Sample pretreatment method and detection method for carbohydrates

Abstract

The invention provides a sample pretreatment method and a detection method for carbohydrates, and belongs to the technical field of chemical substance detection. The sample pretreatment method for the carbohydrates comprises the following steps: contacting a sample solution containing the carbohydrates with a graphitized carbon material to realize selective adsorption of the carbohydrates in the sample solution on the graphitized carbon material, and carrying out solid-liquid separation to obtain the graphitized carbon material adsorbed with the carbohydrates; a derivative solution is in contact with the graphitized carbon material adsorbed with the carbohydrates, after solid-liquid separation, the graphitized carbon material adsorbed with derivative products is obtained, and the derivative solution is an aprotic solution containing a boric acid derivative reagent; and contacting the graphitized carbon material adsorbed with the derivative product with an eluent to realize separation of the derivative product and the graphitized carbon material to obtain the eluent containing the derivative product. Finally, the eluent of the derivative product is used for mass spectrometry detection or liquid chromatography-mass spectrometry detection.

Description

Technical Field

[0001] This application relates to the field of chemical substance detection technology, and in particular to a sample pretreatment method and detection method for carbohydrate substances. Background Technology

[0002] Natural carbohydrates, including small-molecule sugars, glycosides, polysaccharides, glycolipids, and glycopeptides, are not only key biomolecules in living systems but also important natural food additives and nutrients. Therefore, the detection of carbohydrates in complex biological samples, microbial samples, and food products is of great significance in biomedical research, botanical research, microbiological research, and the food industry.

[0003] In complex sample systems, mass spectrometry is a commonly used tool for accurate qualitative and quantitative analysis of target carbohydrate molecules. However, carbohydrate molecules present significant detection challenges: their complex structures and predominantly neutral nature result in weak charge in solution, leading to low ionization efficiency in mass spectrometry ion sources and directly causing insufficient detection sensitivity. More importantly, the high abundance of biological matrices in complex samples generates strong interference, further exacerbating the ion suppression effect on carbohydrates and severely weakening the carbohydrate signal, posing a challenge to accurate detection. Summary of the Invention

[0004] In view of this, this application provides a sample pretreatment method and a detection method for carbohydrate substances, in order to at least partially solve at least one of the aforementioned technical problems.

[0005] According to one embodiment of this application, a sample pretreatment method for carbohydrate substances is provided, comprising the following steps:

[0006] A sample solution containing sugars is brought into contact with a graphitized carbon material to achieve selective adsorption of sugars in the sample solution onto the graphitized carbon material. After solid-liquid separation, the graphitized carbon material adsorbed with sugars is obtained.

[0007] The derivatization solution was contacted with the graphitized carbon material adsorbed with sugars. After solid-liquid separation, the graphitized carbon material adsorbed with the derivatized products was obtained. The derivatization solution was an aprotic solution containing boric acid derivatizing reagent.

[0008] The graphitized carbon material adsorbed with the derivatives is contacted with the eluent to separate the derivatives from the graphitized carbon material, resulting in an eluent containing the derivatives. This eluent containing the derivatives is then used for mass spectrometry or liquid chromatography-mass spectrometry (LC-MS) detection.

[0009] According to an embodiment of the second aspect of this application, a sample detection method for carbohydrates is provided, wherein the eluent containing derivatives obtained by the above-described sample pretreatment method for carbohydrates is injected into a mass spectrometer or liquid chromatography-mass spectrometry (LC-MS) for detection.

[0010] The sample pretreatment method for carbohydrates provided in this application utilizes graphitized carbon materials to achieve selective adsorption and enrichment of carbohydrates, while simultaneously completing the derivatization reaction on the surface of the graphitized carbon material. This single sample treatment enhances the mass spectrometry signal of carbohydrates by combining enrichment and derivatization. Furthermore, by leveraging the different selective adsorption properties of graphitized carbon materials for derivatized products and unreacted derivatizing reagents, excess derivatizing reagents are separated, solving the problem of suppression and interference from large amounts of derivatizing reagent ions in current chemical derivatization methods. The eluent obtained by this sample pretreatment method can be directly used for mass spectrometry or liquid chromatography-mass spectrometry (LC-MS) detection. In addition, this invention has the potential to achieve online sample processing integrating enrichment and derivatization. The adsorption effect on the material surface enables an online derivatization process that effectively controls the contact time between the derivatizing reagent and the carbohydrates, and selective enrichment before derivatization reduces the interference of the biological matrix in the sample on the derivatization reaction during the online derivatization reaction. This simplifies the process while improving detection sensitivity and accuracy, thereby significantly enhancing the mass spectrometry detection performance of carbohydrates in complex samples. Attached Figure Description

[0011] Figure 1 This is a schematic diagram illustrating the principle of the sample pretreatment method in this application;

[0012] Figure 2A A comparison of the adsorption efficiency of sugars on the surface of graphene oxide under different solvent conditions;

[0013] Figure 2B A comparative graph showing the effects of different solvents and pH conditions on the boric acid derivatization reaction of sugars;

[0014] Figure 3 A comparison of the adsorption efficiencies of sugar derivatives and derivatizing reagents on the surface of graphene oxide under different solvent ratios;

[0015] Figure 4 This is a comparison graph of the standard curves for mass spectrometry detection in the embodiments and comparative examples of this application;

[0016] Figure 5 This is a signal comparison diagram of the derivatives and derivatizing reagents in the embodiments and comparative examples of this application;

[0017] Figure 6A This is a mass spectrum of a glucose sample with a concentration of 5 μg / mL, as shown in the embodiments of this application.

