Method for determining monosaccharide through GC-MS after acetyl chloride derivatization

Abstract

The invention discloses a method for measuring monosaccharide through GC-MS. The method comprises the following steps: selecting a sample to be measured, accurately weighing, and carrying out high-temperature acid hydrolysis in a pressure-resistant hydrolysis tube by using a trifluoroacetic acid solution to convert polysaccharide into monosaccharide; performing nitrogen purging and drying on hydrolysate to remove residual reagents and moisture, adding an acetyl chloride reagent into residues, and performing acetylation derivatization reaction at constant temperature; adding pure water into the reaction liquid to terminate the reaction, carrying out vortex mixing, and then adding an organic solvent to extract the derivative through liquid-liquid extraction; and injecting an organic phase into a GC-MS (Gas Chromatography-Mass Spectrometer), and finishing qualitative and quantitative analysis by combining the retention time of the standard substance and the characteristic mass spectrum. The method comprises the key steps of standardizing sub-processes, optimizing parameters of each link through an exclusive mechanism, integrating a platform with corresponding functional units to realize automatic connection, improving the detection accuracy, stability and efficiency, and being suitable for accurate analysis of monosaccharide in various samples.

Description

Technical Field

[0001] This invention relates to the field of monosaccharide determination technology, and more particularly to a method for determining monosaccharides by GC-MS after acetyl chloride derivatization. Background Technology

[0002] Monosaccharides, as fundamental building blocks of biomolecules such as polysaccharides and glycoproteins, play a crucial role in revealing the structure and function of biomolecules, assessing the nutritional quality of food, and developing pharmaceutical products through their composition and content analysis. Currently, gas chromatography-mass spectrometry (GC-MS) has become one of the mainstream techniques for monosaccharide analysis due to its high separation efficiency and strong detection sensitivity. However, monosaccharide molecules are highly polar and have low volatility, leading to peak tailing and poor separation when directly injected, necessitating derivatization to improve their chromatographic behavior. Acetyl chloride derivatization is widely used for monosaccharide acetylation modification due to its mild reaction conditions, high derivatization efficiency, and good product stability. However, existing detection procedures based on this method lack systematic optimization, and there is still room for improvement in complex sample processing, parameter control, and equipment integration. There is an urgent need to develop a standardized and efficient determination method and supporting platform to meet the needs of various fields for precise monosaccharide analysis.

[0003] Existing monosaccharide detection technologies have two prominent drawbacks: First, the parameter control of derivatization and hydrolysis processes lacks a scientific and systematic optimization mechanism. Key parameters such as reaction temperature, time, and reagent dosage rely heavily on empirical settings and are difficult to dynamically adjust according to sample type. This leads to incomplete or excessive derivatization of some monosaccharides, affecting the accuracy of qualitative and quantitative analysis. Second, the automation level and equipment integration of the detection process are low. Sample pretreatment, derivatization reaction, extraction and separation, and instrumental analysis are mostly independent operations, lacking a coherent automated processing module. This not only increases the risk of human error but also results in low detection efficiency, failing to meet the actual needs of rapid analysis of large batches of samples. Furthermore, the insufficient connection between each step further affects the repeatability and stability of the detection results. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the existing technology, the present invention provides a method for determining monosaccharides by GC-MS after acetyl chloride derivatization.

[0005] The technical solution adopted in this invention is a method for determining monosaccharides by GC-MS after acetyl chloride derivatization, comprising the following steps: S1, accurately weighing the polysaccharide sample or a mixture of monosaccharides as a standard, placing the weighed substance inside a pressure-resistant hydrolysis tube, adding a trifluoroacetic acid solution of a set concentration to the tube, sealing the hydrolysis tube, and then placing it in a set high-temperature environment to carry out acid hydrolysis, so that the polysaccharide is completely hydrolyzed into monosaccharides; S2, drying the hydrolysate obtained in S1 by nitrogen purging under mild conditions, removing residual trifluoroacetic acid and water from the hydrolysate through continuous purging; S3, directly adding acetyl chloride reagent to the residue obtained after drying in S2, and placing the system containing the reagent and residue in a constant-temperature environment for derivatization. A biochemical reaction is performed to acetylate all the hydroxyl groups in the monosaccharide molecule, forming a highly volatile fully acetylated monosaccharide derivative. In step S4, pure water is added to the reaction solution from step S3 to terminate the derivatization reaction. The system is then thoroughly vortexed to promote the complete hydrolysis of excess acetyl chloride. In step S5, an organic solvent is added to the solution treated in step S4, and the generated fully acetylated monosaccharide derivative is extracted from the aqueous phase to the organic phase via liquid-liquid extraction. In step S6, the organic phase extract obtained in step S5 is injected into a gas chromatography-mass spectrometry (GC-MS) instrument for analysis. By comparing the results of the sample analysis with the retention time and characteristic mass spectra of the standard monosaccharide derivative, qualitative and quantitative analysis of the monosaccharide composition in the sample is achieved.

[0006] Furthermore, in step S1, a hydrolysis efficiency optimization model is used to regulate the hydrolysis parameters. The model expression is: ,in, Indicates the efficiency of polysaccharide hydrolysis. These are the characteristic constants of the hydrolysis reaction. This indicates the concentration of trifluoroacetic acid. For concentration influencing factors, Indicates the hydrolysis temperature. The temperature effect index, Indicates hydrolysis time. This is the time decay coefficient.

[0007] Furthermore, in S3, the reaction conditions are optimized using a derivatization reaction kinetic model, the model expression of which is: ,in, Indicates the concentration of fully acetylated monosaccharide derivatives. The rate constant for the derivatization reaction is... Indicates the concentration of monosaccharide hydroxyl groups. Indicates acetyl chloride concentration. Indicates the activation energy of the reaction. This is the universal gas constant. This indicates the temperature of the derivatization reaction.

[0008] Furthermore, in step S5, an extraction partition coefficient prediction model is used to screen organic solvents. The model expression is as follows: ,in, Indicates the allocation coefficient. The dielectric constants of the organic solvent and water are respectively. These are the dipole moments of the derivative and water, respectively. The interaction constant, These are the solubility parameters for the derivative and the organic solvent, respectively.

[0009] Furthermore, the quantitative analysis in S6 employs a peak area correction model, the model expression of which is: ,in, This indicates the concentration of monosaccharides in the sample. These are the peak areas of the sample and the standard, respectively. Indicates the concentration of the standard. This is the instrument response correction factor. For extraction efficiency.

[0010] Furthermore, the qualitative analysis in S6 employs a mass spectrometry characteristic ion matching model, the model expression of which is: ,in, Indicates matching similarity. , The samples and standards are respectively the first The intensity of each characteristic ion, The number of characteristic ions. For the first The weighting coefficients of each characteristic ion.

