate, including the reasons for the adverse physiological effects such as toxicity, and to meet the increasing demand for the quality and quantity of the drug, we urgently need to provide a method for separating and purifying polymyxin B sulfate to obtain high-yield and high-purity polymyxin sulfates polymyxin B1, B2, B3, B1-I, and suitable for preparative chromatographic separation, which can efficiently and massively produce the pure products of these polymyxin sulfates. However, due to the similar structures and large differences in content of these four polymyxin sulfates (B1, B2, B3, B1-I), the separation and purification process has always been a key technical difficulty in industry. At present, the methods of the prior art are mainly focused on the separation and purification of a small amount of polymyxin sulfate, usually using high performance liquid chromatography and ultraviolet detectors, wherein the mobile phase in the high performance liquid chromatography uses acetonitrile and sodium sulfate solution (adjusting pH with dilute phosphoric acid); however, since the method uses a salt-containing solution as the mobile phase, it is necessary to thoroughly and frequently clean the chromatographic column during the test or after use, otherwise it is easy to cause the blockage of the chromatographic column, the problem of short life of the chromatographic column, and the operation of the method is cumbersome and time-consuming; another method is to use high performance liquid chromatography and evaporative light scatterer, wherein the evaporative light scatterer for detection requires control and setting of multiple parameter values, such as drift tube temperature, carrier gas pressure and atomization port temperature, and there are strict requirements for the selection of mobile phase, and there are also problems such as long equilibrium time, poor detection stability, and easy clogging of evaporative light detectors. In addition, these two methods also have defects such as small sample loading, and are therefore more suitable for the analysis and detection of a small amount of samples, but not suitable for the separation and purification of a large number of samples, especially not suitable for the separation and purification of polymyxin sulfate prepared on a large scale.

[0007] Therefore, it is necessary to provide a method for separating and purifying polymyxin sulfate, which can be applicable to preparative chromatography, and the obtained product has a high yield and high purity to meet the growing market demand and scientific research requirements. Summary of the invention

[0008] In view of the above technical problems, the object of the present invention is to provide a method for separating polymyxin sulfate B1, B2, B3 and / or B1-I, which has the advantages of large sample loading, high purity and high yield of separated polymyxin sulfate B1, B2, B3, B1-I, and can completely separate and remove impurities M and J, simple operation, low cost, etc., and is suitable for industrial scale.

[0009] In a first aspect of the present invention, a method for separating polymyxin sulfate B1, B2, B3 and / or B1-I is provided, comprising:

[0010] The polymyxin sulfate is passed through a preparative high performance liquid chromatograph to obtain polymyxin sulfate B1, B2, B3 and / or B1-I, wherein the preparative high performance liquid chromatograph comprises an ultraviolet detector, the mobile phase A is acetonitrile, the mobile phase B is a trifluoroacetic acid aqueous solution, the volume percentage of trifluoroacetic acid in the trifluoroacetic acid aqueous solution is 0.01%-1.0%, preferably 0.02%-0.5%, more preferably 0.1%, and the volume ratio of mobile phase A to B is 5:95 to 40:60.

[0011] Compared with the prior art, the beneficial technical effects of the present invention are:

[0012] The present invention uses an ultraviolet detector for detection, which has the advantages of simple operation, good specificity, good system stability, and a wide detection range. In contrast, the evaporative light scattering detector requires setting multiple parameter values, such as drift tube temperature, carrier gas pressure, and atomization port temperature, and has strict requirements for the selection of solvents.

[0013] The invention adopts acetonitrile and trifluoroacetic acid aqueous solution as mobile phases for gradient elution, and only needs two steps of separation and purification to completely separate polymyxin sulfates B1, B2, B3 and B1-I, thereby obtaining polymyxin sulfates with high yield and high purity.

[0014] In addition, the preparation method of the present invention also allows a larger mass loading amount, which is suitable for industrial scale; and can improve the separation effect of polymyxin sulfate and impurities M and J, and can even separate impurity M with a purity greater than 95%, and impurity J with a purity greater than 85%, thereby achieving efficient separation of high-purity impurities M and J from polymyxin sulfate, and providing a basis for further research on the pharmacology and toxicology of impurities for polymyxin sulfate.

[0015] Therefore, the separation method of the present invention has important application value, is suitable for preparative liquid chromatography with a large sample load, and the separated polymyxin sulfate B1, B2, B3, and B1-I can be used as standard products due to their high purity, and are applied to preparation and analysis and detection, which is also conducive to more accurate research on their pharmacological effects without interference from other impurities; at the same time, it also provides an experimental basis for establishing a large-scale chromatographic separation and purification method with low input and high output. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The figure shows a high performance liquid chromatogram (HPLC chart) obtained for polymyxin sulfate spray powder using the standard analytical method provided in the Chinese Pharmacopoeia 2020 Edition.

[0017] Figure 2 The HPLC chart of polymyxin B2 separated by (2) described in Comparative Example 1 is shown.

[0018] Figure 3 The HPLC chart of polymyxin sulfate B1-I separated by (4) described in Comparative Example 1 is shown.

[0019] Figure 4 The HPLC chart of polymyxin sulfate B1 separated by (1) described in Example 1 is shown.

[0020] Figure 5 The HPLC chart of polymyxin B2 separated in (2) described in Example 1 is shown.

[0021] Figure 6 The HPLC chart of polymyxin B3 sulfate separated in (3) described in Example 1 is shown.

[0022] Figure 7 The HPLC chart of polymyxin sulfate B1-I separated by (4) described in Example 1 is shown.

[0023] Figure 8 For impurity M 1 HNMR spectrum.

[0024] Fig. 9 For impurity J 1 HNMR spectrum. DETAILED DESCRIPTION

[0025] In the present invention, the general process of preparative high performance liquid chromatography (preparative HPLC) operation is:

[0026] The substance to be separated is prepared into a column liquid, and the column liquid is allowed to enter a chromatographic column (working pressure greater than 2 MPa), acetonitrile and trifluoroacetic acid aqueous solution are used as a mixed mobile phase, and elution is performed at a total flow rate of 150-250 ml / min, and an ultraviolet detector (detection wavelength is 215 nm) is observed. When a signal begins to appear, 300-600 ml of the eluate are collected, and the purity of the target substance (for example, polymyxin sulfate B1, B2, B3, B1-I; impurities M, J) in each eluate is detected, and then the eluates containing the target substance with a purity greater than a certain value are combined and concentrated to obtain a residue.

[0027] The purity was determined by analytical high performance liquid chromatography (analytical HPLC) according to the method described in 2.1 of Part II of the Examples below. The positioning of polymyxin sulfate B1, B2, B3, B1-I and impurities M and J in the HPLC spectrum was determined by the method described in 2.2 of Part II of the Examples below, and was identified by reverse phase high performance liquid chromatography-high resolution mass spectrometry (RP-HPLC-HRMS); the structures of impurities M and J were determined by high resolution mass spectrometry ESI (+), 1 H-NMR spectrum, 13C-NMR spectrum was obtained.

[0028] Optionally, each eluent is 400 ml, the total flow rate of the mobile phase is 200 ml / min; the working pressure of the chromatographic column is 1-15 MPa, and can be optionally 4-8 MPa, and the column temperature of the chromatographic column is 20-35°C.

[0029] Optionally, observe the UV detector and collect the eluate with a UV absorbance of 1000 or more.

[0030] In the present invention, the concentration can be carried out by a distillation operation known in the art, such as vacuum distillation, rotary evaporator distillation, etc. The distillation temperature is not particularly limited, as long as the solvent (especially the organic solvent) is removed by distillation. Optionally, the distillation temperature is 40-55°C; in a specific embodiment of the present invention, the distillation temperature is 45°C to remove the organic solvent acetonitrile in the eluent.

[0031] The present invention provides a method for separating polymyxin sulfate B1, B2, B3 and / or B1-I, characterized in that the method comprises: passing polymyxin sulfate through a preparative high performance liquid chromatograph to obtain polymyxin sulfate B1, B2, B3 and / or B1-I, wherein the preparative high performance liquid chromatograph comprises an ultraviolet detector, mobile phase A is acetonitrile, mobile phase B is a trifluoroacetic acid aqueous solution, the volume percentage of trifluoroacetic acid in the trifluoroacetic acid aqueous solution is 0.01%-1.0%, preferably 0.02%-0.5%, more preferably 0.1%, the volume ratio of mobile phase A to B is 5:95 to 40:60, optionally, the volume ratio is 8:92-35:65; the total flow rate of the mobile phase is 150-250 ml / min.

[0032] In the preparative HPLC of the present invention, the stationary phase used is 8-50 μm (preferably 10-15 μm) octadecylsilane bonded silica filler (ODS reverse phase C18 filler), and the chromatographic column is prepared using 100, 150 or 200 DAC.

