阅读说明：本技术 一种多黏菌素e组分及其光化学产物和液相色谱-质谱分析方法 (Polymyxin E component and photochemical product thereof and liquid chromatography-mass spectrometry analysis method ) 是由 张含智 刘浩 秦峰 闻宏亮 裘亚 赵敬丹 于 2019-08-08 设计创作，主要内容包括：本发明公开了一种多黏菌素E(Polymyxin E,PME)组分及其光化学产物和液相色谱-质谱分析方法；其中,该多黏菌素E组分包括多黏菌素E双键化组分,具体为包括多黏菌素E1双键化组分、多黏菌素E2双键化组分,该类组分经高效液相色谱-质谱(HPLC-MS)及二级质谱分析验证双键位于N-脂肪酰基链末端。所述双键化组分在丙酮做反应试剂的条件下经光化学反应即帕特诺比希反应(<Image he="60" wi="301" file="DDA0002160799820000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>反应)获得光化学产物,该类产物经HPLC-MS验证为[2+2]环加成产物；上述双键化组分被命名为7’-烯基多黏菌素E1和6’-烯基多黏菌素E2。多黏菌素E双键化组分可能具有不同于常见多黏菌素E组分的抗菌活性或毒性,有潜在的应用价值,并且该类化合物的结构确证也为严格控制多黏菌素E的质量提供了研究基础。(The invention discloses a Polymyxin E (Polymyxin E, PME) component and a photochemical product thereof and a liquid chromatography-mass spectrometry analysis method; the polymyxin E component comprises a polymyxin E double-bonded component, specifically comprises a polymyxin E1 double-bonded component and a polymyxin E2 double-bonded component, and the double bonds of the components are verified to be positioned at the tail end of an N-fatty acyl chain through high performance liquid chromatography-mass spectrometry (HPLC-MS) and secondary mass spectrometry. The double-bonded component is subjected to photochemical reaction, namely, a Patinophiki reaction (in) under the condition that acetone is used as a reaction reagent Reaction) to obtain photochemical products, which are verified as [2+2] by HPLC-MS]A cycloaddition product; the double-bonded components are named 7 '-alkenyl polymyxin E1 and 6' -alkenyl polymyxin E2. The double-bonded component of polymyxin E can beThe compound has antibacterial activity or toxicity different from that of common polymyxin E components, has potential application value, and the structural confirmation of the compound also provides a research basis for strictly controlling the quality of polymyxin E.)

1. A polymyxin E component comprising a polymyxin E double-bonded component.

2. The polymyxin E component of claim 1, wherein the polymyxin E double-bonded component comprises polymyxin E1 double-bonded component and polymyxin E2 double-bonded component.

3. The polymyxin E component of claim 2, wherein the polymyxin E1 double-bonded component is 4 '-alkenyl polymyxin E1, 5' -alkenyl polymyxin E1, 6 '-alkenyl polymyxin E1 or 7' -alkenyl polymyxin E1; the double-bonding component of polymyxin E2 is 4' -alkenyl polymyxin E2, 5' -alkenyl polymyxin E2 or 6' -alkenyl polymyxin E2.

4. The polymyxin E component of claim 2, wherein the double bond is at the end of the N-fatty acyl chain, the polymyxin E1 double-bonded component is 7 '-alkenyl polymyxin E1, the polymyxin E2 double-bonded component is 6' -alkenyl polymyxin E2;

wherein the structural formula of the 7' -alkenyl polymyxin E1 is as follows:

wherein the structural formula of the 6' -alkenyl polymyxin E2 is as follows:

5. the polymyxin E component of claim 1, wherein the polymyxin E component is derived from a polymyxin E drug substance, a polymyxin E sulfate formulation, a polymyxin E sulfonate formulation, or a polymyxin E sodium methanesulfonate formulation.

6. The polymyxin E component of claim 1, wherein the building blocks of polymyxin E component comprise N-fatty acyl chain α, gamma-diaminobutyric acid, threonine, leucine or isoleucine, valine or norvaline, serine, methionine.