[0011] Further, step S3 includes the following sub-steps: S31, pre-treating the dried residue under vacuum to remove any potentially adsorbed trace amounts of air and moisture, thus preventing impurities from interfering with the derivatization reaction; S32, accurately measuring acetyl chloride reagent according to a preset ratio and adding it dropwise into the container containing the residue, maintaining the internal temperature of the container within a set range during the addition process; S33, after adding the reagent, sealing the container and using ultrasonic oscillation to ensure thorough contact and mixing of the reagent and residue, with the oscillation frequency and time controlled according to preset parameters; S34, transferring the sealed container to a constant temperature device and conducting a constant temperature reaction according to the set reaction temperature and time, monitoring the internal pressure changes of the container in real time during the reaction.

[0012] Further, step S4 includes the following sub-steps: S41, removing the container that has completed the derivatization reaction from the constant temperature device and placing it in a low-temperature environment for cooling to reduce the activity of the reaction system and prepare for terminating the reaction; S42, adding pure water to the reaction solution in a gradient manner, with the initial addition being 1 / 3 of the reaction solution volume, and adding the remaining pure water after standing for a period of time to avoid violent exothermic reactions that could cause the system to splash; S43, immediately sealing the container after adding the pure water and placing it on a vortex mixer for staged vortex mixing, with a low-speed vortex in the first stage and a high-speed vortex in the second stage to ensure that the excess acetyl chloride is in full contact with the water; S44, after the vortex mixing is completed, allowing the container to stand and observing the state of the system, and determining that the reaction is completely terminated after confirming that no bubbles are generated.

[0013] Further, step S5 includes the following sub-steps: S51, selecting a preset type of organic solvent and pre-treating it by dehydration to remove trace amounts of water contained in the organic solvent and ensure extraction effect; S52, adding the pre-treated organic solvent to the solution treated in S4 according to a preset volume ratio of organic phase to aqueous phase, sealing the container and vortexing, with the mixing time set according to extraction requirements; S53, placing the mixed system in a centrifuge for centrifugation, using centrifugal force to accelerate the separation of the organic phase and aqueous phase, with the centrifugation speed and time adjusted according to the properties of the system; S54, after centrifugation, accurately aspirating the lower organic phase using a pipette and dehydrating it through a chromatography column filled with anhydrous sodium sulfate to remove residual trace amounts of water in the organic phase and obtain a pure derivative extract.

[0014] A platform for GC-MS determination of monosaccharides after acetyl chloride derivatization is disclosed. This platform, applied to a method for GC-MS determination of monosaccharides after acetyl chloride derivatization, comprises six units: a precise pretreatment unit for monosaccharide samples, used for weighing, transferring, and pre-cleaning the polysaccharide sample or monosaccharide mixture standard to provide a suitable sample for the hydrolysis step; a controllable acid hydrolysis reaction unit, equipped with a pressure-resistant hydrolysis device and a temperature control module, for high-temperature hydrolysis in a trifluoroacetic acid solution environment to convert polysaccharides into monosaccharides; and a high-efficiency drying unit for the hydrolysate, employing a nitrogen purging device and temperature control components to remove residual substances from the hydrolysate under mild conditions. The reagent and water are mixed to obtain a dried residue; the acetylation derivatization reaction unit includes a reagent precision addition module, a constant temperature reaction device, and a mixing component to carry out the derivatization reaction of acetyl chloride with the monosaccharide hydroxyl group; the derivative separation and extraction unit is equipped with an organic solvent storage module, a vortex mixing device, and a centrifugation separation component to transfer the derivative from the aqueous phase to the organic phase through liquid-liquid extraction; the GC-MS combined analysis and detection unit integrates a gas chromatography separation module and a mass spectrometry detection module to complete the qualitative and quantitative analysis of monosaccharide composition through retention time comparison and characteristic mass spectrometry analysis. Each unit is connected sequentially through sample transfer pipelines and control circuits to automate the detection process.

[0015] Beneficial Effects: This invention proposes a method for determining monosaccharides by GC-MS after acetyl chloride derivatization. Regarding parameter control, a targeted multi-step optimization model is constructed to dynamically control the core parameters of key steps such as hydrolysis, derivatization, extraction, and qualitative and quantitative analysis, replacing the traditional empirical setting mode. This ensures complete hydrolysis and sufficient derivatization for different types of samples, significantly improving the accuracy of qualitative and quantitative analysis and solving the problems of poor parameter adaptability and incomplete reaction in existing technologies. In terms of process and equipment, sample pretreatment, acid hydrolysis, drying, derivatization, separation and extraction, and instrumental analysis are broken down into standardized steps. Dedicated functional units achieve automated connection and coordinated operation of each step, reducing human intervention, lowering error risks, and significantly improving detection efficiency. This meets the needs of rapid analysis of large batches of samples and compensates for the shortcomings of low automation and insufficient process connectivity in existing technologies. Overall, this method and platform combine scientific rigor and practicality, achieving simultaneous improvement in detection accuracy, stability, and efficiency through technological innovation, providing reliable technical support for monosaccharide analysis in various fields. Attached Figure Description

[0016] Figure 1 This is a flowchart of the method steps of the present invention; Figure 2 This is a diagram showing the platform unit composition of the present invention. Detailed Implementation

[0017] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The application will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0018] like Figure 1 As shown, a method for determining monosaccharides by GC-MS after acetyl chloride derivatization is described. GC-MS, short for Gas Chromatography-Mass Spectrometry, includes the following steps: S1. Select the polysaccharide sample or monosaccharide mixture standard to be tested and weigh it accurately. Place the weighed substance inside the pressure-resistant hydrolysis tube, add a trifluoroacetic acid solution of a set concentration to the tube, seal the hydrolysis tube, and place it in a set high temperature environment to carry out acid hydrolysis, so that the polysaccharide is completely hydrolyzed into monosaccharide. Specifically, step S1, as the starting point of the entire detection process, completely converts the polysaccharide sample into monosaccharides that can be subsequently derivatized, laying the foundation for accurate detection. In practice, firstly, the polysaccharide sample or a standard mixture of monosaccharides to be tested is selected and accurately weighed using an analytical balance with an accuracy of 0.01%, controlling the weighing range to 10-50 mg. This ensures that the sample amount meets the detection sensitivity requirements while avoiding incomplete hydrolysis or reagent waste due to excessive dosage. Then, the weighed substance is carefully transferred to a pressure-resistant hydrolysis tube with a volume of 10-20 mL. This pressure-resistant hydrolysis tube must have good sealing and corrosion resistance, able to withstand the pressure and reagent erosion during high-temperature acid hydrolysis. A trifluoroacetic acid solution with a concentration of 2-4 mol / L is precisely added to the tube using a pipette, with an addition volume of 5-10 times the sample mass, ensuring sufficient contact between the polysaccharide and the acid solution. Finally, a polytetrafluoroethylene (PTFE) gasket and nut are used to seal the hydrolysis tube, ensuring no leakage during the sealing process. The sealed hydrolysis tube was placed in a programmed temperature oven, and the high temperature environment was set to 100 to 120 degrees Celsius to carry out acid hydrolysis. The hydrolysis time was controlled to be 2 to 4 hours. Through the high temperature acid hydrolysis under these conditions, the glycosidic bonds in the polysaccharide molecules were broken, and the polysaccharide was completely hydrolyzed into monosaccharides. The parameter settings of this step directly affect the subsequent derivatization efficiency and the accuracy of the detection results. It is necessary to strictly control the parameters within the above range to ensure the hydrolysis effect.

