阅读说明：本技术 一种多黏菌素b的氨基酸构型分析方法和n-多肽端序列测序方法 (Polymyxin B amino acid configuration analysis method and N-polypeptide terminal sequence sequencing method ) 是由 张含智 刘浩 罗文燕 顿俊玲 于 2020-04-26 设计创作，主要内容包括：本发明公开了一种多黏菌素B的氨基酸构型分析方法和N-多肽端序列测序方法,建立了多黏菌素B组分的液相纯化方法,可以快速从多黏菌素B组分中纯化分离出多黏菌素B1、多黏菌素B2和多黏菌素B1-I；建立了多黏菌素B酶解组分的制备方法和液相纯化方法,可快速纯化分离出多黏菌素B1、多黏菌素B2和多黏菌素B1-I的酶解组分；采用多黏菌素B组分水解-氨基酸手性衍生-液质联用测定氨基酸构型；采用多黏菌素B组分酶解-液相色谱纯化-蛋白质测序联用测定N-多肽端序列,为严格控制多黏菌素B的质量提供了研究基础。(The invention discloses an amino acid configuration analysis method and an N-polypeptide terminal sequence sequencing method of polymyxin B, and establishes a liquid phase purification method of a polymyxin B component, so that polymyxin B1, polymyxin B2 and polymyxin B1-I can be quickly purified and separated from the polymyxin B component; a preparation method and a liquid phase purification method of the polymyxin B enzymatic component are established, and the polymyxin B1, polymyxin B2 and polymyxin B1-I enzymatic components can be quickly purified and separated; the amino acid configuration is determined by adopting polymyxin B component hydrolysis-amino acid chiral derivation-liquid chromatography-mass spectrometry; the N-polypeptide terminal sequence is determined by adopting the combination of polymyxin B component enzymolysis, liquid chromatography purification and protein sequencing, and a research basis is provided for strictly controlling the quality of polymyxin B.)

1. A method for analyzing the amino acid configuration of polymyxin B, which is characterized by comprising the following steps:

s1: separating and purifying polymyxin B1, polymyxin B2 and polymyxin B1-I from the mixed components of polymyxin B by liquid chromatography;

s2: respectively heating and hydrolyzing the purified polymyxin B1, polymyxin B2 and polymyxin B1-I in a heavy water solution of deuterated hydrochloric acid to obtain a test solution, and then respectively adopting chiral Marfey's reagents to perform derivatization reaction on the reference solution and the hydrolyzed test solution;

s3: and (4) analyzing the reference substance solution and the test solution subjected to derivatization in the step S2 by high performance liquid chromatography-mass spectrometry.

2. The method for analyzing the amino acid configuration of polymyxin B according to claim 1, comprising the steps of:

s1: separating and purifying polymyxin B1, polymyxin B2 and polymyxin B1-I from the polymyxin B mixed component;

weighing mixed components of polymyxin B, adding a solvent to prepare a solution with the concentration of 2-40mg/m L, and purifying by liquid chromatography;

octadecyl bonded silica gel chromatographic column under liquid phase chromatographic condition, the sample injection amount is 100-;

s2: respectively heating and hydrolyzing the purified polymyxin B1, polymyxin B2 and polymyxin B1-I in a heavy water solution of deuterated hydrochloric acid to obtain a test solution, and then respectively adopting chiral Marfey's reagents to perform derivatization reaction on the reference solution and the hydrolyzed test solution;

s3: performing high performance liquid chromatography-mass spectrometry on the reference solution and the test solution subjected to derivatization in the step S2;

the liquid chromatography condition is that a conventional reversed phase chromatographic column is adopted, the column temperature is 25-40 ℃, the detection wavelength is 340nm, the mobile phase A is water and contains 0.01-0.2 percent of formic acid (v/v), the mobile phase B is acetonitrile or methanol, the flow rate is 0.8-1.8m L/min, the split flow is carried out before the mass spectrum detection, the split flow ratio is 2:1, and gradient elution is carried out;

mass spectrum condition mass spectrum ion source is ESI or APCI, scanning mode is both positive and negative, and mass detector is space/linear ion hydrazine, flight time or triple quadrupole mass spectrum.