[0033] The present invention adopts acetonitrile and trifluoroacetic acid aqueous solution as a mixed mobile phase, which is conducive to overcoming the disadvantages caused by the salt (such as sodium sulfate) contained in the mobile phase adopted in the prior art, including that the chromatographic column is easily blocked, resulting in a short life, a long equilibrium time, frequent cleaning, etc.; moreover, the separation method of the present invention can improve the separation effect, obtain polymyxin sulfate B1, B2, B3 and / or B1-I with higher purity and yield, and remove the impurities M and J that are difficult to separate.

[0034] In the preparative HPLC of the present invention, the preparative chromatographic column can be purchased commercially or prepared by itself, and the preparation process includes: after mixing 8-50 μm, ODS reverse phase C18 filler (0.8-1.5 kg) with isopropanol (4000 ml), pouring into a homogenization tank, stirring, and then homogenizing while ultrasonicating to obtain a homogenate; pouring the homogenate into a preparative liquid phase column tube (for example, a column tube of a 50DAC, 100DAC, 150DAC or 200DAC preparative liquid chromatographic column), compacting, and washing with methanol to obtain a preparative chromatographic column. The column efficiency of the chromatographic column is tested by using a naphthalene standard, and the column efficiency is judged to be qualified by the measured number of plates; a qualified preparative chromatographic column has a plate number of 5000 to 15000, preferably 7000-10000 or 6500-8500; the loading amount of the preparative chromatographic column is determined according to actual needs, for example 0.5-0.7 kg / L, such as 0.62 kg / L.

[0035] In the present invention, the preparation method of the initial raw material polymyxin sulfate spray powder used for separating polymyxin sulfate B1, B2, B3 and / or B1-I is well known to those skilled in the art, such as CN114984182A.

[0036] In an embodiment of the present invention, the starting material is polymyxin sulfate spray powder, wherein based on the total mass of polymyxin sulfate spray powder, the mass fractions of polymyxin sulfate B1, B2, B3, and B1-I are respectively B1: 45-53%, B2: 15-20%, B3: 1.2-1.8%, and B1-I: 8-15%; the mass fractions are determined by analytical HPLC according to the method described in 2.1 in Part II of the Examples below. In a specific embodiment of the present invention, the mass fractions of polymyxin sulfate B1, B2, B3, and B1-I in polymyxin sulfate spray powder are respectively B1: 49.65%, B2: 17.38%, B3: 1.5%, and B1-I: 11.86%. Thus, the mass fractions of the polymyxin sulfate B1, B2, B3, and B1-I in the polymyxin sulfate spray powder measured above are each multiplied by the mass of the initial raw material polymyxin sulfate spray powder to obtain the theoretical mass of each of the polymyxin sulfates B1, B2, B3, and B1-I, which is used to calculate the corresponding yield of polymyxin sulfate.

[0037] In the present invention, the upper column liquid refers to the liquid sample injected into the chromatographic column in the liquid chromatograph, and these samples can be the substance to be separated or analyzed itself, or can be the solution of the substance to be separated or analyzed. If not otherwise specified, the upper column liquid is added to the chromatographic column in its entirety. The concentration of the upper column liquid is the sample concentration, and the sample can be the residue obtained by concentrating the eluent under reduced pressure distillation. In some embodiments, these residues are also referred to as crude products.

[0038] According to the present invention, in a specific embodiment, the method for separating polymyxin sulfate B1, B2, B3 and / or B1-I comprises crude fractionation of the polymyxin sulfate to obtain crude products B1, B2, B3 and / or B1-I, wherein the crude fraction is eluted by a mobile phase gradient, and the gradient is set as follows:

[0039] Table 1

[0040]

[0041] The eluate is collected in batches and the purity of polymyxin sulfate B1, B2, B3, and B1-I in each batch of eluate is detected; the volume percentage of trifluoroacetic acid in the trifluoroacetic acid aqueous solution is 0.01%-1.0%, preferably 0.02%-0.5%, and more preferably 0.1%. By adopting the gradient elution procedure, the efficiency of crude separation is improved, the purity of the obtained polymyxin sulfate B1, B2, B3, and B1-I is higher, and the separation effect of these polymyxin sulfates and impurities M and J is improved.

[0042] Preferably, in the above-mentioned step of roughly fractionating polymyxin sulfate, it also includes:

[0043] The eluates collected from 220 to 300 min, preferably the eluate containing polymyxin sulfate B1 with a purity of more than 80%, are combined and concentrated to obtain a crude product B1;

[0044] The eluate collected at 51-60 min, preferably the eluate containing polymyxin B2 with a purity of more than 80%, is combined and concentrated to obtain a crude product B2;

[0045] The eluates collected during 60-120 min, preferably the eluate containing polymyxin B3 with a purity of 80% or more, are combined and concentrated to obtain a crude product B3;

[0046] The eluates collected during 120-180 min, preferably the eluate containing polymyxin sulfate B1-I with a purity of 80% or more, are combined and concentrated to obtain a crude product B1-I.

[0047] Optionally, the method for separating polymyxin sulfate B1, B2, B3 and / or B1-I further comprises refining the crude product B1, B2, B3 and / or B1-I to obtain polymyxin sulfate B1, B2, B3 and / or B1-I, wherein the refining comprises:

[0048] The crude products B1, B2, B3 and / or B1-1 are passed through a preparative high performance liquid chromatograph, wherein:

[0049] The crude product B1 was purified by isocratic elution, in which the volume ratio of mobile phase A to mobile phase B was

[0050] 25:75 to 35:65, preferably 30:70;

[0051] The crude product B2 was purified by isocratic elution, in which the volume ratio of mobile phase A to mobile phase B was

[0052] 12:88 to 17:83, preferably 15:85;

[0053] The crude product B3 was purified by isocratic elution, in which the volume ratio of mobile phase A to mobile phase B was

[0054] 18:82 to 22:78, preferably 20:80; and / or

[0055] The crude product B1-I is purified by isocratic elution, wherein the volume ratio of mobile phase A to mobile phase B is 20:80 to 25:75, preferably 22:78.

[0056] Preferably, the refining of crude products B1, B2, B3 and / or B1-I comprises:

[0057] The crude products B1, B2, B3 and / or B1-I are each dissolved in purified water to obtain a column liquid with a concentration of 50-150 mg / ml, preferably 100 mg / ml; the column liquids are each passed through a preparative high performance liquid chromatograph, eluted with an eluent, the eluents are collected in batches and the purity of polymyxin sulfate B1, B2, B3 and / or B1-I in each batch of eluents is detected by an analytical high performance liquid chromatograph; the eluents containing polymyxin sulfate B1, B2, B3 and / or B1-I with a purity of more than 95% are each combined and concentrated under reduced pressure to obtain polymyxin sulfate B1, B2, B3 and / or B1-I.

[0058] In a specific embodiment of the present invention, the method for obtaining polymyxin sulfate B1 comprises the steps of crude fractionation and refining:

[0059] Crude fractionation step: As described above, the gradient elution method shown in Table 1 is used to combine the eluates containing polymyxin sulfate B1 collected from 220 to 300 min, and then concentrate under reduced pressure to obtain the crude product B1;

[0060] Refining steps: ① Preparation of the upper column liquid: dissolve the crude product B1 with a solvent (such as purified water) to obtain an upper column liquid with a concentration of 50-150 mg / ml, optionally, the concentration of the upper column liquid is 100 mg / ml; ② add all the upper column liquid to the preparative chromatographic column of the present invention, adopt isocratic elution, the mobile phase A is acetonitrile, and the mobile phase B is 0.02-0.5 volume % trifluoroacetic acid aqueous solution; the volume ratio of mobile phase A to B is 25:75 to 35:65; ③ collect the eluate in batches and detect the purity of polymyxin sulfate B1 contained in each batch of eluate by analytical HPLC, combine the eluates containing polymyxin sulfate B1 with a purity of more than 95%, and concentrate under reduced pressure to obtain polymyxin sulfate B1.

[0061] Optionally, in the preparation of the upper column liquid, the eluate containing polymyxin sulfate B1 with an HPLC purity of 80% or more collected in the crude fractionation step is combined; in the isocratic elution, the mobile phase B is a 0.02 volume %, 0.1 volume % or 0.5 volume % trifluoroacetic acid aqueous solution, and the volume ratio of mobile phase A to B is 30:70; thereby further purifying, improving the purity and yield of polymyxin sulfate B1, no impurities M and J are observed, and Figure 4 shown.

[0062] In another specific embodiment, the method for obtaining polymyxin B2 sulfate comprises the steps of crude fractionation and purification:

[0063] Crude fractionation step: As described above, the gradient elution method shown in Table 1 is used to combine the eluates containing polymyxin sulfate B2 collected from 51 to 60 minutes, and then concentrate under reduced pressure to obtain the residue as the crude product B2;

[0064] Refining steps: ① Preparation of the upper column liquid: dissolve the crude product B2 with a solvent (such as purified water) to obtain an upper column liquid with a concentration of 50-150 mg / ml, optionally, the concentration of the upper column liquid is 100 mg / ml; ② add all the upper column liquid to the preparative chromatographic column of the present invention, adopt isocratic elution, the mobile phase A is acetonitrile, and the mobile phase B is 0.02-0.5 volume % trifluoroacetic acid aqueous solution; the volume ratio of mobile phase A to B is 12:88 to 17:83; ③ collect the eluate in batches and detect the purity of polymyxin B2 contained in each batch of eluate by analytical HPLC, combine the eluates containing polymyxin B2 with a purity of more than 95%, and concentrate under reduced pressure to obtain polymyxin B2 sulfate.

