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High-performance liquid chromatography-tandem mass spectrometry method used for qualitatively and quantitatively determining plant active polysaccharide

A high-performance liquid chromatography and active polysaccharide technology, applied in the field of qualitative and quantitative analysis, can solve the problems of reduced chromatographic detection sensitivity, low analysis results, complex reactions, etc., and achieve the effects of improving sensitivity, good separation effect and strong selectivity.

Active Publication Date: 2013-04-24
GUANGZHOU YI JIAN BIOMEDICAL TECH DEV CO LTD
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

At present, the most commonly used derivatization reagent is primary amino (Amino Derivatization Reagent), but the derivation of primary amino reagent has disadvantages. First, the generated Schiff Base (Schiff Base) must undergo a reduction reaction of 8h (90°C) It can generate stable tertiary amine derivatives that can be used for analysis; secondly, acid catalysis is required in the derivatization reaction process of primary amino reagents, and the reaction is complicated and takes a long time, which may lead to a decrease in chromatographic detection sensitivity; in addition, primary amino reagents cannot be used for Derivatization of reducing ketoses, resulting in low analytical results

Method used

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  • High-performance liquid chromatography-tandem mass spectrometry method used for qualitatively and quantitatively determining plant active polysaccharide
  • High-performance liquid chromatography-tandem mass spectrometry method used for qualitatively and quantitatively determining plant active polysaccharide
  • High-performance liquid chromatography-tandem mass spectrometry method used for qualitatively and quantitatively determining plant active polysaccharide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] (1) The polysaccharide sample to be tested is a mixed plant extract: extracted from tremella, shiitake mushroom, chrysanthemum, mulberry, medlar, Poria cocos, Flammulina velutipes and other natural hot plants through water extraction.

[0048] (2) Derivatization treatment of standard substance: the standard substance is Dextran Standard DXT1K Mw1200 (referred to as mixed polysaccharide reference substance, American Polymer Standards Company) and glucose standard substance, the former is used for qualitative and the latter is used for quantitative. Take 200 μL of 0.01mol / L standard product solution in a 15ml EP tube, add 200 μL of AEC-containing methanol solution (the final concentration of AEC is 0.04mol / L), 200 μL of 0.2mol / L sodium cyanoborohydride solution and 60 μL glacial acetic acid, shake well, heat in 70°C water bath for 1 hour, take out and cool to room temperature, add 500 μL of ultrapure water, shake well, add 1.5 mL of chloroform, shake well, and centrifuge a...

Embodiment 2

[0059] (1) The polysaccharide sample to be tested is Borojo powder: Borojo is peeled and pitted, then ground with water, enzymatically hydrolyzed by pectinase, filtered to remove impurities, concentrated in vacuum at low temperature, adjusted to pH, and spray-dried. become.

[0060] (2) Derivatization treatment of standard substance: same as step (2) of Example 1.

[0061] (3) Sample derivatization treatment: Weigh 10mg of sample into a 15mL EP tube, add 1ml of ultrapure water into the EP tube, shake well, take 200μL of the sample solution into a 15ml EP tube after the dissolution is complete, and the rest of the steps are the same as in the example 1 step (3).

[0062] (4) Qualitative determination: same as step (4) of Example 1.

[0063] The result obtained is as Figure 6 , 7 As shown in and 8, the qualification is carried out by the retention time and the characteristic peaks of the fragment ions of each component.

[0064] (5) Quantitative determination: the determin...

Embodiment 3

[0069] (1) The polysaccharide sample to be tested is pine needle extract: it is obtained by crushing pine needles and extracting them with water.

[0070] (2) Derivatization treatment of standard substance: same as step (2) of Example 1.

[0071] (3) Sample derivatization treatment: same as step (3) of Example 1.

[0072] (4) Qualitative determination: same as step (4) of Example 1.

[0073] The result obtained is as Figures 11 to 13 Qualitative analysis was carried out by retention time and characteristic peaks of fragment ions of each component.

[0074] (5) Quantitative determination: the determination conditions are the same as step (5) of Example 1.

[0075] Make a standard curve: take the peak area of ​​the standard glucose solution derivatized by AEC as the abscissa, and the concentration of the standard series as the ordinate, and perform linear regression to obtain the standard curve equation:

[0076] y=2.5e+006x+1.94e+006R=0.9996.

[0077] (6) Result analysis:...

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Abstract

The invention discloses a high-performance liquid chromatography-tandem mass spectrometry method used for qualitatively and quantitatively determining plant active polysaccharide. According to the invention, 3-amino-9-ethyl carbazole derivatization treatment is carried out upon a polysaccharide sample solution to be tested, a mixed polysaccharide sample solution, and a standard glucose solution, such that AEC-derivatized polysaccharide to be tested, mixed polysaccharide solution and standard glucose solution are obtained; mass spectrometry analysis is carried out, wherein AEC-derivatized mixed polysaccharide control is adopted as a reference, and the polysaccharide sample solution to be tested is subjected to polysaccharide ion fragment peak determination, such that reducing polysaccharide polymerization degree is determined, and the type of reducing polymeric polysaccharide contained in the polysaccharide to be tested is qualitatively identified; and high-performance liquid chromatography analysis is carried out, wherein AEC-derivatized standard monosaccharide is adopted as a reference, and a standard glucose curve is made after analysis, such that indirect quantitation is carried out upon various reducing polymeric polysaccharides in the polysaccharide to be tested.

Description

technical field [0001] The invention belongs to a qualitative and quantitative analysis method for active polysaccharides in plants and their extracts, in particular to a high performance liquid chromatography-tandem mass spectrometry method for qualitative and quantitative determination of plant active polysaccharides. Background technique [0002] In recent years, the analysis method of polysaccharides is mainly detected by high performance liquid chromatography (HPLC) with different special chromatographic detectors. The differential detection method is based on the difference in refractive index between the test components and the mobile phase. Zhang Jinjie et al. (Determination of reducing sugar, phosphorylated monosaccharide and sucrose in aquatic products by HPLC differential refractometry, Journal of Food and Biotechnology, 2011, 30 (4)) established a method for the determination of reducing sugar and phosphorylated monosaccharide in aquatic products by HPLC differen...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
Inventor 潘若微李丹胡烨敏吴军孟平王家骥陈骁熠
Owner GUANGZHOU YI JIAN BIOMEDICAL TECH DEV CO LTD
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