3. The method for analyzing the amino acid configuration of polymyxin B according to claim 2, wherein in S1, the polymyxin B mixed component is a polymyxin B bulk drug, an injection or an ointment; the solvent is water and acetonitrile, and the volume ratio of the water to the acetonitrile is 50:50-90: 10.

4. The method for analyzing the amino acid configuration of polymyxin B according to claim 2, wherein polymyxin B1, polymyxin B2 and polymyxin B1-I purified by S1 are tested for their precise molecular weight and purity by HP L C-Q/TOF-MS;

the chromatographic conditions are that the mobile phase A is water and contains 0.01-0.2% formic acid (v/v), the mobile phase B is acetonitrile and contains 0.01-0.2% formic acid (v/v), the mobile phase A and the mobile phase B are 80:20(v/v), the column temperature is 25-60 ℃, the flow rate is 0.8-1.8m L/min, and the sample injection amount is 2-10 mu L.

The mass spectrum conditions are as follows: positive ion scan mode of electrospray-single quadrupole mass spectrometry, mass to charge ratio 300-.

5. The method for analyzing the amino acid configuration of polymyxin B according to claim 2, wherein the polymyxin B1, polymyxin B2 and polymyxin B1-I are fed at concentrations of 1-10mg/m L, wherein the solvent is water and acetonitrile, and the volume ratio of the water to the acetonitrile is 50:50-90: 10.

6. The method for analyzing the amino acid configuration of polymyxin B as claimed in claim 2, wherein in S2, the thermal hydrolysis is specifically that polymyxin B1, polymyxin B2 and polymyxin B1-I are respectively dissolved in deuterium-substituted hydrochloric acid heavy water solution with the molar concentration of 4-8 mol/L, so that the concentrations of polymyxin B1, polymyxin B2 and polymyxin B1-I are all 0.5-2mg/m L, nitrogen gas is filled, sealing is carried out, pyrolysis is carried out at the temperature of 100-140 ℃ for 5-24h, cooling is carried out to the room temperature, 4-8 mol/L sodium hydroxide solution is respectively added, and neutralization is carried out to neutrality, thus obtaining the sample solution.

7. The method for analyzing amino acid configuration of polymyxin B according to claim 2, wherein in S2, the Marfey ' S reagent is L/D-FDAA, FDVA, FD L A or FDPA, the solvent used for dissolving the Marfey ' S reagent is dimethylsulfoxide, acetone or ethanol, and the concentration of the Marfey ' S reagent solution is 0.5-4mg/m L.

8. The method for analyzing the amino acid configuration of polymyxin B as claimed in claim 2, wherein in S2, the derivatization reaction is specifically that polymyxin B1, polymyxin B2 and polymyxin B1-I test solution 20-80 μ L and control solution 10-60 μ L of 0.1-1mg/m L are respectively measured, put into a sample bottle, Marfey' S reagent solution 30-80 μ L and alkaline solution 10-50 μ L are added, mixed uniformly, put into a water bath for reaction for 30-90min, taken out, cooled to room temperature, and added with acetic acid solution 10-50 μ L of 0.02-0.08 mol/L to make the solution neutral.

9. The method for analyzing the amino acid configuration of polymyxin B according to claim 2, wherein the control is D/L-leucine, D/L-isoleucine, D/L-phenylalanine, D/L-threonine and D/L-2, 4-diaminobutyric acid, the alkaline solution is a triethylamine solution or sodium bicarbonate solution at 0.02-0.08 mol/L, and the temperature of the water bath is 40-60 ℃.