[0065] Optionally, in the preparation of the upper column liquid, the eluate containing polymyxin sulfate B2 with a purity of 80% or more collected in the crude fractionation step is combined; in the isocratic elution, the mobile phase B is a 0.02 volume %, 0.1 volume % or 0.5 volume % trifluoroacetic acid aqueous solution, and the volume ratio of mobile phase A to B is 15:85; thereby further purifying, improving the purity and yield of polymyxin sulfate B2, no impurities M and J are observed, and Figure 5 shown.

[0066] In yet another specific embodiment, the method for obtaining polymyxin B3 sulfate comprises crude fractionation and purification steps:

[0067] Crude fractionation step: As described above, the gradient elution method shown in Table 1 is used to combine the eluates containing polymyxin sulfate B3 collected from 60 to 120 minutes, and the residue obtained after reduced pressure concentration is the crude product B3;

[0068] Refining steps: ① Preparation of the upper column liquid: dissolve the crude product B3 with a solvent (such as purified water) to obtain an upper column liquid with a concentration of 50-150 mg / ml, optionally, the concentration of the upper column liquid is 100 mg / ml; ② add all the upper column liquid to the preparative chromatographic column of the present invention, adopt isocratic elution, the mobile phase A is acetonitrile, and the mobile phase B is 0.02-0.5 volume % trifluoroacetic acid aqueous solution; the volume ratio of mobile phase A to B is 18:82 to 22:78; ③ collect the eluate in batches and detect the purity of polymyxin B3 contained in each batch of eluate by analytical HPLC, combine the eluates containing polymyxin B3 with a purity of more than 95%, and concentrate under reduced pressure to obtain polymyxin B3.

[0069] Optionally, in the preparation of the upper column liquid, the eluate containing polymyxin sulfate B3 with a purity of 80% or more collected in the crude fractionation step is combined; in the isocratic elution, the mobile phase B is a 0.02 volume %, 0.1 volume % or 0.5 volume % trifluoroacetic acid aqueous solution, and the volume ratio of mobile phase A to B is 20:80; thereby further purifying, improving the purity and yield of polymyxin sulfate B3, no impurities M and J are observed, and Figure 6 shown.

[0070] In yet another specific embodiment of the present invention, the method for obtaining polymyxin sulfate B1-I comprises the steps of crude fractionation and refining:

[0071] Crude fractionation step: As described above, the gradient elution method shown in Table 1 is used to combine the eluates containing polymyxin sulfate B1-I collected from 120 to 180 min, and then concentrate under reduced pressure to obtain the residue as the crude product B1-I;

[0072] Refining steps: ① Preparation of the upper column liquid: dissolve the crude product B1-I with a solvent (such as purified water) to obtain an upper column liquid with a concentration of 50-150 mg / ml, optionally, the concentration of the upper column liquid is 100 mg / ml; ② add all the upper column liquid to the preparative chromatographic column of the present invention, adopt isocratic elution, the mobile phase A is acetonitrile, and the mobile phase B is 0.02-0.5 volume % trifluoroacetic acid aqueous solution; the volume ratio of mobile phase A to B is 20:80 to 25:75; ③ collect the eluate in batches and use analytical HPLC to detect the purity of polymyxin sulfate B1-I contained in each batch of eluate, and combine the eluates containing polymyxin sulfate B1-I with a purity of more than 95%, and concentrate under reduced pressure to obtain polymyxin sulfate B1-I.

[0073] Optionally, in the preparation of the upper column liquid, the eluate containing polymyxin sulfate B1-I with a purity of 80% or more collected in the crude fractionation step is combined; in the isocratic elution, the mobile phase B is a 0.02 volume %, 0.1 volume % or 0.5 volume % trifluoroacetic acid aqueous solution, and the volume ratio of the mobile phase A to B is 22:78; thereby, further purification can be performed to improve the purity and yield of polymyxin sulfate B1-I, and impurities M and J are not observed. Figure 7 shown.

[0074] The polymyxin sulfates B1, B2, B3 and / or B1-I obtained by the method for separating polymyxin sulfates B1, B2, B3 and B1-I according to the present invention do not contain or contain less than 1% by weight of compound M and / or compound J.

[0075] In the separation method of the present invention, the refining step only needs to carry out a separation and purification process once to obtain polymyxin sulfate B1, B2, B3, B1-I with high purity and high yield, and the presence of impurities M and J is not detected. In contrast, for the method of using acetonitrile and sodium sulfate solution as a mixed mobile phase in the prior art, multiple separation and purification processes must be carried out, and finally a desalting separation and purification process is required to obtain polymyxin sulfate B1, B2, B3, B1-I with the highest possible purity. The purity and yield of polymyxin sulfate B1, B2, B3, B1-I obtained by the prior art method are lower than those of the present invention.

[0076] According to the present invention, in order to identify the structures of impurities M and J, the present invention also provides a method for separating and purifying these two impurities, and characterizes their structures. This is described in detail below.

[0077] According to the method for separating polymyxin sulfate B1, B2, B3 and / or B1-I of the present invention, compound M and / or compound J are also separated and obtained.

[0078]

[0079]

[0080] The above structures of compounds M and J were determined by high resolution mass spectrometry ESI (+), 1 H-NMR spectrum ( Figure 8 ), 13 C-NMR spectrum analysis.

[0081] In the method of the present invention, a step of isolating compound M is included, the step comprising:

[0082] Crude fractionation step: As described above, the gradient elution method shown in Table 1 is used to combine the eluates collected at 60-80 min, preferably the eluate containing compound M with a purity of more than 40%, and then distill under reduced pressure to obtain a crude product M;

[0083] Purification step: dissolving the crude product M with purified water to obtain a column liquid with a concentration of 50-150 mg / ml, preferably 100 mg / ml; passing the column liquid through a preparative high performance liquid chromatograph, using mobile phases A and B for gradient elution, and the gradient setting is as follows:

[0084] time Mobile phase A:B volume ratio 0-40min 8:92 40-80min 12:88 80-120min 15:85 120-160min 18:82

[0085] Collect the eluate eluted with the mobile phase A:B volume ratio of 15:85; then concentrate the eluate under reduced pressure to obtain a residue;

[0086] The residue is eluted repeatedly according to the above steps for multiple times, preferably 2-4 times, until the purity of compound M in the collected eluate reaches more than 95%.

[0087] Among them, in all the above steps: the stationary phase is 8-50 μm, octadecylsilane bonded silica gel, and the chromatographic column is prepared by using 100, 150 or 200 DAC; the detection wavelength is 215 nm.

[0088] In the method of the present invention, a step of isolating compound J is included, the step comprising:

[0089] Crude fractionation step: As described above, the gradient elution method shown in Table 1 is used to combine the eluates collected at 160-210 min, preferably the eluate containing compound J with a purity of more than 20%, and then distill under reduced pressure to obtain a crude product J;

[0090] Purification step: Dissolve the crude product J with purified water to obtain a column liquid with a concentration of 50-150 mg / ml, preferably 100 mg / ml; pass the column liquid through a preparative high performance liquid chromatograph, using mobile phases A and B for gradient elution, and the gradient setting is as follows:

[0091] Time Mobile phase A:B volume ratio 0-40min 20:80 40-80min 21:79 80-120min 22:78 120-160min 23:77

[0092] The eluate eluted with the mobile phase A:B volume ratio of 22:78 was collected, and then the eluate was concentrated under reduced pressure to obtain a residue;

[0093] The residue is eluted repeatedly according to the above steps for multiple times, preferably 5-8 times, until the purity of compound J in the collected eluate reaches 85%.

[0094] In all the above steps, preparative HPLC was used; the stationary phase was 8-50 μm, octadecylsilane bonded silica gel, and 100, 150 or 200 DAC was used to prepare the chromatographic column; and the detection wavelength was 215 nm.

[0095] In the method for separating compounds M and J of the present invention, the refining step is to obtain the compound M or J of maximum purity, so the separation step performed therein can be repeated multiple times until the maximum purity of the target substance (compound M or J) is not significantly improved. In a specific embodiment, the purity of the target substance (compound M or J) contained in the eluent is determined by analytical HPLC. If the difference between the maximum purity determined and the maximum purity obtained in the adjacent previous separation step is not greater than 1%, such as 0.2%, 0.5% or 0.8%, it is considered that the purity of the target substance is not significantly improved, and the refining step ends here. This description also applies to the refining steps of other target substances, such as the comparative examples of the present invention.