[0018] Figure 6BThis is the mass spectrum of a glucose sample with a concentration of 5 μg / mL, used in the comparative example. Detailed Implementation

[0019] The embodiments of this application will be described below. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of this application. In the following detailed description, many specific details are set forth to provide a thorough understanding of the embodiments of this application for ease of explanation. However, it will be apparent that one or more embodiments may be implemented without these specific details. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concepts of this application.

[0020] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. The term "comprising" as used herein indicates the presence of features, steps, or operations, but does not exclude the presence or addition of one or more other features.

[0021] When using expressions such as "at least one of A, B, and C," the expression should generally be interpreted in accordance with the meaning commonly understood by a person skilled in the art (e.g., "a system having at least one of A, B, and C" should include, but is not limited to, systems having A alone, having B alone, having C alone, having A and B, having A and C, having B and C, and / or having A, B, and C, etc.). When using expressions such as "at least one of A, B, or C," the expression should generally be interpreted in accordance with the meaning commonly understood by a person skilled in the art (e.g., "a system having at least one of A, B, or C" should include, but is not limited to, systems having A alone, having B alone, having C alone, having A and B, having A and C, having B and C, and / or having A, B, and C, etc.).

[0022] In the process of developing this application, it was discovered that carbohydrate molecules have complex structures and are mostly neutral, resulting in weak charge in solution. This leads to low ionization efficiency and insufficient detection sensitivity in mass spectrometry ion sources. Therefore, sample pretreatment techniques are needed to improve detection sensitivity. However, while commonly used carbohydrate pretreatment methods can separate carbohydrates from the matrix and increase their concentration by using specific adsorbent materials, they cannot address the inherently weak ionization ability of carbohydrates. Introducing specific groups through reactions can enhance ionization efficiency, but this increases sample complexity and can cause ion suppression due to the use of large amounts of reagents.

[0023] In view of this, this application provides a sample pretreatment method and a detection method for carbohydrates. It utilizes the hydrophilic adsorption of carbohydrates by the surface groups of graphitized carbon material in an aprotic solvent to achieve efficient enrichment. At the same time, the surface of graphitized carbon material is used as an immobilized reaction site to promote the reaction of carbohydrates with boric acid derivatizing reagents in an alkaline aprotic solvent. Purification is completed simultaneously by taking advantage of the adsorption difference between the graphitized carbon material and the excess reagent. Finally, high-purity derivatized products are obtained by elution with a high-water-content alkaline solvent and then detected. During the pretreatment process, the enrichment of carbohydrates provides a high-concentration reaction environment for derivatization. Derivatization enhances the ionization ability of carbohydrates by introducing strong ionization groups. The selective adsorption of graphitized carbon materials eliminates interference from the matrix and excess reagents. The three work synergistically to effectively solve the core problems of traditional enrichment failing to improve ionization defects and traditional derivatization being susceptible to reagent interference. This enables high-sensitivity and high-specificity mass spectrometry detection of natural carbohydrates in complex biological and food samples, providing efficient and convenient technical support for carbohydrate analysis in fields such as biomedicine, botany, microbiology research, and the food industry. It can be directly applied to rapid mass spectrometry detection or mass spectrometry imaging scenarios.

[0024] In one embodiment of this application, a sample pretreatment method for carbohydrate substances is provided, including the following steps S1 to S3.

[0025] Step S1: Contact the sample solution containing sugars with graphitized carbon material to achieve selective adsorption of sugars in the sample solution on the graphitized carbon material. After solid-liquid separation, graphitized carbon material with adsorbed sugars is obtained.

[0026] Step S2: The derivatization solution is contacted with the graphitized carbon material adsorbed with sugars. After solid-liquid separation, the graphitized carbon material adsorbed with the derivatized products is obtained. The derivatization solution is an aprotic solution including boric acid derivatization reagent.

[0027] Step S3: Contact the graphitized carbon material adsorbed with the derivative with the eluent to separate the derivative and the graphitized carbon material, and obtain an eluent containing the derivative. The eluent containing the derivative is used for mass spectrometry detection or liquid chromatography-mass spectrometry detection.

[0028] According to embodiments of this application, the sample pretreatment method for carbohydrates provided in this application significantly improves the mass spectrometry detection performance of carbohydrates in complex samples through the synergistic design of the characteristics of graphitized carbon materials and derivation reactions.

