Disaccharide isomer analysis reagent and analysis method thereof

Abstract

The invention relates to the technical field of analysis and testing, in particular to a chemical derivatization reagent for identifying two disaccharide isomeride types, namely whether disaccharide is lactose or maltose, and an analysis method of the chemical derivatization reagent. The invention provides a disaccharide isomer analysis reagent. The disaccharide isomer analysis reagent comprises 1-(4-carboxyphenyl)-3-methyl-5-pyrazolone; the invention provides a disaccharide isomer analysis method, which comprises: S1, mixing a disaccharide to be detected with an analysis reagent, carrying out a derivatization chemical reaction to produce a molecular product for ion mobility spectrometry analysis, and preparing the molecular product into a chemical solution; s2, treating the chemical solution by adopting an ionization device to generate gas-phase ions; s3, measuring the ion mobility of gas phase ions by adopting an ion mobility spectrometry-mass spectrometry device, or calculating an ion collision cross section through the ion mobility, so as to obtain the type, structure or content of disaccharide molecules; compared with a traditional disaccharide structure analysis method, the analysis method is simple, rapid and accurate.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and testing, and in particular to a chemical derivatization reagent for identifying two disaccharide isomers, namely, whether the disaccharide is lactose or maltose, and an analysis method thereof. Background Art

[0002] Sugar is a class of molecules that exist widely in nature and play an irreplaceable role in the life process. It is the main component of all animal foods, provides energy for almost all animal activities, and participates in almost all important life activities. Therefore, understanding the types and effects of sugars, especially the process of action in animals, is of great significance to the life science process and daily life. At the same time, since sugar molecules exist in most foods and beverages, such as rice, wheat, various vegetables and fruits, honey, various fruit juices, etc., accurate measurement of the types and contents of disaccharides in different types of food or beverages can not only provide a reference for people's daily healthy life, but also can be used for food and beverage origin traceability and quality monitoring.

[0003] According to the different molecular compositions, sugars are divided into monosaccharides, disaccharides and polysaccharides. Disaccharides are formed by two molecules of monosaccharides through glycosidic bonds. The most common disaccharides in nature are sucrose, lactose and maltose. Disaccharides are not only the energy source of various organisms, but also often used as the material raw materials of the composition of organisms, and they play an important role in the storage or transportation of necessary sugars. In short, accurate analysis of the types of disaccharides is not only of great scientific significance, but also of great application value.

[0004] Lactose and maltose are two types of disaccharides. Lactose is composed of one molecule of galactose and one molecule of glucose. Maltose is composed of two molecules of glucose. Lactose and maltose have exactly the same chemical composition. Their molecular formulas are C 12 H 22 O 11 However, due to the different monosaccharides that make up them, they have slightly different chemical structures and are isomers, as shown in Figure 1. Precisely because of their identical chemical composition and slight differences in chemical structure, it is difficult to distinguish them using conventional chemical analysis methods such as mass spectrometry, infrared spectroscopy, and visible / ultraviolet absorption spectroscopy. The chromatography, optical polarimetry, and enzymatic methods currently used for disaccharide analysis are not only complex processes, but also often require sample pretreatment, such as purification, etc., making the analysis of mixed complex samples even more difficult. Therefore, the development of new analytical methods to accurately and quickly obtain information on the composition and structure of disaccharides is of great value in both scientific research and practical applications. Summary of the invention

[0005] The purpose of the present invention is to provide a highly sensitive, rapid and accurate method for analyzing the structure and type of disaccharide molecules in view of the deficiencies in the prior art.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention is to provide a disaccharide isomer analysis reagent, comprising: 1-(4-carboxyphenyl)-3-methyl-5-pyrazolone.

[0008] Preferably, the solvent of the analytical reagent includes: acetonitrile and ammonia water.

[0009] The second aspect of the present invention is to provide a method for analyzing disaccharide isomers, using the above-mentioned analytical reagent, the steps comprising:

[0010] S1, mixing the disaccharide to be tested with the analytical reagent to carry out a derivatization chemical reaction, producing a molecular product for ion mobility spectrometry analysis and configuring the molecular product into a chemical solution;

[0011] S2, treating the chemical solution with an ionizing device to generate gas phase ions;

[0012] S3. Measuring the ion mobility of the gas phase ions using an ion mobility spectrometer-mass spectrometer, or calculating the ion collision cross section using the ion mobility, thereby obtaining the type, structure or content of the disaccharide molecule.

[0013] Preferably, the disaccharides to be detected include: lactose and maltose.

[0014] Preferably, the conditions of the derivatization chemical reaction are: the reaction temperature of the derivatization chemical reaction is 60-80° C. and the reaction time is 2-4 h.

[0015] Preferably, the concentration of the chemical solution is 10 -2 M-10 -8 M.

[0016] Preferably, the solvent of the chemical solution includes: methanol and formic acid water.

[0017] Preferably, the ionization device comprises: an electrospray ionization source, a laser-assisted desorption ionization source, and a desorption electrospray ionization source.

