CN111505142B - Polymyxin B amino acid configuration analysis method and N-polypeptide terminal sequence sequencing method - Google Patents
Polymyxin B amino acid configuration analysis method and N-polypeptide terminal sequence sequencing method Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
Abstract
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; determining the configuration of the amino acid 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.
Description
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, and is the last line of defense for clinically treating multi-drug-resistant gram-negative bacilli infection. The main components are 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, and L-leucine in B1-I is substituted by L-isoleucine; also, polymyxin is a fermentation product, and configurational transformation of amino acids may occur during production. Amino acid changes and D-/L-configuration differences can cause major changes in antimicrobial activity and nephrotoxicity, and the amino acids and their configurations must be monitored to ensure drug safety. In addition, it is necessary to confirm whether the sequence of the polypeptide, i.e., the sequence of amino acid linkage, is correct.
At present, the analysis method for the configuration of the amino acid comprises chiral derivation-chromatography/mass spectrometry after hydrolysis and directly adopts chiral chromatography analysis, but the method determines the amino acid mixture, and the amino acid and the configuration thereof at each position cannot be determined. N-terminal sequencing (Edman degradation) is a common method for determining amino acid sequences, and can compensate for the above defect that the amino acid at each position is determined from the N-terminal, but the alpha-amino group of the N-terminal amino acid needs to be exposed.
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 aqueous solution of deuterated hydrochloric acid to obtain a test solution, and then respectively carrying out derivatization reaction on the reference solution and the hydrolyzed test solution by adopting chiral Marfey's reagent;
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 mixed components of polymyxin B;
weighing mixed components of polymyxin B, adding a solvent to prepare a solution with the concentration of 2-40mg/mL, and purifying by adopting a liquid chromatography;
octadecyl bonded silica gel chromatographic column under liquid chromatographic condition; the sample injection amount is 100-; mobile phase a is water containing 0.01-1% formic acid (v/v), mobile phase B is acetonitrile or methanol, mobile phase a: mobile phase B is 50:50-90:10 (v/v); the flow rate is 5-20 mL/min; the ultraviolet absorption wavelength is 190-280 nm; the supplementary liquid is 50-90% methanol water solution containing 0.01-1% formic acid, and the flow rate of the supplementary liquid is 0.2-0.8 mL/min;
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: analyzing the reference substance solution and the test solution derivatized in the step S2 by high performance liquid chromatography-mass spectrometry;
liquid chromatography conditions conventional reverse phase chromatography columns; the column temperature is 25-40 ℃; the detection wavelength is 340 nm; the mobile phase A is water containing 0.01-0.2% formic acid (v/v), and the mobile phase B is acetonitrile or methanol; the flow rate is 0.8-1.8mL/min, and the flow is divided before mass spectrum detection, wherein the division ratio is 2: 1; gradient elution;
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, polymyxin B1, polymyxin B2 and polymyxin B1-I purified by S1 can be tested for accurate molecular weight and purity by HPLC-Q/TOF-MS;
the chromatographic conditions are as follows: 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 column temperature is 25-60 ℃, the flow rate is 0.8-1.8mL/min, and the sample injection amount is 2-10 muL.
The mass spectrum conditions are as follows: positive ion scan mode of electrospray-single quadrupole mass spectrometry, mass to charge ratio 300-.
Furthermore, the polymyxin B1, polymyxin B2 and polymyxin B1-I are fed at concentrations of 1-10mg/mL, 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 specifically includes: respectively dissolving polymyxin B1, polymyxin B2 and polymyxin B1-I in deuterated hydrochloric acid heavy water solution with the molar concentration of 4-8mol/L to ensure that the concentrations of polymyxin B1, polymyxin B2 and polymyxin B1-I are all 0.5-2mg/mL, charging nitrogen, sealing, carrying out pyrolysis at the temperature of 100-140 ℃ for 5-24h, cooling to room temperature, respectively adding 4-8mol/L sodium hydroxide solution, and neutralizing to neutrality to obtain the test solution.
Further, in S2, the Marfey ' S reagent is L/D-FDAA, FDVA, FDLA 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 0.5-4 mg/mL.