[0029] First, the hydroxyl and carboxyl groups on the surface of graphitized carbon materials can specifically bind to glycosyl groups through hydrophilic interactions, achieving efficient separation of carbohydrates from complex sample matrices (such as proteins and lipids) in an aprotic solvent environment. This enables the separation and concentration enrichment of carbohydrates from complex matrices, effectively reducing matrix interference. Furthermore, carbohydrates immobilized on the surface of graphitized carbon materials are reacted with boric acid derivatizing reagents containing strongly ionizing groups (such as tertiary amines or quaternary ammonium compounds) in an aprotic solution, directly achieving derivatization on the graphitized carbon material surface. This introduces strong ionization sites into the carbohydrate molecules, enhancing the ion signal and fundamentally improving the weak ionization ability caused by their neutral structure. Finally, utilizing the adsorption difference between derivatized products and unreacted reagents on graphitized carbon materials, in an aprotic solvent system, derivatized products can be stably adsorbed on the material surface, while excess derivatizing reagents, due to their weak adsorption capacity, can be effectively removed through solid-liquid separation. This solves the ion suppression problem caused by free reagents in traditional derivatization, resulting in a more stable and accurate mass spectrometric response of the derivatized products. By synergistically combining enrichment, immobilization derivatization, and elution processes, this method achieves efficient enrichment while mitigating the ionization defects of carbohydrates. It also avoids interference from unreacted reagents in the system, enabling highly sensitive and specific mass spectrometry detection of carbohydrates in complex samples.

[0030] Figure 1 This is a schematic diagram illustrating the principle of the sample pretreatment method of this application.

[0031] like Figure 1 As shown, the basic principle of this application is to utilize the selective trapping effect of graphitized carbon materials on sugars and sugar derivatives to achieve the enrichment of sugars on the surface of the adsorbent material, the immobilization and derivatization process of the enriched sugars on the material surface, and the rapid elution and detection of the derivatized products. Specifically, considering the high polarity and strong hydrophilicity of carbohydrates, graphitized carbon material is selected as the adsorption carrier. Its abundant hydroxyl and carboxyl groups interact specifically with the sugar groups, achieving selective enrichment of carbohydrates in aprotic solvents. Subsequently, the material adsorbed with carbohydrates is contacted with an alkaline aprotic solvent containing a boric acid derivatizing reagent. The phenylboronic acid groups in the reagent undergo dehydration condensation with the ortho-dihydroxy groups of the carbohydrates to form stable borate ester bonds, causing the carbohydrates to covalently bind to strongly ionizing groups such as tertiary amines / quaternary ammoniums, completing the immobilization and derivatization, converting neutral, poorly ionized carbohydrates into derivatized products with strong ionization sites. After the derivatization reaction, the derivatized products are stably adsorbed on the material surface due to their structural characteristics, while unreacted excess reagent is separated and removed with the solution due to its weak interaction with the material. Finally, the eluent is used to contact the material, disrupting the hydrophilic interaction between the material and the derivatized products, causing them to desorb into the eluent. The final result is an eluent containing only high-purity carbohydrate derivatized products, achieving complete separation from the sample matrix and excess reagent.

[0032] According to embodiments of this application, the solvent used in the sample solution is an aprotic solvent. Aprotic solvents can weaken the interaction between hydrophobic components (such as proteins and lipids) in the sample matrix and the graphitized carbon material, reducing non-specific adsorption. Simultaneously, they enhance the hydrophilic interaction between polar groups such as hydroxyl and carboxyl groups on the surface of the graphitized carbon material and sugar groups, thereby ensuring the selective adsorption efficiency of carbohydrates on the graphitized carbon material and achieving effective separation of carbohydrates from the complex matrix. The aprotic solvent used in the sample solution includes at least one of acetonitrile, chloroform, dimethyl sulfoxide, and tetrahydrofuran, preferably acetonitrile.

[0033] According to embodiments of this application, the sample solution further includes one of an acidic additive and a first alkaline additive. The first alkaline additive includes at least one of ammonia, ammonium carbonate, and ammonium bicarbonate; the acidic additive includes at least one of acetic acid, formic acid, and hydrochloric acid. Preferably, an acidic additive is used.

[0034] Based on the mass of the sample solution, the mass content of the acidic additive or the first basic additive is less than 10%, and the mass content can be, for example, 9%, 7%, 5%, 3%, 1%, 0.1%, etc. Preferably, based on the mass of the sample solution, the sample solution includes formic acid at a mass content of 0.1% to 1%.

[0035] According to the embodiments of this application, adding appropriate acidic or alkaline additives to the aprotic solvent used in the sample solution can adjust the microenvironment of the solvent system, enhance the hydrophilic interaction between the polar groups on the surface of the graphitized carbon material and the sugar groups, thereby improving the adsorption effect of sugars.

[0036] According to embodiments of this application, the graphitized carbon material can be selected from at least one of graphene, fullerene, carbon nanotubes, carbon nanorods, porous graphitized carbon, and mesoporous graphitized carbon. Although graphitized carbon materials with different morphologies have different structures, they all achieve hydrophilic interactions with sugar groups on their surfaces through hydroxyl and carboxyl groups, ensuring selective adsorption of sugars and thus meeting the diverse needs of sample pretreatment. In practical applications, different morphologies of graphitized carbon materials can be selected according to requirements to adapt to different sample contact methods and processing scenarios.

[0037] According to embodiments of this application, the contact method between the sample solution containing carbohydrates and the graphitized carbon material includes at least one of the following: directly dispersing the graphitized carbon material in the sample solution, passing the sample solution into a solid-phase extraction column filled with graphitized carbon material, passing the sample solution into a capillary with an inner wall coated with graphitized carbon material, and dropping the sample solution onto a glass plate modified with graphitized carbon material.

