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Amino acid configuration analysis method and N-polypeptide terminal sequence sequencing method of polymyxin B

A technology for polymyxin and configuration analysis, which can be used in analytical materials, measuring devices, material separation, etc., and can solve problems such as inability to determine amino acid configuration.

Active Publication Date: 2020-08-07
SHANGHAI INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the analysis methods for amino acid configuration include chiral derivatization after hydrolysis-chromatography / mass spectrometry, and direct chiral chromatography analysis. However, this method measures the mixture of amino acids and cannot determine the amino acid and its structure at each site. type

Method used

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  • Amino acid configuration analysis method and N-polypeptide terminal sequence sequencing method of polymyxin B
  • Amino acid configuration analysis method and N-polypeptide terminal sequence sequencing method of polymyxin B
  • Amino acid configuration analysis method and N-polypeptide terminal sequence sequencing method of polymyxin B

Examples

Experimental program
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Effect test

Embodiment 1

[0086] This embodiment provides a method for separating and purifying polymyxin B1, polymyxin B2 and polymyxin B1-I from polymyxin B mixed components:

[0087] Take polymyxin B mixed component and add solvent, be mixed with the solution that concentration is 10mg / mL, solvent is water: acetonitrile=80:20 (v / v), adopt liquid chromatography to purify;

[0088] Octadecyl bonded silica gel chromatographic column; injection volume 500 μ L; mobile phase A is water, contains 0.1% formic acid (v / v), mobile phase B is acetonitrile, mobile phase A: mobile phase B=85:15 (v / v); The flow rate is 15mL / min; the ultraviolet absorption wavelength is 215nm; the replenishment liquid is 90% aqueous methanol containing 0.1% formic acid, and the flow rate of the replenishment liquid is 0.45mL / min;

[0089] Select the positive ion scan mode of the electrospray-single quadrupole mass spectrometer, and the mass-to-charge ratio range: 300-1200. Collection of polymyxin B1 (m / z 602.4, [M+2H] 2+ ), poly...

Embodiment 2

[0094] This embodiment provides a method for analyzing the amino acid configuration of polymyxin B:

[0095]The polymyxin B1, polymyxin B2 and polymyxin B1-I that embodiment 1 is purified are dissolved in the deuterated hydrochloric acid heavy aqueous solution that molar concentration is 6mol / L respectively, make described polymyxin The concentrations of B1, polymyxin B2 and polymyxin B1-I are all 2mg / mL, filled with nitrogen, sealed, pyrolyzed at 110°C for 7h, cooled to room temperature, and added with 6mol / L sodium hydroxide solution respectively , neutralized to neutral, that is, the test solution.

[0096] Measure respectively the reference substance (D / L-leucine, D / L-isoleucine) of polymyxin B1, polymyxin B2 and polymyxin B1-I need testing solution 50 μ L and 0.5 mg / mL amino acid, D / L-phenylalanine, D / L-threonine and D / L-2,4-diaminobutyric acid) solution 30μL, put in the liquid phase sample bottle, add Marfey's reagent solution 50 μL and 0.02-0.08 mol / L triethylamine so...

Embodiment 3

[0111] This example provides the preparation method of polymyxin B1, polymyxin B2 and polymyxin B1-I enzymatic hydrolyzate:

[0112] Take respectively the polymyxin B1, polymyxin B2 and polymyxin B1-150mg that embodiment 1 purifies, be dissolved in the 0.1mol / L potassium dihydrogen phosphate buffer of 7.5mL (pH adjusted by phosphoric acid to 7.0); weigh 12.5mg of ficin and dissolve it in 2.5ml of sodium chloride solution (3mol / L), which is equivalent to 1U / mL of ficin, and incubate in a water bath at 37°C for 30min; mix the above polymyxin B1 , polymyxin B2 and polymyxin B1-I solutions were added to the preheated ficin solution, 37 ° C water bath for 16 hours, boiled for 10 minutes, centrifuged for 10 minutes (eppendorf centrifuge, 13400rpm), filtered to obtain enzymolysis The product solution was used for later use.

[0113] The above enzymatic hydrolysis product solution was used to prepare a chromatographic column using octadecyl bonded silica gel, mobile phase A was 0.1% ...

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Abstract

The invention discloses an amino acid configuration analysis method and an N-polypeptide end sequence sequencing method of polymyxin B. A liquid phase purification method of a polymyxin B component isestablished, and polymyxin B1, polymyxin B2 and polymyxin B1-I can be rapidly purified and separated from the polymyxin B component. A preparation method and a liquid phase purification method of thepolymyxin B enzymolysis component are established, and the enzymolysis components of polymyxin B1, polymyxin B2 and polymyxin B1-I can be rapidly purified and separated; polymyxin B component hydrolysis-amino acid chiral derivation-liquid chromatography-mass spectrometry are adopted to determine the amino acid configuration; the N-polypeptide terminal sequence is determined by combining enzymolysis of the polymyxin B component, liquid chromatography purification and protein sequencing, so that a research basis is provided for strictly controlling the quality of polymyxin B.

Description

technical field [0001] 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 technique [0002] Polymyxin B is a lipopeptide antibacterial peptide, which has a strong bactericidal effect on Gram-negative bacilli, and is the "last line of defense" in the clinical treatment of multidrug-resistant Gram-negative bacilli. The main components are polymyxin B1, B2 and B1-I, the structural formula is shown in the appendix figure 1 , the first two amino acids include L-2,4-diaminobutyric acid, L-threonine, L-leucine and D-phenylalanine, and the L-leucine in B1-I is composed of L-iso Leucine substitution; and, colistin is a fermentation product, and amino acid configuration changes may occur during the production process. Changes in amino acids and differences in D- / L-configurations will cause large changes in antibacterial...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06G01N30/34
CPCG01N30/02G01N30/06G01N30/34G01N2030/067
Inventor 张含智刘浩罗文燕顿俊玲
Owner SHANGHAI INST FOR FOOD & DRUG CONTROL
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