7. The polymyxin E component of claim 6, wherein the amino acid is in the L-or D-configuration.

8. The polymyxin E component of claim 6, wherein the N-fatty acyl chains comprise 6' -methyloctanoyl, 6' -methylheptanoyl, 7' -methyloctanoyl, octanoyl, heptanoyl, 3' -hydroxy 6' -methyloctanoyl, 3' -hydroxy 6' -methylheptanoyl, hexanoyl.

9. A polymyxin E component of claim 6, wherein the N-fatty acyl chains are each terminally double bonded.

10. The polymyxin E component of claim 1, wherein the polymyxin E comprises polymyxin E1 and polymyxin E2 as main components, and other components comprise polymyxin E3, polymyxin E4, polymyxin E6, polymyxin E1-7' MOA, polymyxin E1-I, polymyxin E2-I, polymyxin E1-Val, polymyxin E2-Val, polymyxin E1-Ser and polymyxin E2-Ser.

11. The polymyxin E component of claim 1, wherein the double-bonded component of the polymyxin E component comprises polymyxin E oxidation products, epimerization products, hydrolysis products, sulfation products, sulfonation products, methanesulfonic acid products.

12. A photochemical product of a polymyxin E component as claimed in any one of claims 1 to 11 wherein the polymyxin E component is reacted with a carbonyl compound photochemically to give a [2+2] cycloaddition product.

13. A photochemical product according to claim 12 wherein the carbonyl compound comprises acetone, butanone, 2-pentanone, 3-pentanone, hexanone, benzophenone, acetophenone, formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, valeraldehyde.

14. The photochemical product defined in claim 12 wherein polymyxin E component is 7 '-alkenylpolymyxin E1 and the cycloaddition product comprises two regioselective products of addition of a carbonyl oxygen to the 7' -carbon or addition of a carbonyl oxygen to the 8 '-carbon, the chirality of the 7' -carbon of the addition product being either R-type or S-type.

15. The photochemical product defined in claim 14 wherein the cycloaddition product of 7' -alkenylpolymyxin E1 is of the formula

16. The photochemical product defined in claim 12 wherein polymyxin E component is 6 '-alkenylpolymyxin E2 and the cycloaddition product comprises two regioselective products of addition of a carbonyl oxygen to the 6' -carbon or addition of a carbonyl oxygen to the 7 '-carbon, the chirality of the 6' -carbon of the addition product being either R-type or S-type.

17. A photochemical product according to claim 12, characterized in that the photochemical reaction is carried out using water, water: acetonitrile or water: methanol.

18. A photochemical product according to claim 17, characterized in that in the photochemical reaction the carbonyl compound is acetone and the volume ratio of solvent to acetone is 1:9-9: 1.

19. A liquid chromatography-mass spectrometry analysis method for analyzing polymyxin components or polypeptide antibiotic components is characterized in that high-resolution primary mass spectrometry, secondary mass spectrometry fragment ions and amino acid residue ion information are adopted to infer component structures.

20. The liquid chromatography-mass spectrometry method of claim 19, wherein the polymyxin component is a polymyxin E1 double-bonded component and a polymyxin E2 double-bonded component; wherein the double-bonded component of polymyxin E1 is 4 '-alkenyl polymyxin E1, 5' -alkenyl polymyxin E1, 6 '-alkenyl polymyxin E14 or 7' -alkenyl polymyxin E1; the double-bonding component of polymyxin E2 is 4' -alkenyl polymyxin E2, 5' -alkenyl polymyxin E2 or 6' -alkenyl polymyxin E2.

21. The method of claim 19, wherein the liquid chromatography is performed using a reverse phase chromatography column, the packing is octadecyl bonded silica gel matrix, octaalkyl bonded silica gel matrix, or tetraalkyl bonded silica gel matrix, the column length is 5-30cm, the diameter is 1-10mm, and the packing particle size is 1-10 μm.

22. The method of liquid chromatography-mass spectrometry of claim 19, wherein the final concentration of polymyxin E is 0.1mg/mL to 20 mg/mL.

23. The liquid chromatography-mass spectrometry method of claim 19, wherein the mobile phase a is an aqueous solution containing trifluoroacetic acid: acetonitrile with the volume ratio of 99:1-80:20 and the trifluoroacetic acid content of 0.01-1% v/v; the mobile phase B is acetic acid, methanol or acetonitrile solution containing formic acid, and the content of the formic acid is 0.01-1% v/v.