[0038] In practical applications, for example, powdered materials such as graphene and fullerene can be directly dispersed in the sample solution to directly contact sugars; porous graphitized carbon or mesoporous graphitized carbon can be filled into solid-phase extraction columns, or carbon nanotubes or carbon nanorods can be coated on capillary walls to achieve online dynamic adsorption and processing of sample solutions; graphitized carbon materials can also be modified onto glass plates to facilitate contact with the added sugar samples.

[0039] According to embodiments of this application, the aprotic solvent used in the derivatization solution includes at least one of acetonitrile, chloroform, dimethyl sulfoxide, and tetrahydrofuran. The derivatization solution also includes a second basic additive, which includes at least one of ammonia, ammonium carbonate, and ammonium bicarbonate, preferably an ammonia-ammonium carbonate buffer solution with a pH of 11. The alkaline environment promotes the efficient dehydration condensation of the phenylboronic acid groups in the boric acid derivatizing reagent with the ortho-dihydroxyl groups of the sugar groups to form stable borate ester bonds, thereby improving the efficiency and specificity of the derivatization reaction and ensuring sufficient derivatization of sugars on the graphitized carbon material surface.

[0040] Based on the volume of the derivatized solution, the volume content of the second basic additive is less than 10%, for example, it can be 9%, 7%, 5%, 3%, 1%, 0.1%, etc. An appropriate content of the second basic additive can ensure an alkaline environment to promote the derivatization reaction while avoiding the impact of excessive alkalinity or additives on the stability of the aprotic solvent system and the selective adsorption effect of graphitized carbon materials on sugar derivatives.

[0041] According to embodiments of this application, in the derivatization solution, the boric acid derivatizing reagent is a reagent containing phenylboronic acid reactive groups and organic amine groups, wherein the organic amine groups include at least one of tertiary amine groups and quaternary ammonium ion groups. The concentration of the boric acid derivatizing reagent is 10 μg / mL to 1000 μg / mL, for example, the concentration can be 10 μg / mL, 50 μg / mL, 100 μg / mL, 500 μg / mL, 1000 μg / mL, etc. An appropriate concentration of boric acid derivatizing reagent can ensure sufficient reaction with the sugars adsorbed on the surface of the graphitized carbon material to achieve efficient derivatization, while avoiding the problem that excessive reagent may be difficult to completely remove through the selective adsorption of the material due to excessive concentration. This reduces the ion suppression interference of the derivatizing reagent on the derivatized product in subsequent detection, ensuring a balance between derivatization reaction efficiency and detection accuracy.

[0042] According to embodiments of this application, the boric acid derivatizing reagent is a reagent containing a phenylboronic acid reactive group and an organic ammonium ion, wherein the organic ammonium ion includes at least one of tertiary ammonium ions and quaternary ammonium ions. The phenylboronic acid reactive group can undergo dehydration condensation with the ortho-dihydroxyl group of a sugar saccharide under alkaline conditions to form a stable boric acid ester bond, achieving derivatization labeling of carbohydrates. Tertiary ammonium ions, quaternary ammonium ions, and other organic ammonium ions possess strong basicity and good ionization ability. After being introduced into carbohydrate molecules through derivatization reactions, they can significantly enhance the charging ability and ionization efficiency of originally neutral carbohydrates in the mass spectrometry ion source. Simultaneously, as reporter ions, they can improve the signal recognition of the derivatized product in mass spectrometry detection.

[0043] According to embodiments of this application, the derivatization solution contacts the graphitized carbon material adsorbed with sugars in at least one of the following ways: directly dispersing the graphitized carbon material adsorbed with sugars in the derivatization solution; passing the derivatization solution into a solid-phase extraction column filled with graphitized carbon material adsorbed with sugars; passing the derivatization solution into a capillary with an inner wall coated with graphitized carbon material adsorbed with sugars; or dropping the derivatization solution onto a glass plate modified with graphitized carbon material adsorbed with sugars. In practical applications, an appropriate contact method can be selected according to sample processing requirements and detection scenarios. Passing the derivatization solution into a capillary with an inner wall coated with graphitized carbon material adsorbed with sugars allows the derivatization solution to flow within the capillary and fully contact the sugars adsorbed on the surface of the graphitized carbon material, enabling online dynamic immobilization derivatization. The derivatized product, after elution, can directly enter a mass spectrometry ion source, allowing for online real-time coupling with mass spectrometry detection, improving detection efficiency and continuity.

[0044] According to embodiments of this application, the eluent comprises water, a polar solvent, and a third basic additive. Based on the volume of the eluent, the water volume content is greater than 50%. This high water content can disrupt the hydrophilic interaction between the graphitized carbon material and the derivative, allowing the derivative to desorb from the surface of the graphitized carbon material and enter the eluent.

[0045] Polar solvents include at least one of methanol and acetonitrile. Polar solvents can help adjust the polarity of the eluent, enhance the solubility of the derivatized products, and promote the efficient elution of the derivatized products. The volume content of the polar solvent is 5%-50% based on the volume of the eluent, for example, 5%, 10%, 20%, 30%, 40%, 50%, etc.

[0046] The third alkaline additive includes at least one of ammonia, ammonium carbonate, and ammonium bicarbonate, preferably an ammonia-ammonium carbonate buffer solution with a pH of 11. The alkaline additive helps maintain the stability of the borate ester bonds and prevents the decomposition of the derivatives during elution.