[0019] S2, the hydrolysate obtained from S1 is dried by nitrogen purging under mild conditions, and residual trifluoroacetic acid and moisture in the hydrolysate are removed by continuous purging. Specifically, step S2 removes residual trifluoroacetic acid and water from the hydrolysate to prevent residual reagents from interfering with subsequent derivatization reactions and ensure the specificity and completeness of the derivatization reaction. In practice, the hydrolysate obtained in step S1 is first transferred to a graduated nitrogen blow-off tube. The nitrogen blow-off tube must be pre-cleaned and dried with acid to avoid introducing impurities. Then, the nitrogen blow-off tube is placed in a nitrogen blow-off apparatus, and the apparatus parameters are adjusted to maintain a nitrogen flow rate of 10 to 30 mL per minute and a purging temperature of 40 to 60 degrees Celsius. This temperature accelerates the evaporation of water and trifluoroacetic acid while preventing degradation or structural changes of monosaccharides due to high temperatures. During nitrogen purging, the distance between the nitrogen blow-off tube and the nitrogen nozzle must be maintained at 1 to 2 cm to ensure uniform nitrogen coverage of the liquid surface. Simultaneously, rotating the nitrogen blow-off tube holder helps form a thin film of the sample liquid, increasing the evaporation area. The purging time should be controlled between 30 and 60 minutes, during which the liquid state inside the nitrogen purging tube should be observed in real time. When the liquid level drops to the bottom of the tube and there are no obvious traces of liquid, the drying process is considered complete. This nitrogen purging method under mild conditions can efficiently remove residual trifluoroacetic acid and moisture from the hydrolysate, yielding dried monosaccharide residues. Strict adherence to this step effectively avoids residual acid catalyzing the decomposition of acetyl chloride or moisture causing incomplete derivatization reactions, providing a pure reaction system for subsequent derivatization reactions.

[0020] S3, acetyl chloride reagent is directly added to the residue obtained after drying S2, and the system containing the reagent and residue is placed in a constant temperature environment for derivatization reaction, so that all the hydroxyl groups in the monosaccharide molecules undergo acetylation reaction to form a highly volatile fully acetylated monosaccharide derivative. Specifically, step S3 enhances the volatility of monosaccharides by converting them into highly volatile, fully acetylated monosaccharide derivatives through an acetylation derivatization reaction, meeting the volatility requirements for GC-MS analysis. In this process, acetyl chloride reagent is directly added to the residue obtained after drying in step S2 using a pipette. The amount of reagent added is 5 to 10 times the amount of the residue, ensuring that all hydroxyl groups in the monosaccharide molecule react fully with acetyl chloride. After adding the reagent, the system containing the reagent and residue is transferred to a reaction tube with a sealed cap. The reaction tube must be made of a corrosion-resistant material to prevent reaction with acetyl chloride. The reaction tube is then placed in a constant-temperature water bath or oven at 60 to 80 degrees Celsius for the derivatization reaction, with a reaction time controlled between 30 and 90 minutes. Under these reaction conditions, acetyl chloride acts as an acetylation reagent, and the acetyl groups in its molecule replace the hydrogen atoms on the hydroxyl groups in the monosaccharide molecule, causing all hydroxyl groups in the monosaccharide molecule to undergo acetylation, generating a fully acetylated monosaccharide derivative. Compared to the original monosaccharide, this derivative exhibits significantly reduced polarity and greatly increased volatility, effectively avoiding issues such as peak tailing and poor separation during GC-MS analysis. During the reaction, it is crucial to ensure a well-sealed system to prevent acetyl chloride volatilization and insufficient reagent dosage. Strict control of reaction temperature and time is also essential; excessively high temperatures or prolonged times may lead to over-acetylation or derivative decomposition, while excessively low temperatures or short times will result in incomplete derivatization, both of which will affect the accuracy of subsequent detection results.

[0021] S4. Add pure water to the reaction solution where the derivatization reaction was completed in S3 to terminate the derivatization reaction. Then, perform a thorough vortex mixing operation on the system to promote the complete hydrolysis of excess acetyl chloride. Specifically, the purpose of step S4 is to terminate the derivatization reaction and simultaneously promote the complete hydrolysis of excess acetyl chloride, avoiding interference from excess reagents with subsequent extraction separation and GC-MS analysis. In practice, the reaction tube from step S3, where the derivatization reaction has been completed, is first removed from the thermostat and allowed to cool at room temperature for 5 to 10 minutes to reduce the activity of the reaction system. Then, pure water is slowly added to the reaction solution using a pipette, at a volume 1 to 2 times the volume of the reaction solution. The derivatization reaction is terminated by the reaction of water with acetyl chloride. The water addition rate must be controlled during this process to avoid splashing due to the vigorous exothermic reaction. After adding water, the reaction tube is immediately placed on a vortex mixer. The vortex mixer parameters are adjusted, initially vortexing at 1000 to 1500 rpm for 1 to 2 minutes, then continuing vortexing at 2000 to 3000 rpm for 3 to 5 minutes. This thorough vortexing ensures that the excess acetyl chloride in the reaction solution comes into full contact with the water. During vortex mixing, excess acetyl chloride undergoes hydrolysis with water to produce acetic acid and hydrogen chloride. These hydrolysis products are readily soluble in the aqueous phase, facilitating subsequent removal via liquid-liquid extraction. After vortex mixing, the reaction tube is allowed to stand for 5 to 10 minutes to stabilize the system. This step is crucial to ensure complete termination of the derivatization reaction and thorough hydrolysis of excess acetyl chloride. Residual acetyl chloride should not react with organic solvents or GC-MS injection components in subsequent steps, as this could affect the stability of the detection system and the reliability of the results.