[0096] The separation method of the present invention can also be used for other types of polymyxin sulfates, such as polymyxin sulfate nanofiltrate, acidified solution or crystallization solution, to separate high-purity and high-yield polymyxin sulfates polymyxin sulfates B1, B2, B3, B1-I, and impurities M and J.

[0097] Example

[0098] The method of the present invention will be described and illustrated in more detail below in conjunction with specific embodiments. It will be understood by those skilled in the art that the purpose of providing these embodiments is only for illustrative purposes and does not constitute any limitation to the scope of the present invention.

[0099] I. General Description

[0100] 1.1 Analytical High Performance Liquid Chromatography (Analytical HPLC)

[0101] Chromatographic column: Thermo Hypersil GOLD TM C 18 ,4.6mm×250mm,5μm;

[0102] Working pressure: 12MPa;

[0103] Detector: UV detector;

[0104] Detection wavelength: 215nm;

[0105] Column temperature: 30℃.

[0106] 1.2 Preparative High Performance Liquid Chromatography (Preparative HPLC)

[0107] Chromatographic column: It is filled with ODS reverse phase C18 filler (model: SP-120-10-ODS-RPS, manufacturer: Japan OSAKA SODACo.LTD.), and the specific preparation process is as follows:

[0108] ① Homogenization: Mix the reversed-phase C18 filler (particle size of 10 μm) and isopropanol at a ratio of 1 g (reverse-phase C18 filler): 4 ml (isopropanol) and put them into a homogenization tank. Stir for 30 minutes, then add it to an ultrasonic cleaner and homogenize it for 5 minutes while ultrasonicating to obtain a homogenate.

[0109] ② Column loading: The bottom of the column is blocked with a flange plug, and the homogenate is poured into the preparative liquid phase column tube (100DAC) using a funnel under normal pressure, and then compacted under a pressure of 118 bar. The whole process is completed within 4 minutes;

[0110] After the chromatographic column was loaded, it was allowed to stand for 1 hour, and then pure methanol was used as the mobile phase and the column was flushed at a low flow rate (20 ml / min) for 1 hour to obtain a 100DAC preparative chromatographic column (reverse phase C18 filler 1.3 kg, column length 60 cm, column inner diameter 15 cm).

[0111] ③ Test column efficiency: The 100DAC prepared chromatographic column obtained above was tested for column efficiency. Naphthalene was used as the standard solution (concentration was 1 mg / ml) as the standard product. Acetonitrile: water = 80:20 (volume ratio) was selected as the mobile phase. The mobile phase was used for isocratic elution for 30 minutes. After the main chromatographic peak of naphthalene was observed, the plate number of the chromatographic column was read as 8000.

[0112] Working pressure: 5MPa;

[0113] Detector: UV detector;

[0114] Detection wavelength: 215nm.

[0115] 1.3 Reverse-phase high performance liquid chromatography-high resolution mass spectrometry (RP-HPLC-HRMS)

[0116] Reversed Phase High Performance Liquid Chromatography

[0117] Model: U3000;

[0118] Column: Ultimate LP-C18, 4.6×250 mm, 5 μm;

[0119] Detector: UV detector;

[0120] Detection wavelength: 215nm;

[0121] Column temperature: 30℃.

[0122] High Resolution Mass Spectrometer

[0123] Model:Q EXACTIVE;

[0124] Spray voltage: 3.9KV;

[0125] Spray temperature: 320℃;

[0126] Ion transfer tube temperature: 320°C;

[0127] Sheath gas 60 bar, nitrogen;

[0128] Auxiliary gas 10bar, nitrogen;

[0129] Lens voltage: 55V;

[0130] Scan mode: FULL MS / dd MS2;

[0131] Ionization mode: ESI;

[0132] Scan range: 150-2000m / z;

[0133] Resolution:70000.

[0134] 1.4 Preparation of purified water

[0135] A 0.25T / h two-stage reverse osmosis water purification system (Guangzhou Kangjing Water Treatment Technology Co., Ltd.) is used.

[0136] 1.5 Preparation of polymyxin sulfate spray powder (mixture)

[0137] The polymyxin sulfate spray powder used in the examples of the present application is prepared according to the method described in Example 5 of CN114984182A, which is a mixture containing polymyxin sulfates B1, B2, B3 and B1-I as main components.

[0138] II. Determination of retention time and theoretical content of polymyxin sulfate B1, B2, B3 and B1-I

[0139] According to the standard analytical method provided in the "Chinese Pharmacopoeia 2020 Edition", the theoretical content and retention time of each polymyxin sulfate component in the polymyxin sulfate spray powder are determined. The specific steps are as follows:

[0140] 2.1 Separation of polymyxin sulfate components

[0141] Analytical HPLC was used to separate the polymyxin sulfate components in the polymyxin sulfate spray powder. The elution conditions were as follows:

[0142] Preparation of the column liquid: 300 g of polymyxin sulfate spray powder was mixed with 15000 ml of purified water to prepare a polymyxin sulfate spray powder solution with a concentration of 0.02 g / ml;

[0143] Injection volume: Take 20 μl of the above-prepared column solution and inject it into the chromatographic column;

[0144] Mobile phase A: acetonitrile, mobile phase B: sodium sulfate solution (4.46 g of anhydrous sodium sulfate was dissolved in 900 ml of water, the pH value was adjusted to 2.3 with dilute phosphoric acid, and then diluted to 1000 ml with water); isocratic elution was performed with mobile phase A:B in a ratio of 22:78 (volume ratio) and a total flow rate of 1.0 ml / min, and the elution time was 50 min.

[0145] Separate and obtain each component. The separation results are as follows Figure 1 shown.

[0146] 2.2 Determination of polymyxin sulfate components

[0147] RP-HPLC-HRMS was used to determine the characteristic peaks of each polymyxin sulfate component in the HPLC spectrum. The test conditions were as follows:

[0148] Mobile phase: Mobile phase A is 0.05% formic acid aqueous solution; mobile phase B is acetonitrile; the volume ratio of mobile phase A:B is 10:90, the total flow rate is 1.0 ml / min, and the isocratic elution is 10 min.

[0149] Column loading solution: Prepare each component separated as described in step 2.1 into a 0.1 mg / ml solution with purified water, and take 5 μl of each solution as the column loading solution.

[0150] The test results are as follows:

[0151] The molecular weight of polymyxin B1 sulfate is 1202.74993; the measured values ​​are m / z1203.75647[M+H], m / z1225.73804[M+Na]; the retention time Rt is 37.02min;

[0153] The molecular weight of polymyxin B2 sulfate is 1188.73428; the measured values ​​are m / z1189.74182[M+H], m / z1211.72302[M+Na]; the retention time Rt is 17.49min;

[0155] The molecular weight of polymyxin B3 sulfate is 1188.73428; the measured value is m / z1189.74194[M+H]; the retention time Rt is 19.90min;

[0157] The molecular weight of polymyxin B1-I was 1202.74993; the measured values ​​were m / z 1203.75635 [M+H], m / z 1225.73792 [M+Na]; and the retention time Rt was 29.69 min.

[0158] according to Figure 1 Using the peak area normalization method, it can be determined that the mass fractions of polymyxin sulfate B1, B2, B3, and B1-I in the polymyxin sulfate spray powder are B1: 49.65%, B2: 17.38%, B3: 1.5%, and B1-I: 11.86%, respectively. It is calculated that the masses of polymyxin sulfate B1, B2, B3, and B1-I contained in 300g of polymyxin sulfate spray powder are 148.95, 52.14, 4.5, and 35.58g, respectively, which are recorded as the theoretical mass.

[0159] III. Isolation of polymyxin sulfate B1, B2, B3 and B1-I

[0160] 3.1 Example 1

[0161] Step 1: Rough classification

[0162] Take 300g of polymyxin sulfate spray powder and dissolve it in 5L of purified water by ultrasonic. After it is completely dissolved, all of it is used as the column liquid with a concentration of 0.06g / ml. It is passed through preparative HPLC. The particle size of the stationary phase ODS reverse phase C18 filler is 10μm, the column temperature is 30°C, and the elution conditions are as follows:

[0163] Mobile phase A: acetonitrile, mobile phase B: 0.1 vol% trifluoroacetic acid (TFA) aqueous solution, total flow rate is 200 ml / min, gradient elution method as shown in Table 1 is adopted, and the main components of the collected eluate are also recorded in Table 1.

[0164] Table 1

[0165]

[0166] Observe the UV detector, collect the eluent with UV absorbance of more than 1000, collect 400 ml each, and use RP-HPLC-HRMS to determine the target substances: polymyxin sulfate B1, B2, B3 and B1-I, and impurities M and J. The purity of the target substance is determined by analytical HPLC according to the method described in 2.1, and the eluents of the same polymyxin sulfate component with a purity of more than 80% are combined to obtain fractions 1, 2, 3 and 4. Fractions 1, 2, 3 and 4 are respectively concentrated under reduced pressure using a rotary evaporator to obtain crude products B2, B3, B1-I and B1.