[0047] According to embodiments of this application, the contact methods between the graphitized carbon material adsorbed with derivatives and the eluent include: directly dispersing the graphitized carbon material adsorbed with derivatives in the eluent; passing the eluent into a solid-phase extraction column filled with graphitized carbon material adsorbed with derivatives; passing the eluent into a capillary with an inner wall coated with graphitized carbon material adsorbed with derivatives; and dropping the eluent onto a glass plate modified with graphitized carbon material adsorbed with derivatives. Based on the selection of the aforementioned contact method between the derivative solution and the graphitized carbon material adsorbed with sugars, a suitable eluent contact method can be further selected to ensure the continuity and adaptability of the entire pretreatment process, and improve operational efficiency and detection results.

[0048] In an embodiment of the second aspect of this application, a method for detecting carbohydrates is provided, wherein the eluent containing derivatives obtained by the above-described sample pretreatment method for carbohydrates is injected into a mass spectrometer or liquid chromatography-mass spectrometry (LC-MS) for detection.

[0049] According to embodiments of this application, the eluent obtained by the sample pretreatment method for carbohydrates described in this application contains high-purity carbohydrate derivatives and effectively removes interference from the sample matrix and excess derivatizing reagents. Simultaneously, the strongly ionizing groups introduced by the derivatized products significantly improve ionization efficiency. Therefore, when this eluent is directly injected into mass spectrometry or liquid chromatography-mass spectrometry (LC-MS) for detection, it exhibits higher sensitivity and specificity, enabling more accurate qualitative and quantitative analysis of carbohydrates in complex samples. Furthermore, it simplifies the detection process and improves analytical efficiency.

[0050] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with specific embodiments. Unless otherwise specified, all methods described in the embodiments are conventional and can be performed according to the techniques or conditions described in the literature or the product manual.

[0051] First, in order to explore the optimal selective adsorption and derivatization conditions for glucose standards, the following selective adsorption and derivatization tests were conducted, with the specific steps as follows.

[0052] (1) Prepare glucose standard solutions in different solvents: Dissolve glucose standard samples in pure water, 90% acetonitrile-10% water, 90% acetonitrile-1% formic acid-9% water, and 90% acetonitrile-1% concentrated ammonia-9% water respectively to prepare sample solutions with a concentration of 50µg / mL in different solvents.

[0053] (2) Preparation of glucose derivatization solutions with different solvents and pH: Dissolve glucose standard samples in 30% acetonitrile-100mM ammonium carbonate buffer (pH 11), 50% acetonitrile-100mM ammonium carbonate buffer (pH 11), 90% acetonitrile-100mM ammonium carbonate buffer (pH 11), 50% acetonitrile-100mM ammonium carbonate buffer (pH 9), and 50% acetonitrile-100mM ammonium carbonate buffer (pH 10) to prepare glucose derivatization solutions with different solvents and pH of 50µg / mL.

[0054] (3) Weigh 2 mg of graphene oxide powder and disperse it in 200 µL of the above sample solutions in different solvents. Vortex for one hour to obtain a suspension. Centrifuge at 8000 rpm to obtain graphitized carbon material adsorbed with sugars. Take out the supernatant (solid phase extraction residue) separately for later use.

[0055] (4) Add 2-(dimethylamino)pyridine-5-boric acid, a derivatizing reagent with a concentration of 100 µg / mL, to 200 μL of glucose derivatization solutions in different solvents and pH values ​​respectively. Vortex at room temperature for one hour to carry out the derivatization reaction and obtain the derivatized test solution.

[0056] (5) Mass spectrometry analysis: Solid-phase extraction residues in different solvents and derivatized test solutions in different solvents and pH values ​​were injected into the mass spectrometry detection system. The mass spectrometry was an electrospray ionization source-ion trap tandem time-of-flight mass spectrometer. The ion source operated in positive and negative ion switching mode, with a voltage of 4500V applied to the spray needle, an ion source temperature of 200℃, and a flow rate of 1.5L / min for the nebulizing gas (N2). The mass analyzer was used in a single-stage mass spectrometry full scan mode, with a scan range of 100-500m / z. The sample injection volume was 2µL, and the mobile phase composition was 90% acetonitrile-water. The mobile phase was pushed into the mass spectrometer at a flow rate of 0.4mL / min for detection.

[0057] The glucose adsorption efficiency was calculated by comparing the glucose signal in the solid-phase extraction residue with the signal of the glucose standard.

[0058] Figure 2A This is a comparison of the adsorption efficiency of sugars on the surface of graphene oxide under different solvent conditions.

[0059] like Figure 2A As shown, the aprotic solvent environment affects the adsorption efficiency of glucose on the graphene oxide surface. When 90% acetonitrile is present, glucose has an adsorption efficiency of more than 80%, and the adsorption efficiency of glucose is even higher in the acidic aprotic solvent environment.

[0060] Figure 2B A comparative graph showing the effects of different solvents and pH conditions on the boric acid derivatization reaction of sugars.