10. The method for analyzing amino acid configuration of polymyxin B according to claim 2, wherein the gradient elution is performed under the following elution conditions in S3:

at 0min, the mobile phase A is 73%, and the mobile phase B is 27%;

at 5min, the mobile phase A is 60% and the mobile phase B is 40%;

at 15min, the mobile phase A is 60% and the mobile phase B is 40%;

at 20min, the mobile phase A is 50%, and the mobile phase B is 50%;

at 34min, the mobile phase A is 50%, and the mobile phase B is 50%;

at 36min, the mobile phase A is 20% and the mobile phase B is 80%;

at 44min, the mobile phase A is 20% and the mobile phase B is 80%;

at 45min, the mobile phase A is 73%, and the mobile phase B is 27%;

at 50min, mobile phase a was 73% and mobile phase B was 27%.

11. A method for sequencing an N-polypeptide terminal sequence of polymyxin B, which is characterized by comprising the following steps:

s1: separating and purifying polymyxin B1, polymyxin B2 and polymyxin B1-I from the mixed components of polymyxin B by liquid chromatography;

s2: respectively carrying out enzymolysis on the purified polymyxin B1, polymyxin B2 and polymyxin B1-I to obtain enzymolysis product solutions, and purifying the enzymolysis product solutions by adopting a liquid chromatography to obtain enzymolysis products of polymyxin B1, polymyxin B2 and polymyxin B1-I;

s3: and dissolving the enzymolysis product obtained in the step S2, adding the solution to a polybrene-treated glass fiber membrane, drying, and then installing the membrane on a protein sequencer PPSQ-53A for analysis, wherein the cycle number is set to be 9.

12. The method for sequencing the N-polypeptide terminal sequence of polymyxin B according to claim 11, comprising the steps of:

s1: separating and purifying polymyxin B1, polymyxin B2 and polymyxin B1-I from the polymyxin B mixed component;

weighing mixed components of polymyxin B, adding a solvent to prepare a solution with the concentration of 2-40mg/m L, and purifying by liquid chromatography;

octadecyl bonded silica gel chromatographic column under liquid phase chromatographic condition, the sample injection amount is 100-;

s2: respectively carrying out enzymolysis on the purified polymyxin B1, polymyxin B2 and polymyxin B1-I to obtain enzymolysis product solutions;

the enzymolysis product solution adopts an octadecyl bonded silica gel chromatographic column, wherein a mobile phase A is water and contains 0.01-1% formic acid (v/v), a mobile phase B is acetonitrile or methanol, gradient elution is carried out, the flow rate is 5-20m L/min, the sample injection amount is 100-;

s3: and dissolving the enzymolysis product obtained in the step S2, adding the solution to a polybrene-treated glass fiber membrane, drying, and then installing the membrane on a protein sequencer PPSQ-53A for analysis, wherein the cycle number is set to be 9.

13. The method for sequencing the N-polypeptide terminal sequence of polymyxin B according to claim 12, wherein the enzymatic hydrolysate obtained in step S2 is further subjected to moisture removal by a rotary evaporator and freeze-drying to obtain white powder.

14. The method for sequencing the N-polypeptide terminal sequence of polymyxin B according to claim 12, wherein the gradient elution at S2 is performed under the following conditions:

at 0min, the mobile phase A is 95% and the mobile phase B is 5%;

at 5min, the mobile phase A is 95% and the mobile phase B is 5%;

at 6min, the mobile phase A is 70% and the mobile phase B is 30%;

at 10min, the mobile phase A is 60% and the mobile phase B is 40%;

at 11min, the mobile phase A is 95% and the mobile phase B is 5%;

at 15min, mobile phase a was 95% and mobile phase B was 5%.

15. The N-polypeptide end sequence sequencing method of polymyxin B as claimed in claim 12, wherein in S2, the enzymatic hydrolysis is carried out by weighing 10-100mg of purified polymyxin B1, polymyxin B2 and polymyxin B1-I, dissolving in 5-20m L of 0.1 mol/L potassium dihydrogen phosphate buffer, weighing 10-20mg of protease, dissolving in 2.5ml of sodium chloride solution, corresponding to 1U/m L of protease, incubating in 37 ℃ water bath for 10-60min, adding polymyxin B1, polymyxin B2 and polymyxin B1-I solution into preheated protease solution, carrying out 30-50 ℃ water bath for 10-24h, boiling for 10min, centrifuging for 10min, filtering, and reserving.