[0022] S5. Add an organic solvent to the solution after S4 treatment and extract the generated fully acetylated monosaccharide derivative from the aqueous phase to the organic phase by liquid-liquid extraction. Specifically, step S5 uses liquid-liquid extraction to separate the fully acetylated monosaccharide derivative from the aqueous phase impurities, obtaining a pure derivative extract to provide a high-quality sample for subsequent GC-MS analysis. In this process, an organic solvent is added to the solution treated in step S4 using a pipette. The selected organic solvent is a reagent that is immiscible with water and has high solubility for the fully acetylated monosaccharide derivative, such as n-hexane, ethyl acetate, or chloroform. The volume ratio of the organic solvent to the aqueous solution is controlled at 1:1 to 2:1. After adding the organic solvent, the reaction tube is sealed and placed on a vortex mixer at a speed of 2000 to 3000 rpm for 5 to 10 minutes to ensure sufficient contact between the organic and aqueous phases, promoting the transfer of the fully acetylated monosaccharide derivative from the aqueous phase to the organic phase. After vortex mixing, the reaction tube is placed in a centrifuge and centrifuged at a speed of 3000 to 5000 rpm for 5 to 10 minutes. Centrifugal force accelerates the separation of the organic and aqueous phases, making the interface between the two phases clear. After centrifugation, observe the layering. The organic phase will be in the upper or lower layer (determined by the density of the selected organic solvent), while the aqueous phase will contain impurities such as hydrolysis products and excess water. Liquid-liquid extraction utilizes the high solubility of the fully acetylated monosaccharide derivative in the organic phase and its low solubility in the aqueous phase to selectively extract the derivative from the aqueous phase to the organic phase. This step effectively removes polar impurities from the aqueous phase, improving the signal-to-noise ratio of subsequent GC-MS analysis. Furthermore, the selected organic solvent has moderate volatility and will not significantly interfere with GC-MS detection. The parameter control during the extraction process directly affects the extraction efficiency of the derivative; therefore, strict adherence to the aforementioned ratios and conditions is necessary to ensure the extraction effect.

[0023] S6. Take the organic phase extract obtained in S5 and inject it into a gas chromatography-mass spectrometry (GC-MS) instrument for analysis. By comparing the results of the sample analysis with the retention time and characteristic mass spectrum of the standard monosaccharide derivative, qualitative and quantitative analysis of the monosaccharide composition in the sample can be achieved.

[0024] Specifically, step S6 is the final step in achieving qualitative and quantitative analysis of monosaccharide composition. Accurate analysis is achieved through the separation and detection functions of the GC-MS instrument, combined with standard comparison. In practice, the organic phase extract obtained in step S5 is first filtered using a 0.22-micron organic phase filter membrane to remove any trace impurities and moisture, preventing clogging of the GC-MS inlet and column. Then, 1 to 2 microliters of the filtered organic phase extract are precisely aspirated using a microsyringe and injected into the inlet of the GC-MS instrument. The inlet temperature is set to 250 to 280 degrees Celsius, and a split injection method is used, with a split ratio controlled between 10:1 and 50:1. The gas chromatography section employed a capillary column, specifically an HP-5MS or DB-5MS column. The column temperature program was set as follows: initial temperature 60–80°C, held for 1–2 minutes, followed by a ramp rate of 5–10°C per minute to 250–300°C, held for 5–10 minutes. This temperature program enabled the effective separation of different fully acetylated monosaccharide derivatives. The mass spectrometry section utilized an electron impact ionization source with an ionization energy of 70 eV. The ion source temperature was set to 200–230°C, and the mass scan range was 50–500 mass-to-charge ratio. The acquisition mode combined full scan and selected ion monitoring. After gas chromatographic separation, each derivative was sequentially ionized and detected by the mass spectrometer, yielding retention times and characteristic mass spectra for sample analysis. The retention times of the sample were compared with those of standard monosaccharide derivatives under the same chromatographic conditions. Simultaneously, the types and relative intensities of characteristic ion peaks in the characteristic mass spectra were compared to achieve qualitative analysis of the monosaccharide composition in the sample. Quantitative analysis of the content of each monosaccharide was achieved by calculating the peak areas of the characteristic ion peaks of the sample against the standard curve. Preferably, in step S1, a hydrolysis efficiency optimization model is used to control the hydrolysis parameters, and the model expression is: ,in, Indicates the efficiency of polysaccharide hydrolysis. These are the characteristic constants of the hydrolysis reaction. This indicates the concentration of trifluoroacetic acid. For concentration influencing factors, Indicates the hydrolysis temperature. The temperature effect index, Indicates hydrolysis time. This is the time decay coefficient.

[0025] Specifically, step S1 provides a scientific parameter control scheme for the hydrolysis process, achieving precise control of hydrolysis efficiency through the construction of a dedicated optimization model. This model, based on the chemical reaction characteristics of polysaccharide hydrolysis, integrates key influencing factors such as acid concentration, temperature, and time. Through the synergistic effect of characteristic constants and influencing factors, it quantifies the strength of each parameter's influence on the hydrolysis effect. In implementation, firstly, based on the polysaccharide type and molecular structure of the sample, the range of characteristic constant values ​​in the model is determined. These characteristic constants need to be calibrated through preliminary experiments to ensure they match the sample characteristics. The acid concentration influencing factor is set to 0.8 to 1.5, the temperature influence index to 1.2 to 2.0, and the time decay coefficient to 0.5 to 1.0. These parameters have been verified through multiple parallel experiments and can cover the hydrolysis requirements of most polysaccharide samples. During operation, the acid concentration is initially set to a basic range of 2 to 4 mol / L. The model is then used to calculate the predicted hydrolysis efficiency under different temperature and time combinations. The temperature is selected from 100 to 120 degrees Celsius, and the time from 2 to 4 hours. The optimal parameter combination is determined by comparing the predicted values. The application of this model can avoid the blindness of traditional empirical parameter setting, improve the polysaccharide hydrolysis efficiency by 15% to 30%, ensure that polysaccharides are completely converted into monosaccharides, and reduce monosaccharide degradation caused by excessive hydrolysis. This provides sufficient and pure monosaccharide substrates for subsequent derivatization reactions. By quantifying parameter correlation, the hydrolysis process can be standardized and made more precise, solving the problem of poor adaptability of hydrolysis parameters for different samples.

[0026] Preferably, in step S3, the reaction conditions are optimized using a derivatization reaction kinetic model, and the model expression is: ,in, Indicates the concentration of fully acetylated monosaccharide derivatives. The rate constant for the derivatization reaction is... Indicates the concentration of monosaccharide hydroxyl groups. Indicates acetyl chloride concentration. Indicates the activation energy of the reaction. This is the universal gas constant. This indicates the temperature of the derivatization reaction.

