阅读说明：本技术 一种寡糖的液相色谱分析方法 (Liquid chromatography analysis method of oligosaccharide ) 是由 梁鑫淼 夏东海 郭志谋 金高娃 闫竞宇 于 2019-10-28 设计创作，主要内容包括：本发明涉及一种寡糖的液相色谱分析方法。其特征是：在高pH、高柱温条件下,采用亲水色谱模式对寡糖进行分析。本方法通过在流动相中添加低末端吸收的碱性添加剂,以增强寡糖的紫外信号。本方法在高pH、高柱温条件下进行,加速了还原糖的变旋,使较宽的差向异构分裂峰变成窄的单峰。本发明在增强了寡糖紫外信号的同时,还具有不产生异构峰的优点,这对寡糖的色谱定性、定量分析及其分离纯化具有十分重要的意义。(The invention relates to a liquid chromatography analysis method of oligosaccharide. The method is characterized in that: and (3) analyzing the oligosaccharide by adopting a hydrophilic chromatographic mode under the conditions of high pH and high column temperature. The method enhances the ultraviolet signal of the oligosaccharide by adding a basic additive with low terminal absorption in the mobile phase. The method is carried out under the conditions of high pH and high column temperature, so that the derotation of reducing sugar is accelerated, and a wider epimerization splitting peak is changed into a narrow single peak. The invention enhances the ultraviolet signal of the oligosaccharide and has the advantage of no generation of isomeric peaks, which has important significance for chromatographic qualitative and quantitative analysis and separation and purification of the oligosaccharide.)

1. A method for liquid chromatography analysis of oligosaccharides, characterized by: analyzing the oligosaccharide by adopting a hydrophilic chromatographic mode under the conditions of high pH and high column temperature; the pH is 8-12.

2. The analytical method of claim 1, wherein: the column temperature is 40-90 ℃.

3. The analytical method of claim 1, wherein: the pH is adjusted by adding one or more of basic additives to the mobile phase.

4. The analytical method of claim 3, wherein: the type, concentration and pH value of the alkaline additive are one or more than two of the following components:

a) 0.0001-30% of ammonia water by mass;

b) triethylamine, the volume fraction is 0.0001-100%;

c) ammonium phosphate buffer salt with the concentration of 1-200mM and the pH value of 8-12;

d) triethylamine phosphate buffer salt with the concentration of 1-200mM and the pH value of 8-12;

e) ammonium formate buffer salt, concentration 1-200mM, pH 8-12;

f) ammonium acetate buffer salt at a concentration of 1-200mM, pH 8-12;

g) ammonium bicarbonate buffer salt, concentration 1-200mM, pH 8-12.

5. The analytical method of claim 3, wherein: the mobile phase comprises an organic phase and a water phase; the organic phase is one or more of acetonitrile, ethanol, methanol and the like.

6. The analytical method of claim 5, wherein: the alkaline additive is added in a manner that the alkaline additive is added in each of the organic phase and the aqueous phase or in either one of the organic phase and the aqueous phase.

7. The analytical method of claim 1, wherein: the hydrophilic chromatographic column is silica gel or an alkali-resistant hydrophilic chromatographic column bonded with a polar functional group;

the polar bonding group is one or more of amide, amino acid, amino group, carboxyl group, glycosyl, zwitter ion and the like;

the inner diameter of the hydrophilic chromatographic column is 2.1-10 mm;

the length of the hydrophilic chromatographic column is 50-250 mm;

the grain diameter of the filler is 1.7-10 mu m;

the pore diameter of the filler is

The specific surface area of the filler is 180-350m²/g。

8. The analytical method of claim 1, wherein: the detector is one or more of an ultraviolet detector, an electrospray detector, an evaporative light scattering detector, a refractive index detector and a mass spectrum detector.

9. The analytical method of claim 8, wherein: the ultraviolet detection wavelength is 190-400 nm.

Technical Field

The invention belongs to the field of biochemical analysis and detection, and relates to a liquid chromatography analysis method of oligosaccharide.

Technical Field

Oligosaccharides, also known as oligosaccharides, are carbohydrates formed by the polymerization of 2-10 monosaccharide molecules via glycosidic bonds (Dairy Science and Technology, second edition, 2008.). Oligosaccharide has multiple biological activities of resisting tumor, resisting oxidation, resisting virus infection, reducing blood sugar and blood lipid, regulating intestinal flora, enhancing immunity, etc. (2015, 44: 40-45.). Common oligosaccharides include chitosan oligosaccharide, breast milk oligosaccharide, fructooligosaccharide, acarbose and the like. The differences of different oligosaccharides are mainly the differences of monosaccharide compositions, connection modes and polymerization degrees, and the types and sources of the oligosaccharides directly influence the biological activity of the oligosaccharides, so that the analysis of the oligosaccharides has very important significance on the research of the biological activity of the oligosaccharides.