Further, in S2, the derivatization reaction specifically includes: respectively measuring 20-80 muL of polymyxin B1, polymyxin B2 and polymyxin B1-I test solution and 10-60 muL of 0.1-1mg/mL reference solution, putting the test solution and the reference solution into a liquid phase sample injection bottle, adding 30-80 muL of Marfey's reagent solution and 10-50 muL of alkaline solution, mixing uniformly, putting the mixture into a water bath for reaction for 30-90min, taking out the mixture, cooling the mixture to room temperature, and adding 10-50 muL of 0.02-0.08mol/L acetic acid solution to enable the mixture to be neutral.
Further, 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 0.02-0.08mol/L triethylamine solution or sodium bicarbonate solution; 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%.
In a second aspect of the present invention, there is provided a method for sequencing the N-polypeptide terminal sequence of polymyxin B, comprising the steps of:
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 an enzymolysis product solution, and purifying the enzymolysis product solution 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/mL, and purifying by adopting a liquid chromatography;
octadecyl bonded silica gel chromatographic column under liquid chromatographic condition; the sample injection amount is 100-1000 mu L; mobile phase a is water containing 0.01-1% formic acid (v/v), mobile phase B is acetonitrile or methanol, mobile phase a: mobile phase B is 50:50-90:10 (v/v); the flow rate is 5-20 mL/min; the ultraviolet absorption wavelength is 190-280 nm; the supplementary liquid is 50-90% methanol water solution containing 0.01-1% formic acid, and the flow rate of the supplementary liquid is 0.2-0.8 mL/min;
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; the mobile phase A is water containing 0.01-1% formic acid (v/v), the mobile phase B is acetonitrile or methanol, and gradient elution is carried out; the flow rate is 5-20mL/min, the sample injection amount is 100-; the supplementary liquid is 50-90% methanol water solution containing 0.01-1% formic acid, and the flow rate is 0.2-0.8 mL/min; selecting a positive ion scanning mode of electrospray-single quadrupole mass spectrometry and a mass-to-charge ratio of 300-1200, and respectively collecting the 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 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 enzymatic hydrolysis specifically includes: weighing 10-100mg of purified polymyxin B1, polymyxin B2 and polymyxin B1-I, and dissolving in 5-20mL of 0.1mol/L potassium dihydrogen phosphate buffer solution; weighing 10-20mg protease, dissolving in 2.5mL sodium chloride solution, which is equivalent to 1U/mL protease, and incubating in 37 ℃ water bath for 10-60 min; respectively adding the polymyxin B1, polymyxin B2 and polymyxin B1-I solutions into a preheated protease solution, carrying out water bath at 30-50 ℃ for 10-24h, boiling for 10min, centrifuging for 10min, and filtering for later use.
Further, the protease is subtilisin, ficin, papain or pronase; 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 is
Wakosil PTH-II column of 4.6 × 250mm, mobile phase of 30-50% acetonitrile water solution, isocratic elution, flow rate of 0.5-2.0 mL/min; the column temperature is 25-45 ℃, and the detection wavelength is 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:
agilent model 1290 high performance liquid chromatograph-6550 QTOF-MS (Agilent Technologies, USA). Mass spectrometry guided Waters auto purification system (Waters technologies (shanghai) ltd); lyophilizer (american laboco type vacuum lyophilizer).