24. The method of claim 23, wherein isocratic elution is used, and the elution volume ratio of mobile phase a to mobile phase B is 70:30 to 95: 5.

25. The method of claim 19, wherein the flow rate is 0.5-1.5ml/min, the sample volume is 1-100 μ l, and the column temperature is selected from 20 ℃ to 40 ℃.

26. The method of claim 19, wherein the mass spectrometry ionization is selected from electrospray ionization, atmospheric pressure ionization, or fast atom bombardment ionization.

27. The method of liquid chromatography-mass spectrometry of claim 19, wherein the mass analyzer of the mass spectrometer selects a quadrupole time-of-flight mass spectrometer, a triple quadrupole mass spectrometer, an ion hydrazine mass spectrometer or an orbital ion hydrazine mass spectrometer.

28. The method of claim 19, wherein the mass spectrum and mass spectrum/mass spectrum scan ranges from m/z50-1700, and the secondary mass spectrum collision energy setting ranges from 5-50 eV.

29. The method of liquid chromatography-mass spectrometry of claim 20, wherein the 7' -alkenyl polymyxin E1 has a molecular weight of 1166.75 and the characteristic ions of the double bond component are m/z 239.17, 139.11, 111.11, 69.07 and 55.05.

30. The method of liquid chromatography-mass spectrometry of claim 21, wherein the 6' -alkenyl polymyxin E2 has a molecular weight of 1152.74 and the characteristic ions of the double bond component are m/z 225.16, 125.10, 97.10 and 55.05.

31. The method of liquid chromatography-mass spectrometry of claim 21, wherein the photochemical product of 7' -alkenylpolymyxin E1 has a molecular weight of 1224.80 and the characteristic ions of the epoxidation product are m/z899.56, 799.50, 498.33, 297.22, 171.11, and 157.10.

Technical Field

The invention belongs to the fields of pharmaceutical chemistry and pharmaceutical analytical chemistry, and particularly relates to a polymyxin E (PME) component, a photochemical product thereof and a liquid chromatography-mass spectrometry analysis method.

Background

Polymyxin E (PME) a multicomponent lipopeptide mixture produced by fermentation of Paenibacillus polymyxa, which is clinically mainly applied to treat multiple drug-resistant gram-negative bacterial infection by using PME sulfate (or named Colistin) or polymyxin E sodium methanesulfonate as a 'last line of defense'. Since PME alone requires higher doses, but the renal toxicity increases, the trend of reducing toxicity and improving therapeutic effect is to use combination or aerosol inhalation, and the development of low-toxicity and high-efficiency PME single component is a research focus in recent years (J Med Chem, 2018, 61 (5): 1845-1857).

The general structural formula and information of the main components of PME (E1, E2, E3, E1-I and E1-7' MOA) are as follows:

the main components of PME are composed of N-terminal Fatty acyl chain (FA), linear tripeptide and cyclic heptapeptide, L- α at positions 1, 3, 4, 8 and 9, gamma-diaminobutyric acid (Dab), L-threonine (Thr) at positions 2 and 10, D-leucine (Leu) at position 6, L-Leu or isoleucine (Ile) at position 7. the difference in structure is caused by the difference in Fatty chain substituents or amino acids of the different components, and the analysis methods for polymyxin components are mainly liquid chromatography (European pharmacopoeia 9.0 edition), liquid chromatography-mass spectrometry (J Chromato A, 2002, 976 (2): 65-78) and capillary Electrophoresis (Electrophoresis,2000,21(18): 3199-3204). since PME is a mixture containing a plurality of components, there are certain difficulties in analyzing components and identifying their components because of different fermentation conditions.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention discloses a novel polymyxin E double-bonded component, in particular to the structures of 7 '-alkenyl polymyxin E1 and 6' -alkenyl polymyxin E2 of the polymyxin E double-bonded component, which is confirmed by an HPLC-MS analysis method and a secondary mass spectrum for the first time and is subjected to photochemical reaction, namely, a patrnor-Bischs reaction (

Reaction) to obtain photochemical products, which are verified as [2+2] by HPLC-MS]Cycloaddition product of [2+2] of the first-obtained doubly-bonded component]Cycloaddition products, the structure of which photochemical products were also confirmed by HPLC-MS.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the invention there is provided a polymyxin E component which includes a polymyxin E double-bonded component.