[0167] Step 2: Refining

[0168] (1) Isolation of polymyxin B1 sulfate

[0169] The crude product B1 was dissolved in purified water. After it was completely dissolved, a column liquid with a concentration of 100 mg / ml was obtained. The column was subjected to preparative HPLC. The particle size of the stationary phase ODS reverse phase C18 filler was 10 μm. The column temperature was 30°C. The elution conditions were as follows:

[0170] Mobile phase A: acetonitrile, mobile phase B: 0.1 vol% trifluoroacetic acid (TFA) aqueous solution, isocratic elution was performed with a mobile phase A:B volume ratio of 30:70 and a total flow rate of 200 ml / min.

[0171] The eluate was collected in portions of 400 ml each, and the purity of each portion of the eluate was determined by analytical HPLC according to the method described in 2.1. The eluates with a purity of more than 95% of polymyxin sulfate B1 were combined, concentrated under reduced pressure using a rotary evaporator, and freeze-dried to obtain 78.6 g of polymyxin sulfate B1.

[0172] (2) Isolation of polymyxin B2 sulfate

[0173] The crude product B2 was dissolved in purified water. After it was completely dissolved, a column liquid with a concentration of 100 mg / ml was obtained. The column was subjected to preparative HPLC. The particle size of the stationary phase ODS reverse phase C18 filler was 10 μm. The column temperature was 30°C. The elution conditions were as follows:

[0174] Mobile phase A: acetonitrile, mobile phase B: 0.1 vol% trifluoroacetic acid (TFA) aqueous solution, isocratic elution was performed with a mobile phase A:B volume ratio of 15:85 and a total flow rate of 200 ml / min.

[0175] The eluate was collected in portions of 400 ml each, and the purity of each portion of the eluate was determined by analytical HPLC according to the method described in 2.1. The eluates with a purity of more than 95% of polymyxin B2 sulfate were combined, concentrated under reduced pressure using a rotary evaporator, and freeze-dried to obtain 17.34 g of polymyxin B2 sulfate.

[0176] (3) Separation of polymyxin B3 sulfate

[0177] The crude product B3 was dissolved in purified water. After it was completely dissolved, a column liquid with a concentration of 100 mg / ml was obtained. The column was subjected to preparative HPLC. The particle size of the stationary phase ODS reverse phase C18 filler was 10 μm. The column temperature was 30°C. The elution conditions were as follows:

[0178] Mobile phase A: acetonitrile, mobile phase B: 0.1 vol% trifluoroacetic acid (TFA) aqueous solution, isocratic elution was performed with a mobile phase A:B volume ratio of 20:80 and a total flow rate of 200 ml / min.

[0179] The eluate was collected in portions of 400 ml each, and the purity of each portion of the eluate was determined by analytical HPLC according to the method described in 2.1. The eluates with a purity of more than 95% of polymyxin B3 sulfate were combined, concentrated under reduced pressure using a rotary evaporator, and freeze-dried to obtain 3.0 g of polymyxin B3 sulfate.

[0180] (4) Separation and acquisition of polymyxin B1-I

[0181] The crude product B1-I was dissolved in purified water. After complete dissolution, a column liquid with a concentration of 100 mg / ml was obtained. The column was subjected to preparative HPLC. The particle size of the stationary phase ODS reverse phase C18 filler was 10 μm. The column temperature was 30°C. The elution conditions were as follows:

[0182] Mobile phase A: acetonitrile, mobile phase B: 0.1 vol% trifluoroacetic acid (TFA) aqueous solution, isocratic elution was performed with a mobile phase A:B volume ratio of 22:78 and a total flow rate of 200 ml / min.

[0183] The eluate was collected in 400 ml portions, and the purity of each eluate was determined by analytical HPLC according to the method described in 2.1. The eluates of polymyxin sulfate B1-I with a purity of more than 95% were combined, concentrated under reduced pressure using a rotary evaporator, and freeze-dried to obtain 19 g of polymyxin sulfate B1-I.

[0184] 3.2 Example 2

[0185] The method of Example 1 was followed, except that the mobile phase B was changed to a 0.5 vol% trifluoroacetic acid (TFA) aqueous solution.

[0186] 3.3 Example 3

[0187] The method of Example 1 was followed, except that the mobile phase B was changed to a 0.02 vol% trifluoroacetic acid (TFA) aqueous solution.

[0188] 3.4 Example 4

[0189] The method of Example 1 was followed, except that the particle size of the stationary phase ODS reverse phase C18 filler of the preparative HPLC was changed to 8 μm.

[0190] 3.5 Example 5

[0191] The method of Example 1 was followed, except that the particle size of the stationary phase ODS reverse phase C18 filler of the preparative HPLC was changed to 50 μm.

[0192] 3.6 Example 6

[0193] The method of Example 1 was followed, except that the column temperature was changed to 35°C.

[0194] 3.7 Comparative Example 1

[0195] Step 1: Rough classification

[0196] Take 300g of polymyxin sulfate spray powder and dissolve it in 5L of purified water by ultrasonic. After it is completely dissolved, all of it is used as the column liquid with a concentration of 0.06g / ml. It is passed through preparative HPLC. The particle size of the stationary phase ODS reverse phase C18 filler is 10μm, the column temperature is 30°C, and the elution conditions are as follows:

[0197] Mobile phase A: acetonitrile, mobile phase B: sodium sulfate solution (4.46 g of anhydrous sodium sulfate was dissolved in 900 ml of water, the pH value was adjusted to 2.3 with dilute phosphoric acid and then diluted to 1000 ml with water); isocratic elution was performed with mobile phase A:B of 22:78 (volume ratio) and a total flow rate of 200 ml / min. The elution time was 450 min. The composition of the collected eluate is also recorded in Table 2.

[0198] Table 2

[0199]

[0200]

[0201] Observe the UV detector, collect the eluate with UV absorbance of more than 1000, collect 400 ml each, and use RP-HPLC-HRMS to determine the target substances: polymyxin sulfate B1, B2, B3 and B1-I, and impurities M and J. The purity of the target substance is determined by analytical HPLC according to the method described in 2.1, and the eluate of polymyxin sulfate B2 with a purity of more than 10% is combined to obtain a comparative fraction 1, and the eluate of polymyxin sulfate B3 with a purity of more than 5% and containing impurity M is combined to obtain a comparative fraction 2; the eluate of polymyxin sulfate B3 with a purity of more than 5% but not containing impurity M is combined to obtain a comparative fraction 3; the eluate of polymyxin sulfate B1-I with a purity of more than 15% is combined to obtain a comparative fraction 4; the eluate of polymyxin sulfate B1 with a purity of more than 30% is combined to obtain a comparative fraction 5. The comparative fractions 2 and 3 were combined, and then each comparative fraction was concentrated under reduced pressure using a rotary evaporator to obtain comparative crude products B2, B3, B1-I and B1.

[0202] Step 2: Refining

[0203] (1) Isolation of polymyxin B1 sulfate

[0204] The crude comparative product B1 was dissolved in purified water. After complete dissolution, a column liquid with a concentration of 100 mg / ml was obtained. The column was subjected to preparative HPLC. The particle size of the stationary phase ODS reverse phase C18 filler was 10 μm. The column temperature was 30°C. The elution conditions were as follows:

[0205] Mobile phase A: acetonitrile, mobile phase B: sodium sulfate solution (4.46 g of anhydrous sodium sulfate was dissolved in 900 ml of water, the pH value was adjusted to 2.3 with dilute phosphoric acid, and then diluted to 1000 ml with water); isocratic elution was performed with mobile phase A:B in a ratio of 30:70 (volume ratio) and a total flow rate of 200 ml / min, and the elution time was 160 min.

[0206] 400 ml of the eluate were collected, and the purity of each eluate was determined by analytical HPLC according to the method described in 2.1. The eluates with a purity of more than 60% of polymyxin B1 were combined and concentrated under reduced pressure using a rotary evaporator to obtain a residue.

[0207] To further purify the residue, the above separation step was repeated twice using the residue as the column liquid, and each time the eluates with purities of 80% and 95% or more were combined to obtain a solution of polymyxin B1 sulfate containing sodium sulfate.

[0208] To remove sodium sulfate, the polymyxin B1 sulfate solution containing sodium sulfate was concentrated under reduced pressure using a rotary evaporator to remove acetonitrile to obtain a residue. The residue was subjected to preparative HPLC, the particle size of the stationary phase ODS reverse phase C18 filler was 10 μm, the column temperature was 30° C., and the elution conditions were as follows:

[0209] Mobile phase A was acetonitrile, and mobile phase B was purified water; isocratic elution was performed with a mobile phase A:B ratio of 60:40 (volume ratio) and a total flow rate of 200 ml / min, and the elution time was 40 min.

[0210] The eluate in the period of 20-32 min was collected (observed by ultraviolet detector, the largest signal peak appeared), concentrated under reduced pressure, and dried to obtain 30 g of polymyxin sulfate B1.