Polymyxin B "Chinese pharmacopoeia" reference substance (batch number: 130313201310)
Example 1
This example provides a method for isolation and purification of polymyxin B1, polymyxin B2, and polymyxin B1-I from mixed components of polymyxin B:
weighing mixed components of polymyxin B, adding a solvent to prepare a solution with the concentration of 10mg/mL, and purifying by adopting a liquid chromatography, wherein the solvent is water and acetonitrile (80: 20 (v/v);
octadecyl bonded silica gel chromatographic column; the sample injection amount is 500 mu L; mobile phase a was water containing 0.1% formic acid (v/v), mobile phase B was acetonitrile, mobile phase a: mobile phase B ═ 85:15 (v/v); the flow rate is 15 mL/min; ultraviolet absorption wavelength is 215 nm; the supplementary liquid is 90% methanol water solution containing 0.1% formic acid, and the flow rate of the supplementary liquid is 0.45 mL/min;
selecting a positive ion scanning mode of electrospray-single quadrupole mass spectrometry, wherein the mass-to-charge ratio range is as follows: 300-1200. Polymyxin B1(M/z 602.4, [ M +2H ] was collected] 2+ ) Polymyxin B2(M/z 595.4, [ M +2H ]] 2+ ) Polymyxin B1-I (M/z 602.4, [ M +2H ]] 2+ ) Removing water by a rotary evaporator, and freeze-drying to obtain white powder.
The purified polymyxin B1 was dissolved in solvent water acetonitrile 80:20(v/v) at a concentration of 2mg/mL and tested for accurate molecular weight by HPLC-Q/TOF-MS (M/z 602.3848, [ M +2H ] M] 2+ ) And purity (98.5%), mobile phase A is 0.1% formic acid water solution, mobile phase B is 0.1% formic acid acetonitrile solution, mobile phase A and mobile phase B are 80:20, and column temperature isThe flow rate is 1mL/min at 50 ℃; the sample injection amount is 5 mu L; the total ion flow chart of the polymyxin B1 is shown in FIG. 2, and the mass spectrum is shown in FIG. 3.
The purified polymyxin B2 was dissolved in water at a concentration of 2mg/mL and acetonitrile 80:20(v/v), and the molecular weight was measured by HPLC-Q/TOF-MS (M/z 595.3770, [ M +2H ] M +2H] 2+ ) And purity (98.7%). The total ion flow chart of the polymyxin B2 is shown in FIG. 4, and the mass spectrum is shown in FIG. 5.
The purified polymyxin B1-I was dissolved in water at a concentration of 2mg/mL and acetonitrile 80:20(v/v), and the molecular weight was measured by HPLC-Q/TOF-MS (M/z 602.3847, [ M +2H ] to be precise (M/z 602.3847)] 2+ ) And purity (94.5%). The total ion flow chart of polymyxin B1-I is shown in FIG. 6, and the mass spectrum is shown in FIG. 7.
Example 2
This example provides a method for analyzing the amino acid configuration of polymyxin B:
respectively dissolving polymyxin B1, polymyxin B2 and polymyxin B1-I purified in example 1 in deuterated hydrochloric acid heavy water solution with the molar concentration of 6mol/L to ensure that the concentrations of polymyxin B1, polymyxin B2 and polymyxin B1-I are all 2mg/mL, charging nitrogen, sealing, pyrolyzing at 110 ℃ for 7 hours, cooling to room temperature, respectively adding 6mol/L sodium hydroxide solution, and neutralizing to neutrality to obtain a test solution.
Respectively measuring 50 muL of polymyxin B1, polymyxin B2 and polymyxin B1-I test solution and 30 muL of 0.5mg/mL reference substance (D/L-leucine, D/L-isoleucine, D/L-phenylalanine, D/L-threonine and D/L-2, 4-diaminobutyric acid) solution, placing the solution into a liquid phase sample feeding bottle, adding 50 muL of Marfey's reagent solution and 20 muL of 0.02-0.08mol/L triethylamine solution or sodium bicarbonate solution, uniformly mixing, placing the mixture into a water bath at 40 ℃ for reacting for 60min, taking out the mixture, cooling the mixture to room temperature, and adding 20 muL of 0.05mol/L acetic acid solution to enable the mixture to be neutral; the Marfey's reagent is L/D-FDAA, FDVA, FDLA or FDPA, a solvent for dissolving the Marfey's reagent is dimethyl sulfoxide, acetone or ethanol, and the concentration of the Marfey's reagent solution is 2 mg/mL.