Further, the polymyxin E double-bonded component comprises a polymyxin E1 double-bonded component and a polymyxin E2 double-bonded component.

Further, the double-bonded component of polymyxin E1 is 4 '-alkenyl polymyxin E1, 5' -alkenyl polymyxin E1, 6 '-alkenyl polymyxin E1 or 7' -alkenyl polymyxin E1; the double-bonding component of polymyxin E2 is 4' -alkenyl polymyxin E2, 5' -alkenyl polymyxin E2 or 6' -alkenyl polymyxin E2.

Further, the double bond is located at the end of the N-fatty acyl chain, the double-bonded component of polymyxin E1 is 7 '-alkenyl polymyxin E1, and the double-bonded component of polymyxin E2 is 6' -alkenyl polymyxin E2;

wherein the structural formula of the 7' -alkenyl polymyxin E1 is as follows:

wherein the structural formula of the 6' -alkenyl polymyxin E2 is as follows:

wherein, the position of the double bond is verified to be positioned at the tail end of the N-fatty acyl chain by HPLC-MS and secondary mass spectrum analysis.

Further, the polymyxin E component is derived from a polymyxin E bulk drug, a polymyxin E sulfate preparation, a polymyxin E sulfonate preparation or a polymyxin E mesylate preparation, and can also be derived from other preparations.

Further, the constituent units of the polymyxin E component include N-fatty acyl chains, α, gamma-diaminobutyric acid, threonine, leucine or isoleucine, valine or norvaline, serine, methionine, and may also include other amino acids.

Further, the amino acid is in L-configuration or D-configuration.

Further, the N-fatty acyl chain includes 6 '-methyloctanoyl (N-fatty acyl of PME 1), 6' -methylheptanoyl (N-fatty acyl of PME 2), 7 '-methyloctanoyl (N-fatty acyl of E1-7' MOA), octanoyl (N-fatty acyl of PME 3), heptanoyl (N-fatty acyl of PME 4), 3 '-hydroxy 6' -methyloctanoyl (N-fatty acyl of PME 6), 3 '-hydroxy 6' -methylheptanoyl, hexanoyl, and may also include other N-fatty acyl groups.

Further, the ends of the N-fatty acyl chains may all be double-bonded.

Further, the main components of polymyxin E are polymyxin E1 and polymyxin E2, and other components comprise polymyxin E3, polymyxin E4, polymyxin E6, polymyxin E1-7' MOA, polymyxin E1-I, polymyxin E2-I, polymyxin E1-Val, polymyxin E2-Val, polymyxin E1-Ser and polymyxin E2-Ser, and other components can be included.

Further, the double-bonded component in the polymyxin E component includes polymyxin E oxidation products, epimerization products, hydrolysis products, sulfation products, sulfonation products, methanesulfonic acid products, and other products.

In a second aspect the present invention provides a photochemical product of any one of the polymyxin E components described above, which polymyxin E component is reacted photochemically with a carbonyl compound to give a [2+2] cycloaddition product.

Further, the carbonyl compound includes acetone, butanone, 2-pentanone, 3-pentanone, hexanone, benzophenone, acetophenone, formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, valeraldehyde, and other forms of carbonyl compounds, preferably acetone.

Further, the polymyxin E component is 7 '-alkenyl polymyxin E1, the cycloaddition product comprises two regioselective products of addition of carbonyl oxygen to 7' -carbon or addition of carbonyl oxygen to 8 '-carbon, and the chirality of the 7' -carbon of the addition product is R-type or S-type.

Further, the structural formula of the cycloaddition product of the 7' -alkenyl polymyxin E1 is shown in the specification

Further, the polymyxin E component is 6 '-alkenyl polymyxin E2, the cycloaddition product comprises two regioselective products of addition of carbonyl oxygen to 6' -carbon or addition of carbonyl oxygen to 7 '-carbon, and the chirality of 6' -carbon of the addition product is R type or S type.