[0211] (2) Isolation of polymyxin B2 sulfate

[0212] The crude comparative product B2 was dissolved in purified water. After complete dissolution, a column liquid with a concentration of 100 mg / ml was obtained. The column liquid was subjected to preparative HPLC. The particle size of the stationary phase ODS reverse phase C18 filler was 10 μm. The column temperature was 30° C. and the elution conditions were as follows:

[0213] Mobile phase A: acetonitrile, mobile phase B: sodium sulfate solution (4.46 g of anhydrous sodium sulfate was dissolved in 900 ml of water, the pH value was adjusted to 2.3 with dilute phosphoric acid, and then diluted to 1000 ml with water); isocratic elution was performed with a mobile phase A:B ratio of 15:85 (volume ratio) and a total flow rate of 200 ml / min, and the elution time was 270 min.

[0214] 400 ml of the eluate were collected, and the purity of each eluate was determined by analytical HPLC according to the method described in 2.1. The eluates of polymyxin B2 sulfate with a purity of more than 30% were combined and concentrated under reduced pressure using a rotary evaporator to obtain a residue.

[0215] To further purify the residue, the above separation step was repeated three times using the residue as the column liquid, and each time the eluates with purities of 60%, 80%, and 85% or more were combined to obtain a solution of polymyxin B2 sulfate containing sodium sulfate.

[0216] To remove sodium sulfate, the polymyxin B2 sulfate solution containing sodium sulfate was concentrated under reduced pressure using a rotary evaporator to remove acetonitrile to obtain a residue. The residue was subjected to preparative HPLC, the particle size of the stationary phase ODS reverse phase C18 filler was 10 μm, the column temperature was 30° C., and the elution conditions were as follows:

[0217] Mobile phase A was acetonitrile, and mobile phase B was ultrapure water; isocratic elution was performed with a mobile phase A:B ratio of 60:40 (volume ratio) and a total flow rate of 200 ml / min, and the elution time was 30 min.

[0218] The eluate in the period of 10-16 min was collected (observed by ultraviolet detector, the largest signal peak appeared), concentrated under reduced pressure, and dried to obtain 6.9 g of polymyxin B2.

[0219] (3) Separation of polymyxin B3 sulfate

[0220] The crude comparative product B3 was dissolved in purified water. After complete dissolution, a column liquid with a concentration of 100 mg / ml was obtained. The column liquid was subjected to preparative HPLC. The particle size of the stationary phase ODS reverse phase C18 filler was 10 μm. The column temperature was 30° C. and the elution conditions were as follows:

[0221] Mobile phase A: acetonitrile, mobile phase B: sodium sulfate solution (4.46 g of anhydrous sodium sulfate was dissolved in 900 ml of water, the pH value was adjusted to 2.3 with dilute phosphoric acid, and then diluted to 1000 ml with water); isocratic elution was performed with mobile phase A:B in a ratio of 20:80 (volume ratio) and a total flow rate of 200 ml / min, and the elution time was 240 min.

[0222] 400 ml of the eluate were collected, and the purity of each eluate was determined by analytical HPLC according to the method described in 2.1. The eluates of polymyxin B3 sulfate with a purity of more than 20% were combined and concentrated under reduced pressure using a rotary evaporator to obtain a residue.

[0223] To further purify the residue, the above separation step was repeated 4 times using the residue as the column liquid, and each time the eluates with purities of 30%, 60%, 80%, and 95% or more were combined to obtain a solution of polymyxin B3 sulfate containing sodium sulfate.

[0224] To remove sodium sulfate, the polymyxin B3 sulfate solution containing sodium sulfate was concentrated under reduced pressure using a rotary evaporator to remove acetonitrile to obtain a residue. The residue was subjected to preparative HPLC, the particle size of the stationary phase ODS reverse phase C18 filler was 10 μm, the column temperature was 30° C., and the elution conditions were as follows:

[0225] Mobile phase A was acetonitrile, and mobile phase B was ultrapure water; isocratic elution was performed with a mobile phase A:B ratio of 60:40 (volume ratio) and a total flow rate of 200 ml / min, and the elution time was 30 min.

[0226] The eluate in the period of 10-16 min was collected (observed by ultraviolet detector, the largest signal peak appeared), concentrated under reduced pressure, and dried to obtain 0.4 g of polymyxin B3.

[0227] (4) Separation and acquisition of polymyxin B1-I

[0228] The crude comparative product B1-I was dissolved in purified water, and after being completely dissolved, a column liquid with a concentration of 100 mg / ml was obtained. The column was subjected to preparative HPLC, with a stationary phase ODS reverse phase C18 filler having a particle size of 10 μm, a column temperature of 30° C., and elution conditions as follows:

[0229] Mobile phase A: acetonitrile, mobile phase B: sodium sulfate solution (4.46 g of anhydrous sodium sulfate was dissolved in 900 ml of water, the pH value was adjusted to 2.3 with dilute phosphoric acid, and then diluted to 1000 ml with water); isocratic elution was performed with mobile phase A:B at a ratio of 22:78 (volume ratio) and a total flow rate of 200 ml / min, and the elution time was 200 min.

[0230] 400 ml of the eluate were collected, and the purity of each eluate was determined by analytical HPLC according to the method described in 2.1. The eluates of polymyxin sulfate B1-I with a purity of more than 30% were combined and concentrated under reduced pressure using a rotary evaporator to obtain a residue.

[0231] To further purify the residue, the above separation step was repeated three times using the residue as the column liquid, and each time the eluates with purities of 60%, 80%, and 95% or more were combined to obtain a solution of polymyxin sulfate B1-I containing sodium sulfate.

[0232] To remove sodium sulfate, the polymyxin sulfate B1-I solution containing sodium sulfate was concentrated under reduced pressure using a rotary evaporator to remove acetonitrile to obtain a residue. The residue was subjected to preparative HPLC, the particle size of the stationary phase ODS reverse phase C18 filler was 10 μm, the column temperature was 30° C., and the elution conditions were as follows:

[0233] Mobile phase A was acetonitrile, and mobile phase B was ultrapure water; isocratic elution was performed with a mobile phase A:B ratio of 60:40 (volume ratio) and a total flow rate of 200 ml / min, and the elution time was 35 min.

[0234] The eluate in the period of 17-25 min was collected (observed by ultraviolet detector, the largest signal peak appeared), concentrated under reduced pressure, and dried to obtain 7.2 g of polymyxin sulfate B1-I.

[0235] Result Description

[0236] The purity and yield of polymyxin sulfate B1, B2, B3 and B1-I in Examples 1-6 and Comparative Example 1 were determined as follows:

[0237] The sample to be tested was prepared into a solution with a concentration of 1 mg / ml using a 20% (volume percentage) acetonitrile aqueous solution as the column liquid, and the purity was determined by analytical HPLC according to the method described in 2.1.

[0238] The yield is calculated as follows: [actual mass of the sample to be tested / (mass of the polymyxin sulfate spray powder × theoretical content of the sample to be tested in the polymyxin sulfate spray powder)] × 100%, wherein the theoretical content of the sample to be tested in the polymyxin sulfate spray powder is determined as described in Section II above. For example, in Example 1, the yield of polymyxin sulfate B1 is calculated as follows:

[0239] [78.6g / (300g×49.65%)]×100%=52.8%.

[0240] The purity of impurity M in Comparative Example 1 was determined as follows:

[0241] The sample to be tested was prepared into a solution with a concentration of 1 mg / ml using a 25% (volume percentage) acetonitrile aqueous solution as the column liquid, and the purity was determined by analytical HPLC using the following elution conditions:

[0242] Mobile phase A: acetonitrile, mobile phase B: sodium sulfate solution (4.46 g of anhydrous sodium sulfate was dissolved in 900 ml of water, the pH value was adjusted to 2.3 with dilute phosphoric acid, and then diluted to 1000 ml with water); isocratic elution was performed with mobile phase A:B in a ratio of 25:75 (volume ratio) and a total flow rate of 1.0 ml / min, and the elution time was 18 min.

[0243] The purity of impurity J in Comparative Example 1 was determined as follows:

[0244] The sample to be tested was prepared into a solution with a concentration of 1 mg / ml using a 20% (volume percentage) acetonitrile aqueous solution as the column liquid and analyzed by analytical HPLC (where the chromatographic column was changed to HPLCONE-5C 18 AQ, 4.6×250 mm, 5 μm) was used to determine the purity using the following elution conditions:

[0245] Mobile phase A: acetonitrile, mobile phase B: sodium sulfate solution (4.46 g of anhydrous sodium sulfate was dissolved in 900 ml of water, the pH value was adjusted to 2.3 with dilute phosphoric acid, and then diluted to 1000 ml with water); isocratic elution was performed with mobile phase A:B in a ratio of 20:80 (volume ratio) and a total flow rate of 1.0 ml / min, and the elution time was 175 min.