[0018] The third aspect of the present invention is to provide the use of the above analytical reagent in analyzing disaccharide isomers.

[0019] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0020] (1) Compared with the traditional disaccharide structure analysis method, the analysis method of the present invention is simple, rapid and accurate;

[0021] (2) The analytical method of the present invention reduces the sample pretreatment and separation steps required by the traditional method, and can also perform disaccharide molecular structure analysis and relative content analysis in the mixture;

[0022] (3) The analysis method of the present invention requires a small amount of sample and has a wide range of applicability, and can be used to analyze disaccharide components in a variety of common foods and beverages. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG1 is a schematic diagram of the chemical structures of lactose and maltose molecules; Figure 1A Schematic diagram of the chemical structure of lactose molecule; Figure 1B Schematic diagram of the chemical structure of the maltose molecule.

[0024] Figure 2 It is the chemical structural formula of 1-(4-carboxyphenyl)-3-methyl-5-pyrazolone (4-(3-methyl-5-oxopyrazolin-1-yl)benzoic acid, CPMP).

[0025] Figure 3 Diagram of the chemical reaction process for disaccharide derivatization of CPMP.

[0026] FIG4 is the experimental result of analyzing the ion mobility of disaccharide derivatization products using ion mobility spectrometry-mass spectrometry technology; wherein, Figure 4A Mass spectra of lactose and maltose in positive ion mode; Figure 4B is [M+Na] + Ion mobility spectra of Figure 4C [M+K] + Ion mobility spectra of Figure 4D Mass spectra of lactose and maltose in negative ion mode; Figure 4E is the ion mobility spectrum of [MH]-; Figure 4F This is the ion mobility spectrum of [M-Cl]-. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] It should be noted that the ionization device in the present invention can be any method that can produce gas-phase ions of derivative products, and the equipment for measuring the ion mobility of gas-phase ions can be any ion mobility spectrum measurement device. In the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0029] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but they are not intended to limit the present invention.

[0030] Example

[0031] This embodiment provides a method for analyzing disaccharide isomers, the steps comprising:

[0032] 1-(4-Carboxyphenyl)-3-methyl-5-pyrazolone was dissolved in acetonitrile: ammonia water (v / v=3:1) to prepare an analytical reagent with a concentration of 5 mg / mL; 100-200 μL of lactose, 100-200 μL of maltose and 400-500 μL of the analytical reagent were mixed and reacted in a 70°C metal heating block for 2-4 hours to produce a molecular product for ion mobility spectrometry analysis, and the molecular product was prepared into 10 -5 mol L -1 chemical solution;

[0033] The TIMS-TOFMS instrument was used for detection and analysis, with an ion source voltage of 2.5-4.5 kV and an injection flow rate of 1-5 μL min -1 ; Atomizer pressure 0.3 bar; Dry gas 3 L min at 200 °C -1 , and analyze the test results; analyze the ion mobility spectrometry experimental results to obtain the types and relative contents of disaccharide molecules in the analysis samples.

[0034] In summary, compared with the traditional disaccharide structure analysis method, the analysis method of the present invention is simple, rapid and accurate; the analysis method of the present invention reduces the sample pretreatment and separation steps required by the traditional method, and can also perform disaccharide molecular structure analysis and relative content analysis in the mixture; the analysis method of the present invention requires a small amount of sample and has a wide range of applicability, and can analyze the disaccharide components in a variety of common foods and beverages.

[0035] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A disaccharide isomer analysis reagent, characterized in that: include: 1-(4-Carboxyphenyl)-3-methyl-5-pyrazolone.

2. The analytical reagent according to claim 1, characterized in that The solvent of the analytical reagent includes: acetonitrile and ammonia water.

3. A method for analyzing disaccharide isomers, using the analytical reagent according to any one of claims 1 to 2, characterized in that the steps include: S1, mixing the disaccharide to be tested with the analytical reagent to carry out a derivatization chemical reaction, producing a molecular product for ion mobility spectrometry analysis and configuring the molecular product into a chemical solution; S2, treating the chemical solution with an ionizing device to generate gas phase ions; S3. Measuring the ion mobility of the gas phase ions using an ion mobility spectrometer-mass spectrometer, or calculating the ion collision cross section using the ion mobility, thereby obtaining the type, structure or content of the disaccharide molecule.

4. The analysis method according to claim 3, characterized in that The disaccharides to be tested include: lactose and maltose.

5. The analysis method according to claim 3, characterized in that The reaction temperature of the derivatization chemical reaction is 60-80° C. and the reaction time is 2-4 hours.

6. The analysis method according to claim 3, characterized in that The concentration of the chemical solution is 10 -2 M-10 -8 M.

7. The analysis method according to claim 3, characterized in that The solvent of the chemical solution includes: methanol and formic acid water.

8. The analysis method according to claim 3, characterized in that The ionization device comprises: an electrospray ionization source, a laser-assisted desorption ionization source, and a desorption electrospray ionization source.

9. Use of the analytical reagent according to any one of claims 1 to 2 in analyzing disaccharide isomers.

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