16. The method for sequencing the N-polypeptide terminal sequence of polymyxin B according to claim 12, wherein the protease is subtilisin, ficin, papain or pronase, and the concentration of the sodium chloride solution is 3 mol/L.

17. The method for sequencing the N-polypeptide end sequence of polymyxin B as claimed in claim 12, wherein the reverse phase chromatography is used for separation in S3Wakosil PTH-II column of 4.6 × 250mm, mobile phase of 30-50% acetonitrile water solution, isocratic elution, flow rate of 0.5-2.0m L/min, column temperature of 25-45 deg.C, and detection wavelength of 269 nm.

Technical Field

The invention relates to the field of pharmaceutical analytical chemistry, in particular to a polymyxin B amino acid configuration analysis method and an N-polypeptide terminal sequence sequencing method.

Background

Polymyxin B is lipopeptide antibacterial peptide, has a strong bactericidal effect on gram-negative bacilli, is the 'last line of defense' for clinical treatment of multi-drug resistant gram-negative bacilli infection, mainly comprises polymyxin B1, B2 and B1-I, the structural formula is shown in figure 1, the first two amino acids comprise L-2, 4-diaminobutyric acid, L-threonine, L-leucine and D-phenylalanine, L-leucine in B1-I is replaced by L-isoleucine, polymyxin is a fermentation product, configuration transformation of amino acids can occur in the production process, the change of amino acids and the difference of D-/L-configuration can cause large changes of antibacterial activity and nephrotoxicity, the amino acids and the configuration thereof must be monitored to ensure the safety of medicines, and in addition, whether the polypeptide sequence, namely the connection sequence of the amino acids, is correct or not is necessarily confirmed.

At present, the analysis method of amino acid configuration comprises chiral derivation-chromatography/mass spectrometry after hydrolysis, and chiral chromatography is directly adopted for analysis, but the method measures amino acid mixtures, and the amino acid and the configuration thereof at each position cannot be determined.

Therefore, establishing a simple and easy-to-operate analysis method for the amino acid configuration analysis and N-polypeptide terminal sequence sequencing of polymyxin B provides a research basis for strictly controlling the quality of polymyxin B, and is a problem to be solved by the technical personnel of the invention urgently.

Disclosure of Invention

The invention provides a polymyxin B amino acid configuration analysis method and an N-polypeptide terminal sequence sequencing method for overcoming the defects in the prior art, is simple and easy to operate, is used for the polymyxin B amino acid configuration analysis and the N-polypeptide terminal sequence sequencing, and provides a research basis for strictly controlling the quality of the polymyxin B.

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

the first aspect of the invention provides a polymyxin B amino acid configuration analysis method, which comprises the following steps:

s1: separating and purifying polymyxin B1, polymyxin B2 and polymyxin B1-I from the mixed components of polymyxin B by liquid chromatography;

s2: respectively heating and hydrolyzing the purified polymyxin B1, polymyxin B2 and polymyxin B1-I in a heavy water solution of deuterated hydrochloric acid to obtain a test solution, and then respectively adopting chiral Marfey's reagents to perform derivatization reaction on the reference solution and the hydrolyzed test solution;

s3: and (4) analyzing the reference substance solution and the test solution subjected to derivatization in the step S2 by high performance liquid chromatography-mass spectrometry.

Further, the method for analyzing the amino acid configuration of polymyxin B comprises the following steps:

s1: separating and purifying polymyxin B1, polymyxin B2 and polymyxin B1-I from the polymyxin B mixed component;

weighing mixed components of polymyxin B, adding a solvent to prepare a solution with the concentration of 2-40mg/m L, and purifying by liquid chromatography;

octadecyl bonded silica gel chromatographic column under liquid phase chromatographic condition, the sample injection amount is 100-;

s2: respectively heating and hydrolyzing the purified polymyxin B1, polymyxin B2 and polymyxin B1-I in a heavy water solution of deuterated hydrochloric acid to obtain a test solution, and then respectively adopting chiral Marfey's reagents to perform derivatization reaction on the reference solution and the hydrolyzed test solution;