[0061] like Figure 2B As shown, solvent conditions have little effect on the signal of the derivatized product, while the pH value of the solvent has a certain influence on the level of the derivatized product. Higher pH alkaline conditions are more conducive to the improvement of the derivatized product signal. This is because during the boric acid derivatization reaction, the boric acid group only forms a tetrahedral configuration under alkaline conditions, thereby reacting with the ortho-dihydroxy group to dehydrate and form a five-membered ring.

[0062] Furthermore, to investigate the adsorption behavior of derivatized products and reagents on the surface of graphene oxide under different solvent conditions, the following experiments were conducted:

[0063] (1) Prepare derivatization reagents with the same pH but different solvent contents: Dissolve glucose standard samples in 30% acetonitrile-100mM ammonium carbonate buffer (pH 11), 50% acetonitrile-100mM ammonium carbonate buffer (pH 11), 75% acetonitrile-100mM ammonium carbonate buffer (pH 11), and 90% acetonitrile-100mM ammonium carbonate buffer (pH 11) to prepare derivatization reagents with the same pH but different solvent contents with a concentration of 10µg / mL.

[0064] (2) Add 2-(dimethylamino)pyridine-5-boric acid, a derivatizing reagent with a concentration of 50 µg / mL, to 200 μL of derivatizing reagents with the same pH but different solvent contents. Vortex at room temperature for one hour to carry out the derivatization reaction and obtain the derivatized test solution.

[0065] (3) Add 2 mg of graphene oxide powder to 200 μL of derivatizing reagents with the same pH but different solvent contents, and vortex for 1 hour to fully disperse the material in the solution to obtain a suspension. Centrifuge at 8000 rpm to separate the suspensions and collect the supernatant (solid phase extraction residue) for later use.

[0066] (4) Mass spectrometry analysis: Solid-phase extraction residues with the same pH but different solvent contents and derivatized test solutions were collected and injected into the mass spectrometry detection system. The mass spectrometry was an electrospray ionization source-ion trap tandem time-of-flight mass spectrometer. The ion source operated in positive and negative ion switching mode, the voltage applied to the spray needle was 4500V, the ion source temperature was 200℃, and the flow rate of the nebulizing gas (N2) was 1.5L / min. The mass analyzer was used in a first-stage mass spectrometry full scan mode, with a scan range of 100-500m / z. The sample injection volume was 2µL, the mobile phase composition was 90% acetonitrile-water, and the mobile phase was pushed into the mass spectrometer at a flow rate of 0.4mL / min for detection.

[0067] By comparing the signals of the derivatized products and derivatized solutions with different solvent contents and the derivatized reagents that have not undergone derivatization reaction, the adsorption efficiency of the derivatized products and derivatized reagents on graphene oxide can be obtained.

[0068] Figure 3 This is a comparison of the adsorption efficiencies of sugar derivatives and derivatizing reagents on the surface of graphene oxide under different solvent ratios.

[0069] like Figure 3 As shown, the derivatized products exhibit high adsorption efficiency only when the proportion of aprotic solvent is higher than 75%, and the adsorption efficiency drops to 0 when the proportion of water exceeds 50%. The adsorption of the derivatized solvent is less affected by the proportion of aprotic solvent, maintaining an adsorption efficiency of around 50% in all solvent environments. Although the reason for the adsorption of the derivatized solvent onto graphene oxide remains to be investigated, the experimental results indicate that using 90% acetonitrile-100mM ammonium carbonate buffer for immobilization derivatization can efficiently adsorb the derivatized products and remove some of the derivatizing reagents. Furthermore, using 30% acetonitrile-100mM ammonium carbonate buffer to elute the derivatized products can effectively recover the products and remove some of the derivatizing reagents from the eluent.

[0070] In summary, in the embodiments of this application, a 90% acetonitrile-1% formic acid-9% water environment is used as the aprotic solvent for the glucose sample solution, a 90% acetonitrile-100mM ammonium carbonate buffer solution with pH 11 is used for immobilization derivatization reaction, and a 30% acetonitrile-100mM ammonium carbonate buffer solution with pH 11 is used for elution of the derivatized products.

[0071] Example:

[0072] This embodiment provides a method for detecting glucose samples with gradient concentrations (glucose enrichment-immobilization derivatization), including the following specific steps:

[0073] (1) Dissolve glucose standard samples of different concentrations in 90% acetonitrile-1% formic acid-9% water to prepare a series of glucose samples with concentration gradients of 5μg / mL, 15μg / mL, 35μg / mL and 50μg / mL.

[0074] (2) Weigh 2 mg of graphene oxide powder and disperse it in glucose samples of different concentrations in 500 µL or more. Vortex for one hour to ensure that the material is fully dispersed in the solution to obtain a suspension. Centrifuge at 8000 rpm to separate the suspension, remove the supernatant, and obtain graphene oxide adsorbed with sugars. Wash the graphene oxide three times with 90% acetonitrile-water.

[0075] (3) Add a derivatizing reagent (500 μL of 200 µg / mL 2-(dimethylamino)pyridine-5-boric acid in 90% acetonitrile-100 mM (pH 11) ammonium carbonate buffer) to the graphene oxide adsorbed with sugars. Perform the immobilization derivatization reaction by vortexing at room temperature for one hour. After the reaction is complete, centrifuge at 8000 rpm to separate the components, remove the supernatant, and obtain the graphene oxide adsorbed with the derivatized products.