[0246] The test results are shown in Table 3 below.

[0247]

[0248] From the above results, it can be seen that compared with Comparative Example 1, the separation and purification methods of Examples 1-6 of the present invention provide polymyxin sulfates B1, B2, B3 and B1-I with high yield and high purity.

[0249] Specifically, in Example 1, the purity test results of polymyxin B1 are as follows: Figure 4 As shown in FIG. 1 , the retention time of polymyxin sulfate B1 is 37.29 min, and there is no characteristic peak of impurity J (retention time is 30.61 min); the purity test results of polymyxin sulfate B2 are as follows Figure 5 As shown, the retention time of polymyxin sulfate B2 is 17.41min, and there is no characteristic peak of impurity M (retention time is 18.38min); the purity test results of polymyxin sulfate B3 are as follows Figure 6 As shown, the retention time of polymyxin sulfate B3 is 19.86 min, and there is no characteristic peak of impurity M (retention time is 18.38 min); the purity test results of polymyxin sulfate B1-I are as follows Figure 7 As shown, the retention time of polymyxin sulfate B1-I is 30.11 min, and there is no characteristic peak of impurity J (retention time is 30.61 min). This result shows that the polymyxin sulfates B1, B2, B3 and B1-I obtained in Example 1 of the present application are not mixed with impurities M and J.

[0250] In contrast, in Comparative Example 1, the purity test results of polymyxin B2 sulfate are as follows: Figure 2 As shown, it is mixed with impurities M (retention time R t =8.302min, content is about 9.56%); Purity test of polymyxin B1-I is as follows Figure 3 As shown, it is mixed with impurity J (retention time R t =109.19min, content is about 1.98%).

[0251] IV. Separation of impurities M and J

[0252] 4.1 Example 7 - Separation of impurity M

[0253] Step 1: Rough classification

[0254] The process is carried out in the same manner as step 1 of Example 1, and the eluate is collected during the period of 60-80 min, and each portion is collected in 400 ml. The purity is determined by analytical HPLC according to the method described in 2.1, and the eluates of impurity M with a purity of more than 40% are combined, and the crude product M is obtained by concentrating under reduced pressure using a rotary evaporator.

[0255] Step 2: Refining

[0256] The crude product M was dissolved in purified water. After it was completely dissolved, a column liquid with a concentration of 100 mg / ml was obtained. The column was subjected to preparative HPLC. The particle size of the stationary phase ODS reverse phase C18 filler was 10 μm. The column temperature was 30°C. The elution conditions were as follows:

[0257] Mobile phase A: acetonitrile, mobile phase B: 0.1% by volume trifluoroacetic acid (TFA) aqueous solution, gradient elution was performed at a total flow rate of 200 ml / min, as shown in Table 4 below:

[0258] Table 4

[0259] Time Mobile phase A:B volume ratio 0-40min 8:92 40-80min 12:88 80-120min 15:85 120-160min 18:82

[0260] Collect the eluate eluted with the mobile phase A:B volume ratio of 15:85; then concentrate the eluate under reduced pressure to obtain a residue;

[0261] To further purify the residue, the above separation step was repeated twice using the residue as the column liquid to obtain impurity M. The purity of impurity M was measured in the same manner as in Example 1 and was found to be 96%.

[0262] According to high resolution mass spectrometry ESI(+), 1 H-NMR spectrum, 13 C-NMR spectrum, the structural formula of impurity M is

[0263]

[0264] Its chemical name is 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-heptaazacyclotricosan-21-yl)amino)butan-2-yl)amino)-3-hydroxy-1-oxobutan-2-yl)amino)-1-oxobutan-2-yl)octanamide.

[0265] ESI(+): MS m / z 1211.71631[M+Na]+(C 55 H 96 N 16 NaO 13 + ), m / z1189.73511[M+H]+(C 55 H 97 N 16 O 13 +).

[0266] 1 H-NMR (D2O, 400MHz) spectrum Figure 8 shown.

[0267] 13 C-NMR (D2O, 400MHz): δc 10.7,16.6,18.4,18.6,18.8,19.2,25.6,25.8,28.0,28.5,28.6,28.7,28.9,29.0,29. 6,30.5,33.6,35.4,35.4,36.0,36.0,36.4,36.4,36.5,36.7,36.9,50.6,51.1,51.2,5 1.6,52.2,52.7,55.5,58.9,59.0,59.8,66.3,67.1,127.4,129.0,129.0,129.2,129.2,135.8,171.6,171.8,171.9,172.4,172.9,172.9,173.1,173.4,173.7,173.9,177.8.

[0268] 4.2 Example 8 - Separation of impurity J

[0269] Step 1: Rough classification

[0270] The same method as step 1 of Example 1 was used to collect the eluate from 160 to 210 min, 400 ml of each portion was collected, and the purity was determined by analytical HPLC according to the method described in 2.1. The eluates of impurity J with a purity of more than 20% were combined and concentrated under reduced pressure using a rotary evaporator to obtain a crude product J.

[0271] Step 2: Refining

[0272] The crude product J was dissolved in purified water. After it was completely dissolved, a column liquid with a concentration of 100 mg / ml was obtained. The column was subjected to preparative HPLC. The particle size of the stationary phase ODS reverse phase C18 filler was 10 μm. The column temperature was 30°C. The elution conditions were as follows:

[0273] Mobile phase A: acetonitrile, mobile phase B: 0.1% by volume trifluoroacetic acid (TFA) aqueous solution, gradient elution was performed at a total flow rate of 200 ml / min, as shown in Table 5 below:

[0274] Table 5

[0275] Time Mobile phase A:B volume ratio 0-40min 20:80 40-80min 21:79 80-120min 22:78 120-160min 23:77

[0276] The eluate eluted with the mobile phase A:B volume ratio of 22:78 was collected, and then the eluate was concentrated under reduced pressure to obtain a residue;

[0277] To further purify the residue, the above separation steps were repeated 7 times using the residue as the column liquid to obtain impurity J. The purity of impurity J was determined to be 85% using the same method as in Example 1.

[0278] According to high resolution mass spectrometry ESI(+), 1 H-NMR spectrum, 13 C-NMR spectrum, the structural formula of impurity M is

[0279]

[0280] Its chemical name is N-((2S)-4-amino-1-(((2S)-1-(((2S)-4-amino-1-oxo-1-(((3S,6S,9S,12S,15R,18S)-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-heptaazacyclotricosan-21-yl)amino)butan-2-yl)amino)-3-hydroxy-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)-6-methyloctanamide.

[0281] ESI(+): MS m / z 1189.72510[M+H]+,595.36615[1 / 2M+2H] 2+ .

[0282] 1 H-NMR (D2O, 400MHz) Fig. 9 shown.

[0283] 13C-NMR (D2O, 400MHz): δc 10.7,18.5,19.1,20.3,22.4,23.5,25.5,25.7,28.0,28.4,28.6,28.8,29.8,33.5,35. 3,35.4,35.9,36.0,36.3,36.4,36.5,36.8,36.4,36.5,36.7,39.1,51.2,51.2,51.6,5 1.6,51.8,52.0,52.8,55.6,55.9,59.6,60.9,66.2,114.9,117.8,,129.0,129.2,129.0,135.6,171.1,171.7,172.1,172.4,172.7,172.9,173.0,173.3,173.4,175.1,177.9.

[0284] Comparative Example 2 - Separation of impurity M

[0285] Step 1: Rough classification

[0286] The same method as step 1 of comparative example 1 was used to collect the eluate during the period of 160-210 min, with 400 ml of the eluate collected each time. The purity was determined by analytical HPLC according to the method described in 2.1. The eluates of impurity M with a purity of more than 20% were combined and concentrated under reduced pressure using a rotary evaporator to obtain a comparative crude product M.

[0287] Step 2: Refining

[0288] The crude comparative product M was dissolved in purified water, and after complete dissolution, a column liquid with a concentration of 100 mg / ml was obtained. The column was subjected to preparative HPLC, with a stationary phase ODS reverse phase C18 filler having a particle size of 10 μm, a column temperature of 30°C, and elution conditions as follows:

[0289] Mobile phase A: acetonitrile, mobile phase B: sodium sulfate solution (4.46 g of anhydrous sodium sulfate was dissolved in 900 ml of water, the pH value was adjusted to 2.3 with dilute phosphoric acid and then diluted to 1000 ml with water); gradient elution was performed at a total flow rate of 200 ml / min, as shown in Table 6 below:

[0290] Table 6

[0291] time Mobile phase A:B volume ratio 0-30min 10：90 30-60min 12：88 60-90min 14：86 90-120min 16：84

[0292] To further purify the residue, the above separation step was repeated four times using the residue as the column liquid, and each time the eluates with purities of 40%, 60%, 80%, and 90% or more were combined to obtain a solution containing impurity M.