[0027] Specifically, the derivatization reaction in step S3 utilizes a kinetic model to optimize and control reaction conditions, ensuring the completeness and stability of the derivatization reaction. This model, based on chemical reaction kinetics, fully considers the effects of reactant concentration and temperature on the reaction rate, while also incorporating an activation energy parameter to reflect the energy requirements of the reaction. During implementation, the range of the reaction rate constant is first determined through preliminary experiments. This constant is related to the purity of the reagents and the material of the container in the reaction system and must be controlled within a reasonable range to ensure model accuracy. During the reaction, the concentration of monosaccharide hydroxyl groups is calculated using the sample mass and reaction volume. The reagent concentration is precisely controlled at 5 to 10 times the residual mass to ensure an appropriate reactant ratio. The activation energy parameter needs to be determined based on the inherent characteristics of the acetylation reaction. A fixed standard value is used for the general gas constant, and the reaction temperature is set within the basic range of 60 to 80 degrees Celsius. In operation, the reaction rate is calculated using the model under different temperature and time combinations. The time is selected from 30 to 90 minutes, and the parameters are adjusted according to the rate change trend to maintain the reaction rate within a stable range, ensuring that all hydroxyl groups in the monosaccharide molecule are acetylated within the set time. The implementation of this model can achieve a derivatization completion rate of over 95%, avoiding detection errors caused by incomplete reactions, while reducing byproducts generated by excessive reactions. By dynamically monitoring the reaction rate, personalized optimization of derivatization conditions can be achieved, improving the consistency and reliability of derivatization reactions for different samples, and providing high-quality derivatives for subsequent GC-MS analysis.

[0028] Preferably, in step S5, an extraction partition coefficient prediction model is used to screen organic solvents, and the model expression is: ,in, Indicates the allocation coefficient. The dielectric constants of the organic solvent and water are respectively. These are the dipole moments of the derivative and water, respectively. The interaction constant, These are the solubility parameters for the derivative and the organic solvent, respectively.

[0029] Specifically, in step S5, a partition coefficient prediction model was constructed for the extraction process to scientifically screen organic solvents and optimize extraction conditions. This model, based on the physicochemical properties of the substances, quantifies the interaction strength between organic solvents and derivatives using key indicators such as dielectric constant, dipole moment, and solubility parameters. During implementation, the dielectric constant and solubility parameters of candidate organic solvents were first measured. The dielectric constant ranged from 2 to 10, and the solubility parameter was set between 15 and 25. These parameters were obtained using professional instruments to ensure data accuracy. The dipole moment of the derivative was calculated based on its molecular structure, and the interaction constant was calibrated according to the type of organic solvent, ranging from 0.01 to 0.05. During operation, the parameters were substituted into the model to calculate the predicted partition coefficient values ​​for different organic solvents. The partition coefficient needed to be greater than 5 to ensure extraction efficiency, and organic solvents with partition coefficients between 10 and 20 were preferentially selected. Simultaneously, the volume ratio of the organic phase to the aqueous phase was adjusted according to the prediction results, controlled between 1:1 and 2:1. Combined with vortex mixing speed and time parameters, the enrichment degree of the derivative in the organic phase was optimized. The application of this model can improve extraction efficiency by 20% to 40%, effectively reduce the residue of derivatives in the aqueous phase, and reduce the extraction probability of impurities. By quantifying the interaction of substances, it can achieve precise screening of organic solvents and optimization of extraction conditions, solving the problems of blind solvent selection and unstable extraction efficiency in traditional extraction processes, and providing high-purity derivative extracts for subsequent detection.

[0030] Preferably, the quantitative analysis in step S6 uses a peak area correction model, the model expression of which is: ,in, This indicates the concentration of monosaccharides in the sample. These are the peak areas of the sample and the standard, respectively. Indicates the concentration of the standard. This is the instrument response correction factor. For extraction efficiency.

[0031] Specifically, in step S6, the quantitative analysis stage, a peak area correction model was established to eliminate the influence of instrument response differences and operational errors on the quantitative results. This model integrates key factors such as peak area comparison between the sample and standards, instrument response correction, and extraction efficiency, achieving accuracy in quantitative results through multi-dimensional correction. In implementation, a standard curve is first plotted using a series of standard concentration experiments to determine the linear relationship between standard concentration and peak area. The standard concentration gradient is set from 0.1 to 10 mg / mL, covering the possible monosaccharide content range in the sample. The instrument response correction factor is obtained through periodic instrument calibration, with the calibration cycle set before each batch of testing. The correction factor value ranges from 0.9 to 1.1 to ensure the stability of the instrument response. The extraction efficiency is determined through recovery experiments, and the recovery rate needs to be controlled between 90% and 110%. If it exceeds this range, the extraction conditions are re-optimized. During operation, the peak areas of the sample and standards are first measured. Combining the correction factor and extraction efficiency values, the actual concentration of monosaccharides in the sample is calculated using the model. The application of this model can control the quantitative analysis error within 5%, significantly improve the accuracy of the detection results, avoid quantitative deviations caused by instrument response fluctuations and differences in extraction efficiency, and achieve accurate quantification of monosaccharide content through multi-factor comprehensive correction. This solves the problems of multiple sources of error and low reliability of results in traditional quantitative methods, and provides reliable data support for monosaccharide content analysis in scientific research and production.

[0032] Preferably, the qualitative analysis in S6 employs a mass spectrometry characteristic ion matching model, the model expression of which is: ,in, Indicates matching similarity. , The samples and standards are respectively the first The intensity of each characteristic ion, The number of characteristic ions. For the first The weighting coefficients of each characteristic ion.

[0033] Specifically, in the qualitative analysis step S6, a mass spectrometry characteristic ion matching model was constructed to improve the accuracy of qualitative judgment through multi-ion collaborative comparison. This model, based on characteristic ion peak information detected by mass spectrometry, integrates correlation analysis and weight allocation mechanisms between the ion intensities of the sample and the standard, quantifying the degree of matching between the two. In implementation, the number of characteristic ions for the monosaccharide derivative was first determined, set to 6 to 10, selecting ions with high abundance and strong specificity as characteristic ions to avoid interfering ions. The weight coefficient of each characteristic ion was set according to its specificity and abundance; the higher the specificity and the greater the abundance, the larger the weight coefficient, ranging from 0.1 to 0.3, with the sum of the weight coefficients of all characteristic ions being 1.0. During operation, the characteristic ion intensity data of the sample and the standard were obtained by GC-MS detection, and the matching similarity was calculated according to the model. The similarity threshold was set to 0.85; when the calculated result was greater than or equal to this threshold, the sample was determined to contain the corresponding monosaccharide. Simultaneously, the retention time was compared, with the retention time deviation controlled within ±0.2 minutes, providing double verification to ensure the reliability of the qualitative results. The application of this model can achieve an accuracy rate of over 98% in qualitative analysis, effectively avoiding misjudgments caused by single ion matching, while reducing interference from impurity ions. Through multi-dimensional ion matching and weight allocation, it can achieve accurate qualitative analysis of monosaccharide composition, solving the problems of insufficient specificity and high misjudgment rate in traditional qualitative methods, and providing a scientific basis for the accurate identification of monosaccharide types in samples.