s3: performing high performance liquid chromatography-mass spectrometry on the reference solution and the test solution subjected to derivatization in the step S2;

the liquid chromatography condition is that a conventional reversed phase chromatographic column is adopted, the column temperature is 25-40 ℃, the detection wavelength is 340nm, the mobile phase A is water and contains 0.01-0.2 percent of formic acid (v/v), the mobile phase B is acetonitrile or methanol, the flow rate is 0.8-1.8m L/min, the split flow is carried out before the mass spectrum detection, the split flow ratio is 2:1, and gradient elution is carried out;

mass spectrum condition mass spectrum ion source is ESI or APCI, scanning mode is both positive and negative, and mass detector is space/linear ion hydrazine, flight time or triple quadrupole mass spectrum.

Further, in S1, the polymyxin B mixed component is a polymyxin B bulk drug, an injection or an ointment; the solvent is water and acetonitrile, and the volume ratio of the water to the acetonitrile is 50:50-90: 10.

Further, the polymyxin B1, polymyxin B2 and polymyxin B1-I purified by S1 can be tested for their precise molecular weight and purity by HP L C-Q/TOF-MS;

the chromatographic conditions are that the mobile phase A is water and contains 0.01-0.2% formic acid (v/v), the mobile phase B is acetonitrile and contains 0.01-0.2% formic acid (v/v), the mobile phase A is 80:20(v/v), the column temperature is 25-60 ℃, the flow rate is 0.8-1.8m L/min, and the sample injection amount is 2-10 mu L.

The mass spectrum conditions are as follows: positive ion scan mode of electrospray-single quadrupole mass spectrometry, mass to charge ratio 300-.

Furthermore, the injection concentrations of polymyxin B1, polymyxin B2 and polymyxin B1-I are all 1-10mg/m L, wherein the solvent is water and acetonitrile, and the volume ratio of the water to the acetonitrile is 50:50-90: 10.

Further, in S2, the heating hydrolysis is specifically that polymyxin B1, polymyxin B2 and polymyxin B1-I are respectively dissolved in deuterated hydrochloric acid heavy water solution with the molar concentration of 4-8 mol/L, so that the concentrations of polymyxin B1, polymyxin B2 and polymyxin B1-I are all 0.5-2mg/m L, nitrogen gas is filled in the solution, the solution is sealed and pyrolyzed for 5-24h at the temperature of 100-140 ℃, the solution is cooled to room temperature, 4-8 mol/L sodium hydroxide solution is respectively added into the solution and neutralized to obtain the test solution.

Further, in S2, the Marfey ' S reagent is L/D-FDAA, FDVA, FD L A or FDPA, the solvent used for dissolving the Marfey ' S reagent is dimethyl sulfoxide, acetone or ethanol, and the concentration of the Marfey ' S reagent solution is 0.5-4mg/m L.

Further, in S2, the derivatization reaction specifically comprises the steps of respectively measuring 20-80 mu L of polymyxin B1, polymyxin B2, polymyxin B1-I test solution and 10-60 mu L of 0.1-1mg/m L reference solution, placing the test solutions into a liquid phase sample feeding bottle, adding 30-80 mu L of Marfey' S reagent solution and 10-50 mu L of alkaline solution, mixing uniformly, placing the test solutions into a water bath for reaction for 30-90min, taking out the test solutions, cooling the test solutions to room temperature, and adding 10-50 mu L of 0.02-0.08 mol/L of acetic acid solution to enable the test solutions to be neutral.

Furthermore, the reference substances are D/L-leucine, D/L-isoleucine, D/L-phenylalanine, D/L-threonine and D/L-2, 4-diaminobutyric acid, the alkaline solution is a triethylamine solution or a sodium bicarbonate solution of 0.02-0.08 mol/L, and the temperature of the water bath is 40-60 ℃.