Performing high performance liquid chromatography-mass spectrometry on the derivatized reference substance solution and the derivatized test substance solution:
liquid chromatography conditions conventional reverse phase chromatography columns (e.g., C8, C18); the column temperature is 25 ℃, and the detection wavelength is 340 nm; mobile phase a was water containing 0.1% formic acid (v/v) and mobile phase B was acetonitrile; the flow rate is 1.5mL/min, and the flow is divided before mass spectrum detection, wherein the division ratio is 2: 1; gradient elution; the elution conditions for the gradient elution are shown in table 1:
TABLE 1
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.
FIGS. 9, 10 and 11 are total ion flow diagrams, typical chromatograms and extracted ion flow chromatograms obtained by hydrolysis of purified components polymyxins B1, B2 and B1-I with deuterated hydrochloric acid and derivatization with L-FDAA, respectively.
Specifically, the method comprises the following steps:
FIG. 9 is 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 shows 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.
Comparing (as reference) the ultraviolet typical chromatogram and the total ion flow chart of 10 amino acid-L-FDAA derivatives (figure 8), it can be seen that the method of the invention can realize more ideal separation.
By extracting the amino groups[ M + H ] of acid (threonine, leucine, isoleucine, phenylalanine, 2, 4-diaminobutyric acid) -L-FDAA] + And obtaining an extracted ion current chromatogram of each amino acid, and comparing the extracted ion current chromatogram with a chromatogram (figure 8) of each reference substance-FDAA derivative, so as to judge the configuration of the amino acid in each component of the polymyxin B. According to this example, it was determined that the amino acids in polymyxin B1 were L-threonine, L-leucine, D-phenylalanine, and L-2, 4-diaminobutyric acid, that the amino acids in polymyxin B2 were L-threonine, L-leucine, D-phenylalanine, and L-2, 4-diaminobutyric acid, and that the amino acids in polymyxin B1-I were L-threonine, L-isoleucine, D-phenylalanine, and L-2, 4-diaminobutyric acid.
Example 3
This example provides methods for the preparation of polymyxin B1, polymyxin B2, and polymyxin B1-I enzymatic products:
respectively weighing polymyxin B1, polymyxin B2 and polymyxin B1-I50mg purified in example 1, and dissolving in 7.5mL of 0.1mol/L potassium dihydrogen phosphate buffer (pH is adjusted to 7.0 by phosphoric acid); weighing 12.5mg of ficin, dissolving in 2.5mL of sodium chloride solution (3mol/L), which is equivalent to 1U/mL of ficin, and incubating in 37 ℃ water bath for 30 min; respectively adding the polymyxin B1, polymyxin B2 and polymyxin B1-I solutions into a preheated ficin solution, carrying out water bath at 37 ℃ for 16h, boiling for 10min, centrifuging for 10min (eppendorf centrifuge, 13400rpm), and filtering to obtain an enzymolysis product solution for later use.
Preparing a chromatographic column from the enzymolysis product solution by using octadecyl bonded silica gel, performing gradient elution on a mobile phase A by using 0.1% formic acid aqueous solution and a mobile phase B by using acetonitrile; the elution conditions for the gradient elution are shown in table 2:
TABLE 2
The flow rate is 15 mL/min; the sample injection amount is 100 mu L; the ultraviolet absorption wavelength is 215 nm; the make-up was 90% aqueous methanol containing 0.1% formic acid at a flow rate of 0.45 mL/min. Selecting a positive ion scanning mode of electrospray-single quadrupole mass spectrometry, wherein the mass-to-charge ratio range is as follows: 300-1200. Respectively collecting the enzymolysis products (M/z 963.6, [ M + H ] +) of polymyxin B1, polymyxin B2 and polymyxin B1-I, removing water by a rotary evaporator, and freeze-drying to obtain white powder.
Due to the fact that ficin can specifically remove N-fatty acyl chains and 1-position alpha, gamma-L-diaminobutyric acid, the molecular weights of the enzymolysis products of polymyxin B1, polymyxin B2 and polymyxin B1-I are consistent, the structural formulas of the enzymolysis products of polymyxin B1 and polymyxin B2 are shown in figure 12, and the structural formula of the enzymolysis product of polymyxin B1-I is shown in figure 13. Taking polymyxin B1 enzymolysis product as an example, the polymyxin B1 enzymolysis product has [ M + H ] determined by HPLC-Q/TOF-MS] + M/z is 963.5786, [ M +2H ]] 2+ The m/z is 482.2931, and the total ion flow diagram and the mass spectrum are respectively shown in FIGS. 14 and 15.