Further, the photochemical reaction adopts water and water: acetonitrile or water: methanol; preferably water: 3-5: 1, more preferably water: acetonitrile 4: 1.

Further, in the photochemical reaction, the carbonyl compound is acetone, and the volume ratio of the solvent to the acetone is 1:9-9:1, preferably 1:4-4:1, and more preferably 1: 1.

The third aspect of the present invention is to provide a liquid chromatography-mass spectrometry analysis method for analyzing polymyxin components or polypeptide antibiotic components, which uses high resolution primary mass spectrometry, secondary mass spectrometry fragment ions and amino acid residue ion information to infer component structure.

Further, the polymyxin component is a polymyxin E1 double-bonded component and a polymyxin E2 double-bonded component; wherein the double-bonded component of polymyxin E1 is 4 '-alkenyl polymyxin E1, 5' -alkenyl polymyxin E1, 6 '-alkenyl polymyxin E14 or 7' -alkenyl polymyxin E1; the double-bonding component of polymyxin E2 is 4' -alkenyl polymyxin E2, 5' -alkenyl polymyxin E2 or 6' -alkenyl polymyxin E2.

Furthermore, the liquid chromatogram adopts a reversed phase chromatographic column, the filler is octadecyl bonded silica gel matrix, octaalkyl bonded silica gel matrix or tetraalkyl bonded silica gel matrix, the length of the chromatographic column is 5-30cm, the diameter is 1-10mm, and the particle size of the filler is 1-10 μm; preferably a C18 column, 25cm, 4.6mm, 5 μm.

Further, the final concentration of the polymyxin E is 0.1mg/mL-20mg/mL, preferably 0.5-4 mg/mL, and more preferably 2 mg/mL.

Further, in the liquid chromatography method, the mobile phase A is an aqueous solution containing trifluoroacetic acid: acetonitrile with the volume ratio of 99:1-80:20 and the trifluoroacetic acid content of 0.01-1% (v: v); the mobile phase B is acetic acid, methanol or acetonitrile solution containing formic acid, and the content of the formic acid is 0.01-1% (v: v). Preferably, mobile phase A is an aqueous solution containing 0.1% (v: v) trifluoroacetic acid: acetonitrile 95:5, mobile phase B is acetonitrile solution containing 0.1% (v: v) formic acid.

Further, in the liquid chromatography method, isocratic elution is adopted, and the elution volume ratio of the mobile phase A to the mobile phase B is 70:30-95: 5; preferably 80: 20.

Further, in the liquid chromatography method, the flow rate is 0.5-1.5 ml/min; preferably 1 ml/min.

Further, in the liquid chromatography method, the injection volume is 1-100 μ l; preferably 4. mu.l.

Further, in the liquid chromatography method, the column temperature of the chromatographic column is selected to be 20-40 ℃; preferably 30 deg.c.

Furthermore, the mass spectrum ionization mode is selected from electrospray ionization, atmospheric pressure ionization or fast atom bombardment ionization, and is also other ionization modes; preferably electrospray ionization, positive ion collection mode.

Furthermore, the mass analyzer of the mass spectrum can select a quadrupole time-of-flight mass spectrum, a triple quadrupole mass spectrum, an ion hydrazine mass spectrum or a rail ion hydrazine mass spectrum, and can also be other mass analyzers; preferably a quadrupole time-of-flight mass spectrometer.

Further, the mass spectrum and mass spectrum/mass spectrum scan ranges from m/z 50-1700.

Further, the setting range of the secondary mass spectrum collision energy is 5-50 eV; preferably 20 eV.

Further, the molecular weight of the 7' -alkenyl polymyxin E1 is 1166.75, and the characteristic ions of the double bond component are m/z 239.17, 139.11, 111.11, 69.07 and 55.05.

Further, the molecular weight of the 6' -alkenyl polymyxin E2 is 1152.74, and the characteristic ions of the double bond component are m/z 225.16, 125.10, 97.10 and 55.05.

Further, the photochemical product of 7' -alkenyl polymyxin E1 has a molecular weight of 1224.80 and the characteristic ions of the epoxidation product are m/z899.56, 799.50, 498.33, 297.22, 171.11 and 157.10.