[0076] (4) Add 50 μL of 30% acetonitrile-100mM (pH 11) ammonium carbonate buffer to the graphene oxide adsorbed with the derivative in the previous step, and vortex at room temperature for 1 hour to elute the derivative. After elution, centrifuge at 8000 rpm to obtain eluents of glucose samples with different concentrations.

[0077] (5) The eluents of glucose samples of different concentrations and glucose standards of different concentrations were injected into the mass spectrometry detection system for detection. The mass spectrometry was an electrospray ionization source-ion trap tandem time-of-flight mass spectrometer. The ion source operated in positive and negative ion switching mode, the voltage applied to the spray needle was 4500V, the ion source temperature was 200℃, and the flow rate of the nebulizing gas (N2) was 1.5L / min. The mass analyzer was used in a single-stage mass spectrometry full scan mode, with a scan range of 100-500m / z. The sample injection volume was 2µL, the mobile phase composition was 90% acetonitrile-water, and the mobile phase was pushed into the mass spectrometer at a flow rate of 0.4mL / min for detection.

[0078] Comparative Example

[0079] This comparative example provides a method for detecting glucose samples with gradient concentrations (direct glucose detection), including the following specific steps:

[0080] (1) Dissolve glucose standard samples of different concentrations in 90% acetonitrile-1% formic acid-9% water to prepare a series of glucose samples with concentration gradients of 5μg / mL, 15μg / mL, 35μg / mL and 50μg / mL.

[0081] (2) Take 200 μL of glucose samples with the above concentration gradient, add 100 µg / mL of 2-(dimethylamino)pyridine-5-boronic acid derivatizing reagent to each sample, and vortex at room temperature for 1 hour to carry out the derivatization reaction to obtain derivatized test solutions with different glucose concentrations.

[0082] (3) Derivative solutions of different glucose concentrations were collected and injected into a mass spectrometry detection system for detection. The mass spectrometer was an electrospray ionization source-ion trap tandem time-of-flight mass spectrometer. The ion source operated in positive and negative ion switching mode, the voltage applied to the spray needle was 4500V, the ion source temperature was 200℃, and the flow rate of the nebulizing gas (N2) was 1.5L / min. The mass analyzer was used in a single-stage mass spectrometry full scan mode, with a scan range of 100-500m / z. The sample injection volume was 2µL, the mobile phase composition was 90% acetonitrile-water, and the mobile phase was pushed into the mass spectrometer at a flow rate of 0.4mL / min for detection.

[0083] The signals of glucose standards of different concentrations obtained in the examples and comparative examples were plotted as standard curves.

[0084] Figure 4 This is a comparison graph of the standard curves for mass spectrometry detection in the embodiments and comparative examples of this application.

[0085] like Figure 4 As shown, the glucose derivatives detected by the sample detection method of this application have a good linear relationship with the glucose concentration in the concentration range of 5~50 μg / mL and good experimental repeatability. At the same time, the signal height is more than 10 times higher than that of the glucose standard directly detected in the comparative example, indicating that the sample detection method of this application has a significant improvement in the sensitivity of glucose detection.

[0086] Figure 5 This is a signal comparison diagram of the derivatives and derivatizing reagents in the embodiments and comparative examples of this application.

[0087] like Figure 5 As shown. The detection method of this application can improve the signal intensity of the derivatized product, while reducing the signal intensity of the derivatizing reagent by about 10 times, thus solving the problem of ion suppression of the derivatized product by the derivatizing reagent.

[0088] Figure 6A This is a mass spectrum of a glucose sample with a concentration of 5 μg / mL, as shown in the embodiments of this application. Figure 6B This is the mass spectrum of a glucose sample with a concentration of 5 μg / mL, used in the comparative example.

[0089] like Figure 6A and 6B As shown, in the comparative example, the glucose sample was not pretreated by enrichment-immobilization derivatization, and the glucose signal at a concentration of 5 μg / mL was almost a noise signal. However, after enrichment-immobilization derivatization of the glucose sample in this application, the signal-to-noise ratio of the glucose derivative can reach about 300, indicating that the detection method of this application has a significant improvement in the sensitivity of glucose detection.

[0090] Therefore, this application provides a sample pretreatment method and a detection method for carbohydrate substances, which have at least the following advantages compared with the prior art:

[0091] (1) The sample pretreatment method of this application can simultaneously achieve the separation, enrichment and immobilization derivatization of sugars on the surface of graphitized carbon materials, and integrate the two key steps in a synergistic manner to improve the mass spectrometry signal of sugars from both enrichment and derivatization aspects in a fast and simple way.

[0092] (2) The pretreatment method of this application can separate the unreacted excess derivatizing reagent and the sugar adsorbed on the material by means of the selective adsorption of graphitized carbon material when performing sugar derivatization on the surface of graphitized carbon material, effectively solving the problem of ion inhibition and interference caused by the presence of a large number of derivatizing reagents in the separate derivatization method.

[0093] (3) The detection method of this application can effectively control the contact time between the derivatization reagent and the sugar through the adsorption effect on the surface of graphitized carbon material and the online sample processing system. Furthermore, through selective enrichment before derivatization, the interference of the biological matrix in the sample on the derivatization reaction during the online derivatization reaction can be reduced, which provides potential for achieving more efficient online derivatization of sugars.