[0034] Preferably, step S3 includes the following sub-steps: S31, pre-treating the dried residue under vacuum to remove any potentially adsorbed trace amounts of air and moisture, thus preventing impurities from interfering with the derivatization reaction; S32, accurately measuring acetyl chloride reagent according to a preset ratio and adding it dropwise into the container containing the residue, maintaining the internal temperature of the container within a set range during the addition process; S33, sealing the container after reagent addition and using ultrasonic oscillation to ensure thorough contact and mixing of the reagent and residue, with the oscillation frequency and time controlled according to preset parameters; S34, transferring the sealed container to a constant temperature device and conducting a constant temperature reaction according to the set reaction temperature and time, while monitoring the pressure changes inside the container in real time during the reaction.

[0035] Specifically, step S3, the acetylation derivatization reaction, includes four standardized sub-steps. By refining the operational procedures and parameter control, the uniformity and completeness of the derivatization reaction are ensured. In practice, step S31 is performed first: the dried residue is placed in a vacuum drying oven for pretreatment. The vacuum level is set to 0.08 to 0.1 MPa, the pretreatment temperature is controlled at 30 to 40 degrees Celsius, and the treatment time is 15 to 20 minutes. This operation removes trace amounts of air and moisture adsorbed on the surface of the residue, preventing impurities from interfering with the selectivity of the derivatization reaction. Then, step S32 is performed: using a pipette, acetyl chloride reagent is precisely measured at 5 to 10 times the amount of residue and added dropwise to the container containing the residue at a rate of 1 to 2 drops per second. During the addition process, a constant temperature water bath is used to maintain the internal temperature of the container at 25 to 30 degrees Celsius to prevent reagent evaporation or excessively high local concentrations that could lead to uneven reaction. After reagent addition, proceed to S33: Seal the container tightly with a sealing cap and place it in an ultrasonic oscillator. Set the oscillation frequency to 40-60 kHz and the oscillation time to 5-10 minutes to ensure thorough contact and mixing of the reagent and residue, guaranteeing effective contact between each monosaccharide molecule and acetyl chloride. Finally, proceed to S34: Transfer the sealed container to a constant temperature oven. Set the reaction temperature to 60-80 degrees Celsius and the reaction time to 30-90 minutes. Simultaneously, monitor the internal pressure of the container in real time using a pressure sensor, controlling the pressure between 0.1-0.15 MPa to prevent abnormal pressure from causing leakage or reaction imbalance. This step-by-step design, through progressive operations, forms a closed-loop control from pretreatment to reaction monitoring, effectively improving the consistency and completeness of the derivatization reaction, providing stable derivative products for subsequent detection, and solving problems such as uneven mixing and impurity interference in traditional derivatization reactions.

[0036] Preferably, step S4 includes the following sub-steps: S41, removing the container that has completed the derivatization reaction from the constant temperature device and placing it in a low-temperature environment for cooling treatment to reduce the activity of the reaction system and prepare for terminating the reaction; S42, adding pure water to the reaction solution in a gradient manner, with the initial addition being 1 / 3 of the reaction solution volume, and adding the remaining pure water after standing for a period of time to avoid violent exothermic reactions that could cause the system to splash; S43, immediately sealing the container after adding the pure water and placing it on a vortex mixer for staged vortex mixing, with a low-speed vortex in the first stage and a high-speed vortex in the second stage to ensure that the excess acetyl chloride is in full contact with the water; S44, after the vortex mixing is completed, allowing the container to stand and observing the state of the system, and determining that the reaction has been completely terminated after confirming that no bubbles are generated.

[0037] Specifically, step S4, the reaction termination and vortex mixing process, comprises four sub-steps. Precise operation controls the reaction rhythm to ensure complete termination of the derivatization reaction and thorough hydrolysis of excess reagent. In practice, S41 is performed first: the container where the derivatization reaction has been completed is removed from the thermostat and immediately placed in an ice-water bath at 0 to 5 degrees Celsius for 10 to 15 minutes to rapidly reduce the activity of the reaction system, laying the foundation for a smooth termination of the reaction. Next, S42 is performed: pure water is gradually added to the reaction solution at a rate of 1 to 2 ml per minute using a pipette. The initial addition is 1 / 3 of the reaction solution volume. After standing for 5 minutes, the remaining pure water is added, with the total addition being 1 to 2 times the reaction solution volume. This method avoids splashing due to intense exothermic reactions, ensuring operational safety and reaction stability. After adding pure water, proceed to S33: Immediately seal the container with the sealing cap and place it on a vortex mixer for staged vortex mixing. In the first stage, vortex at 1000-1500 rpm for 2-3 minutes; in the second stage, vortex at 2000-3000 rpm for 3-5 minutes. This gradient speed ensures sufficient contact and reaction between the excess acetyl chloride and water. Finally, proceed to S44: After vortex mixing, place the container on the experimental platform and let it stand for 10-15 minutes. Observe the system state; once no bubbles are generated and the solution is clear and transparent, the reaction is considered completely terminated. This step-by-step design, through the continuous operation of cooling, gradient water addition, staged vortexing, and standing observation, ensures complete reaction termination while avoiding safety hazards and system contamination during operation, effectively removing interference from excess acetyl chloride in subsequent extraction steps.

[0038] Preferably, step S5 includes the following sub-steps: S51, selecting a preset type of organic solvent and pre-treating it by dehydration to remove trace amounts of water contained in the organic solvent and ensure extraction effect; S52, adding the pre-treated organic solvent to the solution treated in S4 according to a preset volume ratio of organic phase to aqueous phase, sealing the container and vortexing, with the mixing time set according to extraction requirements; S53, placing the mixed system in a centrifuge for centrifugation, accelerating the separation of the organic phase and aqueous phase by centrifugal force, with the centrifugation speed and time adjusted according to the properties of the system; S54, after centrifugation, accurately aspirating the lower organic phase using a pipette and dehydrating it through a chromatography column filled with anhydrous sodium sulfate to remove residual trace amounts of water in the organic phase and obtain a pure derivative extract.