The fourth aspect of the invention provides a method for identifying impurities containing double bond structures in a medicament and application thereof, wherein the method adopts the photochemical reaction of the medicament and any one carbonyl compound and carries out analysis by a liquid chromatography-mass spectrometry analysis method.

Compared with the prior art, the invention has the following beneficial effects by adopting the technical scheme:

(1) the invention discovers that polymyxin E contains a component 6' -alkenyl PME2 for the first time, and discloses structures of 7' -alkenyl PME1 and 6' -alkenyl PME2 for the first time;

(2) the invention obtains the epoxidation product of 7' -alkenyl PME1 through photochemical reaction, namely, a petunidin-Bixi reaction for the first time;

(3) the HPLC-MS method can directly analyze the components in the polymyxin E, summarizes the general strategy of component structure analysis, namely comprehensively deducing the component structure by adopting high-resolution primary mass spectrum, secondary mass spectrum fragment ions and amino acid residue ion information, is simple and rapid, and can be popularized to the structure analysis of other polypeptide antibiotics;

(4) the HPLC-MS method comprises a new cracking way which can help to assist in judging the component structure of polymyxin E;

(5) the structure analysis strategy of the double-bonded component and the photochemical reaction product thereof is suitable for other polymyxin double-bonded components or polypeptide antibiotic double-bonded components, and is easy to popularize.

The invention establishes a PME HPLC-MS component analysis method, establishes a general strategy for PME component structure analysis, namely, comprehensively adopts high-resolution primary mass spectrum, secondary mass spectrum fragment ion and amino acid residue ion information to infer the component structure, and discovers a new cracking way. In addition, a photochemical reaction product of a double-bonded component in PME was obtained, and the structure of the double-bonded component was deduced to be 7 '-alkenylpolymyxin E1 in combination with a structure analysis strategy, and 6' -alkenylpolymyxin E2 was found for the first time. The structure analysis strategy can also be applied to structure inference of other lipopeptide compounds, and the photochemical reaction also provides a novel solution for identifying impurities containing double bond structures in the medicine, and has certain guiding significance for improving the product process and improving the quality control level.

Drawings

FIG. 1 is a typical chromatogram obtained by the analytical method in European pharmacopoeia 9.0 for polymyxin E control (European pharmacopoeia control) in one example of the present invention.

FIG. 2 is a mass spectrometric total ion flow diagram of a polymyxin E control analyzed in accordance with an embodiment of the present invention, showing 7' -alkenylpolymyxin E1 for peak 14 and 6' -alkenylpolymyxin E2 for peak 8 '.

FIG. 3 shows a primary mass spectrum and a secondary mass spectrum of polymyxin E1 according to one embodiment of the present invention.

FIG. 4 shows the secondary mass spectrum fragmentation pattern of polymyxin E1 in accordance with one embodiment of the present invention.

FIG. 5 is a secondary mass spectrum of 7' -alkenylpolymyxin E1 according to one embodiment of the present invention.

FIG. 6 is a secondary mass spectrum of 6' -alkenylpolymyxin E2 according to an embodiment of the present invention.

FIG. 7 is a second mass spectrum of the photochemical product of 7' -alkenylpolymyxin E1 in accordance with one embodiment of the present invention.

Detailed Description

The invention relates to a novel polymyxin E component, a photochemical product thereof and an HPLC-MS analysis method. The components comprise a polymyxin E1 double-bonded component and a polymyxin E2 double-bonded component, and the double bonds of the components are verified to be positioned at the tail end of an N-fatty acyl chain by HPLC-MS and secondary mass spectrometry. The double-bonded component is subjected to photochemical reaction, namely, a Partnobishi reaction under the condition that acetone is used as a reaction reagent to obtain a photochemical product, the photochemical product is verified to be a [2+2] cycloaddition product by HPLC-MS, and the double-bonded component is named as 7 '-alkenyl polymyxin E1 and 6' -alkenyl polymyxin E2.

The following description of the embodiments of the present invention will be made with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.

Polymyxin E in the following examples is exemplified by polymyxin E sulfate "European pharmacopoeia" control, but may be used in other polymyxin E controls and preparations.