Example 4
This example provides a method for sequencing the N-polypeptide terminal sequence of polymyxin B:
the enzymatic hydrolysis products of polymyxin B1, polymyxin B2, and polymyxin B1-I obtained in example 3 were dissolved in water (concentration: 5mg/mL), applied to a polybrene-treated glass fiber membrane, dried, and then mounted on a protein sequencer PPSQ-53A for analysis (Shimadzu, Kyoto, Japan) with the cycle number set at 9. Separating by reverse phase chromatography with a separating column of
Wakosil PTH-II column (4.6X 250mm), mobile phase 37% acetonitrile water solution, isocratic elution, flow rate of 1.0 mL/min; the column temperature was 40 ℃ and the detection wavelength was 269 nm.
The amino acid sequence diagram of the enzymatic hydrolysate of polymyxin B1 is shown in FIG. 16, where threonine (Thr) is determined as the amino acid in the first cycle, α, γ -L-diaminobutyric acid (Dab) is determined as the amino acid in the second cycle (with some Thr remaining), Dab is less in the third cycle and may be related to the fact that its γ -amino group is also linked to the subsequent peptide chain, Dab is determined in the fourth cycle, phenylalanine (Phe) is determined in the fifth cycle, leucine (Leu) is determined in the sixth cycle, Dab is determined in the third cycle, Dab is determined in the eighth cycle, and Thr is determined in the ninth cycle.
The amino acid sequence of the polymyxin B2 enzymatic hydrolysate was identical to that of polymyxin B1, as shown in fig. 17.
The amino acid sequence of polymyxin B1-I enzymatic hydrolysate is shown in FIG. 18, and compared to polymyxin B1, isoleucine (Ile) was determined only in the sixth cycle.
In conclusion, the method can judge the amino acid sequence of the polymyxin B component and the amino acid configuration of each site, and establishes a polymyxin B sequence determination and amino acid configuration analysis platform. The method for determining the amino acid sequence and the configuration is simple and sensitive, and is suitable for the quality control of polypeptide medicaments.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Claims (9)
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; the Marfey's reagent is L/D-FDAA;
s3: performing high performance liquid chromatography-mass spectrometry on the reference solution and the test solution subjected to derivatization in the step S2;
wherein the liquid chromatography conditions are conventional reverse phase chromatography columns; the conventional reverse phase chromatography column is C8 or C18; the column temperature is 25-40 ℃; the detection wavelength is 340 nm; mobile phase a is water containing 0.01-0.2% formic acid (v/v), mobile phase B is acetonitrile or methanol; the flow rate is 0.8-1.8mL/min, and the flow is divided before mass spectrum detection, wherein the division ratio is 2: 1; gradient elution; the elution conditions for the gradient elution were as follows:
at 0min, the mobile phase A is 73 percent, and the mobile phase B is 27 percent;
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 content of mobile phase A is 50%, and the content of 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, the mobile phase A is 73%, and the mobile phase B is 27%;
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.