[0094] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of this application. It should be understood that the above descriptions are merely specific embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A sample pretreatment method for carbohydrate substances, characterized in that, Includes the following steps: A sample solution containing sugars is brought into contact with a graphitized carbon material to achieve selective adsorption of sugars in the sample solution onto the graphitized carbon material. After solid-liquid separation, the graphitized carbon material adsorbed with sugars is obtained. The derivatization solution is contacted with graphitized carbon material adsorbed with sugars, and after solid-liquid separation, graphitized carbon material adsorbed with derivatized products is obtained. The derivatization solution is an aprotic solution including boric acid derivatizing reagent. The graphitized carbon material adsorbed with the derivatives is contacted with an eluent to separate the derivatives from the graphitized carbon material, resulting in an eluent containing the derivatives. The eluent containing the derivatives is used for mass spectrometry detection or liquid chromatography-mass spectrometry detection.

2. The sample pretreatment method for carbohydrates according to claim 1, characterized in that, The boric acid derivatizing reagent is a reactive reagent containing phenylboronic acid reactive groups and organic amine groups; The organic amine group includes at least one of a tertiary amine group and a quaternary ammonium ion group; In the derivatized solution, the concentration of the boric acid derivatizing reagent is 10 μg / mL to 1000 μg / mL.

3. The sample pretreatment method for carbohydrates according to claim 1 or 2, characterized in that, The aprotic solvent used in the derivatized solution includes at least one of acetonitrile, chloroform, dimethyl sulfoxide, and tetrahydrofuran; The derivative solution also includes a second alkaline additive, which includes at least one of ammonia, ammonium carbonate, and ammonium bicarbonate, preferably an ammonia-ammonium carbonate buffer solution with a pH of 11. Based on the volume of the derivatized solution, the volume content of the second alkaline additive is less than 10%.

4. The sample pretreatment method for carbohydrates according to claim 1, characterized in that, The solvent used in the sample solution is an aprotic solvent; The aprotic solvent used in the sample solution includes at least one of acetonitrile, chloroform, dimethyl sulfoxide, and tetrahydrofuran, preferably acetonitrile.

5. The sample pretreatment method for carbohydrates according to claim 1 or 4, characterized in that, The sample solution also includes one of an acidic additive and a first alkaline additive, preferably an acidic additive; Based on the mass of the sample solution, the mass content of the acidic additive or the first basic additive is less than 10%; The first alkaline additive includes at least one of ammonia, ammonium carbonate, and ammonium bicarbonate; The acidic additive includes at least one of acetic acid, formic acid, and hydrochloric acid; Preferably, based on the mass of the sample solution, the sample solution further includes formic acid at a mass content of 0.1% to 1%.

6. The sample pretreatment method for carbohydrates according to claim 1, characterized in that, Graphitized carbon materials include at least one of graphene, fullerene, carbon nanotubes, carbon nanorods, porous graphitized carbon, and mesoporous graphitized carbon.

7. The sample pretreatment method for carbohydrates according to claim 1, characterized in that, The eluent includes water, a polar solvent, and a third basic additive.

8. The sample pretreatment method for carbohydrates according to claim 7, characterized in that, Based on the volume of the eluent, the water volume content is greater than 50%; The polar solvent includes at least one of methanol and acetonitrile; Based on the volume of the eluent, the volume content of the polar solvent is 5%-50%; The third alkaline additive includes at least one of ammonia, ammonium carbonate, and ammonium bicarbonate, preferably an ammonia-ammonium carbonate buffer solution with a pH of 11.

9. The sample pretreatment method for carbohydrates according to claim 1, characterized in that, The contact methods between the sample solution containing carbohydrates and the graphitized carbon material include at least one of the following: directly dispersing the graphitized carbon material in the sample solution, passing the sample solution into a solid-phase extraction column filled with graphitized carbon material, passing the sample solution into a capillary with an inner wall coated with graphitized carbon material, and dropping the sample solution onto a glass plate modified with graphitized carbon material. The derivatization solution can be contacted with the graphitized carbon material adsorbed with sugars by at least one of the following methods: directly dispersing the graphitized carbon material adsorbed with sugars in the derivatization solution; passing the derivatization solution into a solid-phase extraction column filled with graphitized carbon material adsorbed with sugars; passing the derivatization solution into a capillary with a coating of graphitized carbon material adsorbed with sugars on its inner wall; or dropping the derivatization solution onto a glass plate modified with graphitized carbon material adsorbed with sugars. The methods for contacting the graphitized carbon material adsorbed with the derivatives with the eluent include: directly dispersing the graphitized carbon material adsorbed with the derivatives in the eluent; passing the eluent into a solid-phase extraction column filled with graphitized carbon material adsorbed with the derivatives; passing the eluent into a capillary with a coating of graphitized carbon material adsorbed with the derivatives on its inner wall; and dropping the eluent onto a glass plate modified with graphitized carbon material adsorbed with the derivatives.

10. A method for detecting carbohydrates, characterized in that the eluent containing derivatives obtained by the sample pretreatment method for carbohydrates according to any one of claims 1 to 9 is injected into a mass spectrometer or liquid chromatography-mass spectrometry for detection.

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