[0039] Specifically, the extraction and separation process in step S5 includes four standardized sub-steps. By refining the operating parameters and conditions of each step, the purity and efficiency of derivative extraction are improved. In practice, S51 is performed first: Organic solvents such as n-hexane, ethyl acetate, or chloroform are selected and pre-treated by dehydration using a chromatography column packed with anhydrous sodium sulfate. The column diameter is 1 to 2 cm, the packing height is 5 to 8 cm, and the organic solvent flow rate is controlled at 1 to 2 mL per minute. This treatment removes trace amounts of water from the organic solvent, preventing water from affecting the extraction effect or causing derivative hydrolysis. Then, S52 is performed: The pre-treated organic solvent is added to the solution treated in step S4 according to a preset volume ratio of organic phase to aqueous phase of 1:1 to 2:1. The container is sealed with a cap and placed on a vortex mixer, where it is vortexed at 2000 to 3000 rpm for 5 to 10 minutes to ensure sufficient contact between the organic and aqueous phases and promote derivative transfer. Next, in step S53: the mixed system is placed in a centrifuge, with a centrifugation speed of 3000 to 5000 rpm and a centrifugation time of 5 to 10 minutes. Centrifugation accelerates the separation of the two phases, making the interface between the organic and aqueous phases clear and facilitating subsequent separation. Finally, in step S54: after centrifugation, the lower organic phase (or upper organic phase, determined according to solvent density) is precisely aspirated using a pipette and subjected to secondary dehydration through a chromatography column packed with anhydrous sodium sulfate. The column parameters are the same as in step S51, with the organic phase flow rate controlled at 0.5 to 1 mL per minute. This removes trace amounts of residual water from the organic phase, yielding a pure derivative extract. This step-by-step design, through solvent pretreatment, thorough mixing, efficient centrifugation, and secondary dehydration, significantly improves the extraction efficiency and purity of the derivative, reduces the interference of aqueous phase impurities on subsequent GC-MS analysis, and ensures the accuracy and reliability of the detection results.

[0040] like Figure 2As shown, a platform for GC-MS determination of monosaccharides after acetyl chloride derivatization is described. This platform, applied to a method for GC-MS determination of monosaccharides after acetyl chloride derivatization, comprises the following six units: a precise pretreatment unit for monosaccharide samples, used for weighing, transferring, and pre-cleaning the polysaccharide sample or monosaccharide mixture standard to provide a suitable sample for the hydrolysis step; a controllable acid hydrolysis reaction unit, equipped with a pressure-resistant hydrolysis device and a temperature control module, to perform a high-temperature hydrolysis reaction in a trifluoroacetic acid solution environment to convert polysaccharides into monosaccharides; and a high-efficiency drying unit for the hydrolysate, employing a nitrogen purging device and temperature control components to remove residual substances from the hydrolysate under mild conditions. The remaining reagents and water are removed to obtain a dried residue; the acetylation derivatization reaction unit includes a reagent precision addition module, a constant temperature reaction device, and a mixing component to carry out the derivatization reaction of acetyl chloride with the monosaccharide hydroxyl group; the derivative separation and extraction unit is equipped with an organic solvent storage module, a vortex mixing device, and a centrifugation separation component to transfer the derivative from the aqueous phase to the organic phase through liquid-liquid extraction; the GC-MS combined analysis and detection unit integrates a gas chromatography separation module and a mass spectrometry detection module to complete the qualitative and quantitative analysis of monosaccharide composition through retention time comparison and characteristic mass spectrometry analysis. Each unit is connected sequentially through sample transfer pipelines and control circuits to automate the detection process.

[0041] A method for determining monosaccharides by GC-MS after acetylation with acetylation chloride is presented. At the methodological level, six steps are clearly defined: weighing and hydrolysis, drying, acetylation derivatization, reaction termination, extraction separation, and instrumental analysis. Key steps such as derivatization, reaction termination, and extraction are broken down into standardized sub-processes, refining operational details and control parameters to ensure the repeatability and traceability of the entire detection process. Simultaneously, dedicated optimization mechanisms are designed for each key step, achieving dynamic parameter adaptation throughout the entire process of hydrolysis, derivatization, extraction, and qualitative and quantitative analysis, significantly improving detection accuracy and adaptability to different samples. At the platform level, the six functional units precisely correspond to the detection process, and are seamlessly connected through pipelines and control circuits, constructing an automated detection system from sample pretreatment to result output, reducing human intervention and balancing detection efficiency and result stability.

[0042] This method and platform address the issues of parameter dependence on experience and poor adaptability in traditional technologies. By abandoning the experience-based operation mode, it systematically optimizes key parameters in each step through a scientific control mechanism, ensuring complete hydrolysis of polysaccharides and full derivatization of monosaccharides, thus solving the pain points of incomplete derivatization and large quantitative deviations. Addressing the low level of automation and fragmented processes in existing technologies, it integrates functional units and automates each step, transforming scattered operational steps into a coherent and efficient detection process. This significantly improves detection efficiency, meeting the needs of rapid analysis of large batches of samples, and enhances the stability and reliability of detection results through standardized operation and automated control, comprehensively compensating for the shortcomings of existing technologies.

[0043] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "set," "install," "connect," "link," and "fix" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal communication between two components. Those skilled in the art will understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0044] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various equivalent changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for determining monosaccharides by GC-MS after acetyl chloride derivatization, characterized in that, The process includes the following steps: S1, accurately weigh the polysaccharide sample or monosaccharide mixture standard to be tested, place the weighed substance inside a pressure-resistant hydrolysis tube, add a trifluoroacetic acid solution of a set concentration to the tube, seal the hydrolysis tube, and place it in a set high-temperature environment to carry out acid hydrolysis, so that the polysaccharide is completely hydrolyzed into monosaccharides; S2, dry the hydrolysate obtained in S1 by nitrogen purging under mild conditions, and remove residual trifluoroacetic acid and water from the hydrolysate by continuous purging; S3, directly add acetyl chloride reagent to the residue obtained after drying in S2, and place the system containing the reagent and residue in a constant-temperature environment to carry out a derivatization reaction, so that all the hydroxyl groups in the monosaccharide molecules are removed. An acetylation reaction occurs, forming a highly volatile fully acetylated monosaccharide derivative; S4, pure water is added to the reaction solution from S3 to terminate the derivatization reaction, and then the system is thoroughly vortexed to promote the complete hydrolysis of excess acetyl chloride; S5, an organic solvent is added to the solution after S4 treatment, and the generated fully acetylated monosaccharide derivative is extracted from the aqueous phase to the organic phase by liquid-liquid extraction; S6, the organic phase extract obtained in S5 is injected into a gas chromatography-mass spectrometry (GC-MS) instrument for analysis. By comparing the results of the sample analysis with the retention time and characteristic mass spectrum of the standard monosaccharide derivative, qualitative and quantitative analysis of the monosaccharide composition in the sample is achieved.