Further, in S3, the elution conditions of the gradient elution are as follows:

at 0min, the mobile phase A is 73%, and the mobile phase B is 27%;

at 5min, the mobile phase A is 60% and the mobile phase B is 40%;

at 15min, the mobile phase A is 60% and the mobile phase B is 40%;

at 20min, the mobile phase A is 50%, and the mobile phase B is 50%;

at 34min, the mobile phase A is 50%, and the mobile phase B is 50%;

at 36min, the mobile phase A is 20% and the mobile phase B is 80%;

at 44min, the mobile phase A is 20% and the mobile phase B is 80%;

at 45min, the mobile phase A is 73%, and the mobile phase B is 27%;

at 50min, mobile phase a was 73% and mobile phase B was 27%.

The second aspect of the present invention provides a method for sequencing the N-polypeptide terminal sequence of polymyxin B, which comprises the following steps:

s1: separating and purifying polymyxin B1, polymyxin B2 and polymyxin B1-I from the mixed components of polymyxin B by liquid chromatography;

s2: respectively carrying out enzymolysis on the purified polymyxin B1, polymyxin B2 and polymyxin B1-I to obtain enzymolysis product solutions, and purifying the enzymolysis product solutions by adopting a liquid chromatography to obtain enzymolysis products of polymyxin B1, polymyxin B2 and polymyxin B1-I;

s3: and dissolving the enzymolysis product obtained in the step S2, adding the solution to a polybrene-treated glass fiber membrane, drying, and then installing the membrane on a protein sequencer PPSQ-53A for analysis, wherein the cycle number is set to be 9.

Further, the N-polypeptide terminal sequence sequencing method of polymyxin B comprises the following steps:

s1: separating and purifying polymyxin B1, polymyxin B2 and polymyxin B1-I from the polymyxin B mixed component;

weighing mixed components of polymyxin B, adding a solvent to prepare a solution with the concentration of 2-40mg/m L, and purifying by liquid chromatography;

octadecyl bonded silica gel chromatographic column under liquid phase chromatographic condition, the sample injection amount is 100-;

s2: respectively carrying out enzymolysis on the purified polymyxin B1, polymyxin B2 and polymyxin B1-I to obtain enzymolysis product solutions;

the enzymolysis product solution adopts an octadecyl bonded silica gel chromatographic column, wherein a mobile phase A is water and contains 0.01-1% formic acid (v/v), a mobile phase B is acetonitrile or methanol, gradient elution is carried out, the flow rate is 5-20m L/min, the sample injection amount is 100-;

s3: and dissolving the enzymolysis product obtained in the step S2, adding the solution to a polybrene-treated glass fiber membrane, drying, and then installing the membrane on a protein sequencer PPSQ-53A for analysis, wherein the cycle number is set to be 9.

Further, the enzymolysis product obtained in S2 is subjected to water removal by a rotary evaporator and freeze-drying to obtain white powder.

Further, in S2, the elution conditions of the gradient elution are as follows:

at 0min, the mobile phase A is 95% and the mobile phase B is 5%;

at 5min, the mobile phase A is 95% and the mobile phase B is 5%;

at 6min, the mobile phase A is 70% and the mobile phase B is 30%;

at 10min, the mobile phase A is 60% and the mobile phase B is 40%;

at 11min, the mobile phase A is 95% and the mobile phase B is 5%;

at 15min, the mobile phase A is 95% and the mobile phase B is 5%;

further, in S2, the enzymolysis specifically comprises weighing 10-100mg of purified polymyxin B1, polymyxin B2 and polymyxin B1-I, dissolving in 5-20m L of 0.1 mol/L of potassium dihydrogen phosphate buffer, weighing 10-20mg of protease, dissolving in 2.5ml of sodium chloride solution, corresponding to 1U/m L of protease, incubating in 37 ℃ water bath for 10-60min, adding polymyxin B1, polymyxin B2 and polymyxin B1-I solution into preheated protease solution, respectively, water-bathing for 10-24h at 30-50 ℃ for 10-24 min, boiling for 10min, centrifuging for 10min, and filtering for later use.