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/mL, and purifying by adopting a liquid chromatography;
octadecyl bonded silica gel chromatographic column under liquid chromatographic condition; the sample injection amount is 100-; the mobile phase A is water containing 0.01-1% formic acid (v/v), the mobile phase B is acetonitrile or methanol, and the mobile phase A and the mobile phase B are 50:50-90:10 (v/v); the flow rate is 5-20 mL/min; the ultraviolet absorption wavelength is 190-280 nm; the supplementary liquid is 50-90% methanol water solution containing 0.01-1% formic acid, and the flow rate of the supplementary liquid is 0.2-0.8 mL/min;
s2: respectively heating and hydrolyzing the purified polymyxin B1, polymyxin B2 and polymyxin B1-I in a heavy aqueous solution of deuterated hydrochloric acid to obtain a test solution, and then respectively carrying out derivatization reaction on the reference solution and the hydrolyzed test solution by adopting chiral Marfey's reagent;
s3: analyzing the reference substance solution and the test solution derivatized in the step S2 by high performance liquid chromatography-mass spectrometry;
liquid chromatography conditions conventional reverse phase chromatography columns; the column temperature is 25-40 ℃; the detection wavelength is 340 nm; mobile phase a is water containing 0.01-0.2% formic acid (v/v), mobile phase B is acetonitrile or methanol; the flow rate is 0.8-1.8mL/min, and the flow is divided before mass spectrum detection, wherein the division ratio is 2: 1; gradient elution;
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 drug substance, 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 HPLC-Q/TOF-MS;
the chromatographic conditions are as follows: 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 ℃, 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/mL, 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 amino acid configuration of polymyxin B according to claim 2, wherein in S2, the thermal hydrolysis is specifically: respectively dissolving polymyxin B1, polymyxin B2 and polymyxin B1-I in deuterated hydrochloric acid heavy water solution with the molar concentration of 4-8mol/L to ensure that the concentrations of polymyxin B1, polymyxin B2 and polymyxin B1-I are all 0.5-2mg/mL, charging nitrogen, sealing, carrying out pyrolysis at the temperature of 100-140 ℃ for 5-24h, cooling to room temperature, respectively adding 4-8mol/L sodium hydroxide solution, and neutralizing to neutrality to obtain the test solution.
7. The method for analyzing amino acid configuration of polymyxin B according to claim 2, wherein the solvent used to dissolve the Marfey 'S reagent in S2 is dimethyl sulfoxide, acetone or ethanol, and the concentration of the Marfey' S reagent solution is 0.5-4 mg/mL.
8. The method for analyzing the amino acid configuration of polymyxin B according to claim 2, wherein in S2, the derivatization reaction specifically comprises: respectively measuring 20-80 mu L of polymyxin B1, polymyxin B2 and polymyxin B1-I test solution and 10-60 mu L of 0.1-1mg/mL reference substance solution, putting the test solution, the polymyxin B2 and the polymyxin B1-I test solution into a liquid phase sample introduction bottle, adding 30-80 mu L of Marfey's reagent solution and 10-50 mu L of alkaline solution, uniformly mixing, putting the mixture into a water bath for reaction for 30-90min, taking out, cooling to room temperature, and adding 10-50 mu L of 0.02-0.08mol/L acetic acid solution to enable the mixture to be neutral.
9. The method for analyzing the amino acid configuration of polymyxin B according to claim 8, wherein the control is D/L-leucine, D/L-isoleucine, D/L-phenylalanine, D/L-threonine or D/L-2, 4-diaminobutyric acid; the alkaline solution is 0.02-0.08mol/L triethylamine solution; the temperature of the water bath is 40-60 ℃.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7319021B2 (en) * | 2002-11-01 | 2008-01-15 | Promega Corporation | Cell lysis composition, methods of use, apparatus and kit |
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-
2020
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7319021B2 (en) * | 2002-11-01 | 2008-01-15 | Promega Corporation | Cell lysis composition, methods of use, apparatus and kit |
| CN107367562A (en) * | 2017-08-03 | 2017-11-21 | 上海上药第生化药业有限公司 | The analyzing detecting method of sulfuric acid Polymyxin B sulfate and application |
Non-Patent Citations (4)
| Title |
|---|
| Total Structures of Colistin Minor Components;YOSHITOMO IKAI等;《THE JOURNAL OF ANTIBIOTICS》;19980531;第51卷(第5期);第493-494页 * |
| YOSHITOMO IKAI等.Total Structures of Colistin Minor Components.《THE JOURNAL OF ANTIBIOTICS》.1998,第51卷(第5期), * |
| 多黏菌素E类似物的纯化和结构鉴定;吴勇等;《中国抗生素杂志》;20180131;第43卷(第1期);第59-63页 * |
| 液质联用技术检测硫酸多黏菌素B中各组分;刘孟娟等;《中国药业》;20111231;第20卷(第19期);第32页第2节 * |
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