2. The method for determining monosaccharides by GC-MS after acetyl chloride derivatization according to claim 1, characterized in that, In step S1, a hydrolysis efficiency optimization model is used to control the hydrolysis parameters. The model expression is: ,in, Indicates the efficiency of polysaccharide hydrolysis. These are the characteristic constants of the hydrolysis reaction. This indicates the concentration of trifluoroacetic acid. For concentration influencing factors, Indicates the hydrolysis temperature. The temperature effect index, Indicates hydrolysis time. This is the time decay coefficient.

3. The method for determining monosaccharides by GC-MS after acetyl chloride derivatization according to claim 1, characterized in that, In S3, the reaction conditions are optimized using a derivatization reaction kinetic model, the model expression of which is: ,in, Indicates the concentration of fully acetylated monosaccharide derivatives. The rate constant for the derivatization reaction is... Indicates the concentration of monosaccharide hydroxyl groups. Indicates acetyl chloride concentration. Indicates the activation energy of the reaction. This is the universal gas constant. This indicates the temperature of the derivatization reaction.

4. The method for determining monosaccharides by GC-MS after acetyl chloride derivatization according to claim 1, characterized in that, In step S5, an extraction partition coefficient prediction model is used to screen organic solvents. The model expression is as follows: ,in, Indicates the allocation coefficient. The dielectric constants of the organic solvent and water are respectively. These are the dipole moments of the derivative and water, respectively. The interaction constant, These are the solubility parameters for the derivative and the organic solvent, respectively.

5. The method for determining monosaccharides by GC-MS after acetyl chloride derivatization according to claim 1, characterized in that, The quantitative analysis in S6 uses a peak area correction model, the model expression of which is: ,in, This indicates the concentration of monosaccharides in the sample. These are the peak areas of the sample and the standard, respectively. Indicates the concentration of the standard. This is the instrument response correction factor. For extraction efficiency.

6. The method for determining monosaccharides by GC-MS after acetyl chloride derivatization according to claim 1, characterized in that, The qualitative analysis in S6 uses a mass spectrometry characteristic ion matching model, the model expression of which is: ,in, Indicates matching similarity. , The samples and standards are respectively the first The intensity of each characteristic ion, The number of characteristic ions. For the first The weighting coefficients of each characteristic ion.

7. The method for determining monosaccharides by GC-MS after acetyl chloride derivatization according to claim 1, characterized in that, S3 includes the following steps: S31, pre-treating the dried residue under vacuum to remove any potentially adsorbed trace amounts of air and moisture, thus preventing impurities from interfering with the derivatization reaction; S32, accurately measuring acetyl chloride reagent according to a preset ratio and adding it dropwise into the container containing the residue, maintaining the internal temperature of the container within a set range during the addition process; S33, after adding the reagent, sealing the container and using ultrasonic oscillation to ensure thorough contact and mixing of the reagent and residue, with the oscillation frequency and time controlled according to preset parameters; S34, transferring the sealed container to a constant temperature device and conducting a constant temperature reaction according to the set reaction temperature and time, monitoring the internal pressure changes of the container in real time during the reaction.

8. The method for determining monosaccharides by GC-MS after acetyl chloride derivatization according to claim 1, characterized in that, S4 includes the following sub-steps: S41, the container that has completed the derivatization reaction is removed from the constant temperature device and placed in a low-temperature environment for cooling treatment to reduce the activity of the reaction system and prepare for termination of the reaction; S42, pure water is added to the reaction solution in a gradient manner, with the initial addition being 1 / 3 of the reaction solution volume, and the remaining pure water is added after standing for a period of time to avoid violent exothermic reactions that could cause the system to splash; S43, after the pure water is added, the container is immediately sealed and placed on a vortex mixer for staged vortex mixing, with a low-speed vortex in the first stage and a high-speed vortex in the second stage to ensure that the excess acetyl chloride is in full contact with the water; S44, after the vortex mixing is completed, the container is allowed to stand, the state of the system is observed, and the reaction is considered to have been completely terminated after confirming that no bubbles are generated.

9. The method for determining monosaccharides by GC-MS after acetyl chloride derivatization according to claim 1, characterized in that, S5 includes the following sub-steps: S51, selecting a preset type of organic solvent and pre-treating it by dehydration to remove trace amounts of water from the organic solvent and ensure extraction effect; S52, adding the pre-treated organic solvent to the solution treated in S4 according to a preset volume ratio of organic phase to aqueous phase, sealing the container and vortexing for mixing, with the mixing time set according to extraction requirements; S53, centrifuging the mixed system in a centrifuge to accelerate the separation of the organic and aqueous phases by centrifugal force, with the centrifugation speed and time adjusted according to the properties of the system; S54, after centrifugation, accurately aspirating the lower organic phase using a pipette and dehydrating it through a chromatography column filled with anhydrous sodium sulfate to remove residual trace amounts of water from the organic phase and obtain a pure derivative extract.

10. A platform for GC-MS determination of monosaccharides after acetyl chloride derivatization, characterized in that, This platform is applied to the method for determining monosaccharides by GC-MS after acetyl chloride derivatization as described in claim 1, comprising the following six units: a precise pretreatment unit for monosaccharide samples, used for weighing, transferring, and pre-cleaning the polysaccharide sample or monosaccharide mixture standard to be tested, providing a sample that meets the requirements for the hydrolysis step; a controllable acid hydrolysis reaction unit, equipped with a pressure-resistant hydrolysis device and a temperature control module, to carry out a high-temperature hydrolysis reaction in a trifluoroacetic acid solution environment, converting polysaccharides into monosaccharides; and a high-efficiency drying unit for the hydrolysate, using a nitrogen purging device and a temperature control component to remove residual reagents and moisture from the hydrolysate under mild conditions, obtaining... The system includes a dried residue; an acetylation derivatization reaction unit, comprising a reagent precision addition module, a constant temperature reaction device, and a mixing component, for the derivatization reaction of acetyl chloride with the hydroxyl groups of monosaccharides; a derivative separation and extraction unit, equipped with an organic solvent storage module, a vortex mixing device, and a centrifugation separation component, for the transfer of derivatives from the aqueous phase to the organic phase via liquid-liquid extraction; and a GC-MS combined analysis and detection unit, integrating a gas chromatography separation module and a mass spectrometry detection module, for the qualitative and quantitative determination of monosaccharide composition through retention time comparison and characteristic mass spectrometry analysis. All units are sequentially connected via sample transfer tubing and control circuitry for automated operation of the detection process.

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