Further, the protease is subtilisin, ficin, papain or pronase, and the concentration of the sodium chloride solution is 3 mol/L.

Further, in S3, the reverse phase chromatography is adopted for separation, and the separation column isWakosil PTH-IIcolumn, 4.6 × 250mm, mobile phase 30-50% acetonitrile water solution, isocratic elution, flow rate 0.5-2.0m L/min, column temperature 25-45 deg.C, and detection wavelength 269 nm.

By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:

the invention establishes a liquid phase purification method of the polymyxin B component, and can quickly purify and separate polymyxin B1, polymyxin B2 and polymyxin B1-I from the polymyxin B component; a preparation method and a liquid phase purification method of the polymyxin B enzymatic hydrolysis component are established, and the polymyxin B1, polymyxin B2 and polymyxin B1-I enzymatic hydrolysis components can be rapidly purified; the amino acid configuration is determined by adopting polymyxin B component hydrolysis-amino acid chiral derivation-liquid chromatography-mass spectrometry; the N-polypeptide terminal sequence is determined by adopting the combination of polymyxin B component enzymolysis, liquid chromatography purification and protein sequencing, and a research basis is provided for strictly controlling the quality of polymyxin B.

The amino acid configuration analysis method and the N-polypeptide terminal sequence sequencing method can also be used for analyzing other polymyxin components, and can comprehensively and finely control the quality of the polypeptide medicine.

Drawings

FIG. 1 shows the structural formulas of polymyxin B1, polymyxin B2, and polymyxin B1-I;

FIG. 2 is a mass spectrometric total ion flow diagram of purified polymyxin B1;

FIG. 3 is a mass spectrum of purified polymyxin B1;

FIG. 4 is a mass spectrometric total ion flow diagram of purified polymyxin B2;

FIG. 5 is a mass spectrum of purified polymyxin B2;

FIG. 6 is a mass spectrometric total ion flow diagram of purified polymyxin B1-I;

FIG. 7 is a mass spectrum of purified polymyxin B1-I;

FIG. 8 is a typical chromatogram (8-A) and total ion flow diagram (8-B) of a 10 amino acid control;

FIG. 9 shows a total ion flow diagram (9-A), a typical chromatogram (9-B) and an extracted ion flow chromatogram (9-C, D, E, F) of each amino acid-FDAA derivative in polymyxin B1;

FIG. 10 is a total ion flow diagram (10-A), a typical chromatogram (10-B) and an extracted ion flow chromatogram (10-C, D, E, F) of each amino acid-FDAA derivative in polymyxin B2;

FIG. 11 is a total ion flow diagram (11-A), a typical chromatogram (11-B) and an extracted ion flow chromatogram (11-C, D, E, F) of each amino acid-FDAA derivative in polymyxin B1-I;

FIG. 12 shows the structural formulas of the enzymatic hydrolysates of polymyxin B1 and polymyxin B2;

FIG. 13 shows the structural formula of the enzymolysis product of polymyxin B1-I;

FIG. 14 is a total ion flow diagram of a polymyxin B1 enzymatic hydrolysate;

FIG. 15 is a mass spectrum of a polymyxin B1 enzymatic hydrolysate;

FIG. 16 is a drawing showing the amino acid sequence determination of the enzymatic product of polymyxin B1;

FIG. 17 is a drawing showing the amino acid sequence determination of the enzymatic product of polymyxin B2;

FIG. 18 is a diagram showing the amino acid sequence determination of the polymyxin B1-I enzymatic hydrolysate.

Detailed Description

The present invention will be described in detail and specifically with reference to the following examples to facilitate better understanding of the present invention, but the following examples do not limit the scope of the present invention.

Instruments and reagents:

model Agilent 1290 high performance liquid chromatograph-6550 QTOF-MS (Agilent Technologies, usa), mass spectrometry-guided Waters automatic purification system (Waters Technologies, japan), lyophilizer (model L abcnco vacuum lyophilizer, usa).

Polymyxin B "Chinese pharmacopoeia" reference substance (batch number: 130313201310)