[0293] To remove sodium sulfate, the solution containing impurity M was concentrated under reduced pressure using a rotary evaporator to remove acetonitrile, thereby obtaining a residue. The residue was subjected to preparative HPLC, with a stationary phase ODS reverse phase C18 filler having a particle size of 10 μm, a column temperature of 30° C., and elution conditions as follows:

[0294] Mobile phase A was acetonitrile, and mobile phase B was purified water; isocratic elution was performed with a mobile phase A:B ratio of 60:40 (volume ratio) and a total flow rate of 200 ml / min, and the elution time was 30 min.

[0295] The eluate with a collection period of 10-16 min (observe the UV detector to see the largest signal peak), was concentrated under reduced pressure and dried to obtain impurity M. The purity of impurity M was determined to be 90% using the same method as in Example 1.

[0296] Comparative Example 3 - Separation of Impurity J

[0297] Step 1: Rough classification

[0298] The same method as step 1 of comparative example 1 was used to collect the eluate during the period of 260-320 min, with 400 ml of the eluate collected each time. The purity was determined by analytical HPLC according to the method described in 2.1. The eluates of impurity J with a purity of more than 2% were combined and concentrated under reduced pressure using a rotary evaporator to obtain a comparative crude product J.

[0299] Step 2: Refining

[0300] The crude comparative product J was dissolved in purified water. After complete dissolution, a column liquid with a concentration of 100 mg / ml was obtained. The column was subjected to preparative HPLC. The particle size of the stationary phase ODS reverse phase C18 filler was 10 μm. The column temperature was 30°C. The elution conditions were as follows:

[0301] Mobile phase A: acetonitrile, mobile phase B: sodium sulfate solution (4.46 g of anhydrous sodium sulfate was dissolved in 900 ml of water, the pH value was adjusted to 2.3 with dilute phosphoric acid and then diluted to 1000 ml with water); gradient elution was performed at a total flow rate of 200 ml / min, as shown in Table 7 below:

[0302] Table 7

[0303] time Mobile phase A:B volume ratio 0min 21：79 30min 23：77 60min 24：76 90min 30：70 120min 35：70

[0304] To further purify the residue, the above separation step was repeated four times using the residue as the column liquid, and each time the eluates with purities of 5%, 15%, 30%, and 40% or more were combined to obtain a solution containing impurity J.

[0305] To remove sodium sulfate, the solution containing impurity J was concentrated under reduced pressure using a rotary evaporator to remove acetonitrile, thereby obtaining a residue. The residue was subjected to preparative HPLC, with a stationary phase ODS reverse phase C18 filler having a particle size of 10 μm, a column temperature of 30° C., and elution conditions as follows:

[0306] Mobile phase A was acetonitrile, and mobile phase B was purified water; isocratic elution was performed with a mobile phase A:B ratio of 60:40 (volume ratio) and a total flow rate of 200 ml / min, and the elution time was 30 min.

[0307] The eluate with a collection period of 10-16 min (observe the UV detector to see the largest signal peak), was concentrated under reduced pressure and dried to obtain impurity J. The purity of impurity J was determined to be 40% using the same method as in Example 1.

[0308] For the above-mentioned Examples 7, 8 and Comparative Examples 2 and 3, the separation step is repeated multiple times in the refining step to further purify the residue until the maximum purity of the impurity M or J contained in the collected eluate does not show a significant improvement, that is, the difference in the maximum purity of the impurity M or J that can be obtained in two adjacent separation steps is not greater than 1%, then the separation step is stopped.

[0309] Comparing the results of Example 7 with Comparative Example 2, it can be seen that the preparative liquid chromatography method of the present invention can obtain high-purity (96%) impurity M. In addition, comparing Example 8 with Comparative Example 3, it can be seen that the preparative liquid chromatography method of the present invention can obtain high-purity (85%) impurity J.

Claims

1. A method for separating polymyxin sulfate B1, B2, B3 and / or B1-I, characterized in that: The method comprises: The polymyxin sulfate is passed through a preparative high performance liquid chromatograph and an ultraviolet detector to obtain polymyxin sulfate B1, B2, B3 and / or B1-I, wherein the mobile phase A is acetonitrile, the mobile phase B is a trifluoroacetic acid aqueous solution, the volume percentage of trifluoroacetic acid in the trifluoroacetic acid aqueous solution is 0.01%-1.0%, preferably 0.02%-0.5%, more preferably 0.1%, and the volume ratio of the mobile phase A to B is 5:95 to 40:

60.

2. The method according to claim 1, characterized in that The method comprises crude fractionation of the polymyxin sulfate to obtain crude products B1, B2, B3 and / or B1-I, wherein the crude fraction is eluted by a mobile phase gradient, and the gradient is set as follows:

3. The method according to claim 2, characterized in that The method comprises: The eluates collected from 220 to 300 min, preferably the eluate containing polymyxin sulfate B1 with a purity of more than 80%, are combined and concentrated to obtain a crude product B1; The eluate collected at 51-60 min, preferably the eluate containing polymyxin B2 with a purity of more than 80%, is combined and concentrated to obtain a crude product B2; The eluates collected during 60-120 min, preferably the eluate containing polymyxin B3 with a purity of 80% or more, are combined and concentrated to obtain a crude product B3; The eluates collected during 120-180 min, preferably the eluate containing polymyxin sulfate B1-I with a purity of 80% or more, are combined and concentrated to obtain a crude product B1-I.

4. The method according to claim 1, characterized in that: The stationary phase of the chromatographic column in the preparative high performance liquid chromatograph is octadecylsilane bonded silica gel with a particle size of 8-50 μm.

5. The method according to claim 2 or 3, characterized in that: The method further comprises refining the crude products B1, B2, B3 and / or B1-I to obtain polymyxin sulfate B1, B2, B3 and / or B1-I, wherein the refining comprises: The crude products B1, B2, B3 and / or B1-I are passed through a preparative high performance liquid chromatograph, wherein The crude product B1 was purified by isocratic elution, wherein the volume ratio of mobile phase A to B was 25:75 to 35:65, preferably 30:70; The crude product B2 is purified by isocratic elution, wherein the volume ratio of mobile phase A to B is 12:88 to 17:83, preferably 15:85; The crude product B3 is purified by isocratic elution, wherein the volume ratio of mobile phase A to mobile phase B is 18:82 to 22:78, preferably 20:80; and / or The crude product B1-I is purified by isocratic elution, wherein the volume ratio of mobile phase A to mobile phase B is 20:80 to 25:75, preferably 22:

78.

6. The method according to claim 5, characterized in that The refining of crude products B1, B2, B3 and / or B1-I comprises: The crude products B1, B2, B3 and / or B1-I are each dissolved in purified water to obtain a column liquid with a concentration of 50-150 mg / ml, preferably 100 mg / ml; the column liquids are each passed through a preparative high performance liquid chromatograph, eluted with an eluent, the eluents are collected in batches and the purity of polymyxin sulfate B1, B2, B3 and / or B1-I in each batch of eluents is detected by an analytical high performance liquid chromatograph; the eluents containing polymyxin sulfate B1, B2, B3 and / or B1-I with a purity of more than 95% are each combined and concentrated under reduced pressure to obtain polymyxin sulfate B1, B2, B3 and / or B1-I.

7. The method according to claim 1, characterized in that The polymyxin sulfate B1, B2, B3 and / or B1-I obtained herein do not contain or contain less than 1% by weight of compound M and / or compound J.

8. The method according to claim 1, characterized in that Compound M and / or Compound J are also isolated 9. The method according to claim 2, characterized in that: The method comprises the step of isolating compound M, the step comprising: The eluates collected at 60-80 min, preferably the eluate containing compound M with a purity of 40% or more, are combined, and then distilled under reduced pressure to obtain a crude product M; The crude product M is dissolved in purified water to obtain a column liquid with a concentration of 50-150 mg / ml, preferably 100 mg / ml; the column liquid is passed through a preparative high performance liquid chromatograph, and mobile phases A and B are used for gradient elution, and the gradient setting is as follows: Collect the eluate eluted with the mobile phase A:B volume ratio of 15:85; then concentrate the eluate under reduced pressure to obtain a residue; The residue is eluted repeatedly according to the above steps for multiple times, preferably 2-4 times, until the purity of compound M in the collected eluate reaches more than 95%.

10. The method according to claim 2, characterized in that The method comprises the step of isolating compound J, the step comprising: The eluates collected at 160-210 min, preferably the eluate containing compound J with a purity of 20% or more, are combined, and then distilled under reduced pressure to obtain a crude product J; The crude product J was dissolved in purified water to obtain a column liquid with a concentration of 50-150 mg / ml, preferably 100 mg / ml; the column liquid was passed through a preparative high performance liquid chromatograph, and mobile phases A and B were used for gradient elution, and the gradient setting was as follows: The eluate eluted with the mobile phase A:B volume ratio of 22:78 was collected, and then the eluate was concentrated under reduced pressure to obtain a residue; The residue is eluted repeatedly according to the above steps for multiple times, preferably 5-8 times, until the purity of compound J in the collected eluate reaches 85%.

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