Liquid chromatography is an important tool for oligosaccharide analysis. Since the oligosaccharide has no or weak ultraviolet absorption, a chromophore is introduced by a derivatization method (biochemistry, 2019,5(3):16-19.) to meet the requirement of ultraviolet detection, and then the oligosaccharide is separated by a hydrophilic chromatographic column or a reverse chromatographic column. However, the derivatization method is complex and time-consuming to operate, and the characteristic fragments of the oligosaccharide isomers tend to disappear after derivatization.

The above disadvantages are overcome by directly analyzing the reducing sugar using a hydrophilic column, but a general-purpose detector such as an electrospray detector, an evaporative light scattering detector, a differential refraction detector, or a mass spectrometry detector is required. Whereas in the case of limited detector conditions, such as the case of a UV detector alone, it may be desirable to select additives with low terminal absorption to enhance the UV signal of the oligosaccharide. In addition, in the analysis of reducing sugar, epimers generated by alpha-type and beta-type mutarotans are separated by a chromatographic column and form a wide splitting peak, which influences the peak capacity of the chromatographic column and the separation degree between the reducing sugar. Therefore, it is required to develop a method for analyzing oligosaccharides by liquid chromatography, which have weak terminal absorption and can control epimer separation at the same time.

Disclosure of Invention

The invention aims to provide a liquid chromatographic analysis method of oligosaccharide, which has the advantage of no generation of isomeric peaks while enhancing the ultraviolet signal of the oligosaccharide, and has very important significance for chromatographic qualitative and quantitative analysis and separation and purification of the oligosaccharide.

In order to achieve the above object, the invention adopts the technical scheme that: and (3) analyzing the oligosaccharide by adopting a hydrophilic chromatographic mode under the conditions of high pH and high column temperature.

The pH value is 8-12, and the adjustment is carried out by adding an alkaline additive into the mobile phase.

Wherein the mobile phase comprises an organic phase and an aqueous phase. The organic phase is one or more of acetonitrile, ethanol, methanol and the like.

Wherein, the adding mode of the alkaline additive is respectively adding in two phases or adding in any phase. The type, concentration and pH of the alkaline additive are as follows:

a) 0.0001-30% of ammonia water by mass;

b) triethylamine, the volume fraction is 0.0001-100%;

c) ammonium phosphate buffer salt with the concentration of 1-200mM and the pH value of 8-12;

d) triethylamine phosphate buffer salt with the concentration of 1-200mM and the pH value of 8-12;

e) ammonium formate buffer salt, concentration 1-200mM, pH 8-12;

f) ammonium acetate buffer salt at a concentration of 1-200mM, pH 8-12;

g) ammonium bicarbonate buffer salt, concentration 1-200mM, pH 8-12.

The column temperature is 40-90 ℃.

The hydrophilic chromatographic column is silica gel or an alkali-resistant hydrophilic chromatographic column bonded with a polar functional group.

Wherein, the polar bonding group is one or more of amide, amino acid, amino group, carboxyl group, glycosyl, zwitter ion and the like.

Wherein, hydrophilic colorThe inner diameter of the spectrum column is 2.1-10 mm; the column length is 50-250 mm; the grain diameter of the filler is 1.7-10 mu m; the pore diameter of the filler isThe specific surface area of the filler is 180-350m²/g。

The detector adopted by the analysis method is one or more of an ultraviolet detector, an electrospray detector, an evaporative light scattering detector, a differential refraction detector and a mass spectrum detector.

Wherein, the ultraviolet detection wavelength is 190-400 nm.

The invention has the following advantages:

1. can be detected by ultraviolet. The alkaline additive added in the mobile phase has weak terminal absorption and weak inhibition effect on the ultraviolet signal of the oligosaccharide, and can directly carry out ultraviolet detection on the oligosaccharide at low wavelength.

2. No isomeric peaks are produced. The method is carried out under the conditions of high pH and high column temperature, so that alpha-type and beta-type epimers of reducing sugar are always in quick rotation change, a chromatographic column cannot be separated, and a wider epimerization splitting peak is changed into a narrow single peak.

Drawings

FIG. 1 is a liquid chromatography analysis of chitooligosaccharide by the Xbridge Amide column of example 1.

FIG. 2 is a liquid chromatography analysis spectrum of chitosan oligosaccharide by the XBridge Amide chromatographic column described in example 2 of the present invention.

Detailed Description

The present invention will be further described with reference to examples. The examples are given solely for the purpose of illustration and are not intended to be limiting.

Example 1

The liquid chromatography analysis of the chitosan oligosaccharide by the Xbridge Amide chromatographic column adopts the following chromatographic conditions:

basic chromatographic conditions:

a chromatographic column: XBridge Amide (Waters,4.6 × 150mm,3.5 μm);

column temperature: 50 ℃; 30 ℃ (comparative);

mobile phase: a, ACN (containing 0.02% ammonia water, namely 0.02mL of 28-30% ammonia water in each 100mL of acetonitrile); b, 0.02% ammonia (pH 10.3, i.e., 0.02mL of 28-30% ammonia per 100mL of water);

flow rate: 1 mL/min;

elution conditions: 0-35 min, 75-55% A, linear;

sample preparation: chitooligosaccharide solution (10mg/mL, solvent: a/B-1/1 (v/v));

sample introduction amount: 10 mu L of the solution;

ultraviolet detection wavelength: 195 nm;

neutral chromatographic conditions (comparative):

a chromatographic column: XBridge Amide (Waters,4.6 × 150mm,3.5 μm);

column temperature: 50 ℃;

mobile phase: a, ACN; b, H₂O；

Flow rate: 1 mL/min;

elution conditions: 0-15 min, 80-60% A, linear;

sample preparation: chitooligosaccharide solution (10mg/mL, solvent: a/B-1/1 (v/v));

sample introduction amount: 10 mu L of the solution;

ultraviolet detection wavelength: 195 nm;

chitosan oligosaccharide contains conjugated amide structure, so that direct ultraviolet detection can be performed, but obvious epimeric splitting peak exists (figure 1 a). Comparing fig. 1c with fig. 1a and 1b, it can be seen that epimerization can be suppressed at high pH and high temperature, and more sample peaks can be separated. Therefore, after the invention is adopted, no isomeric peak is generated in the liquid chromatographic analysis of the chitooligosaccharide, and more sample components can be seen.

Example 2

The liquid chromatography of chitosan oligosaccharide by XBridge Amide column differs from example 1 in that the following chromatographic conditions are used:

basic chromatographic conditions:

sample preparation: a chitosan oligosaccharide solution (10mg/mL, solvent: a/B ═ 1/1 (v/v));

the chitosan oligosaccharide, because it contains a basic amino group, its conventional hydrophilic chromatography was performed under acidic conditions (comparative example):

a chromatographic column: XBridge Amide (Waters,4.6 × 150mm,3.5 μm);

column temperature: 50 ℃;

mobile phase: a, volume fraction 80% ACN (containing 10mM ammonium formate diluted from 100mL of 100mM ammonium formate at pH 3.2, with 100mL water and 800mL acetonitrile); b, 20% ACN by volume (containing 10mM ammonium formate diluted from 100mL of 100mM ammonium formate at pH 3.2, with 700mL water and 200mL acetonitrile);

flow rate: 1 mL/min;

elution conditions: 0-10 min, 80-60% A, linear; 10-30 min, 60-32% A, linear;

sample preparation: a chitosan oligosaccharide solution (10mg/mL, solvent: a/B ═ 1/1 (v/v));

sample introduction amount: 10 mu L of the solution;

ultraviolet detection wavelength: 195 nm;

the conjugated structure of the chitosan oligosaccharide is less, so that ultraviolet detection cannot be directly carried out in conventional hydrophilic chromatographic analysis and an epimerization splitting peak exists (fig. 2a, because the noise is too high and the ultraviolet signal is too poor, the actually existing epimerization phenomenon cannot be obviously observed). Comparing fig. 2c with fig. 2a and 2b (weak epimerization), it can be seen that the high pH and high temperature can inhibit epimerization and allow direct uv detection of chitosan oligosaccharide. Therefore, after the chitosan oligosaccharide preparation is adopted, the chitosan oligosaccharide has better ultraviolet response and does not generate isomeric peaks.

The method enhances the ultraviolet signal of the oligosaccharide by adding a basic additive with low terminal absorption in the mobile phase. The method is carried out under the conditions of high pH and high column temperature, so that the derotation of reducing sugar is accelerated, and a wider epimerization splitting peak is changed into a narrow single peak. The invention enhances the ultraviolet signal of the oligosaccharide and has the advantage of no generation of isomeric peaks, which has important significance for chromatographic qualitative and quantitative analysis and separation and purification of the oligosaccharide.