A method for constructing a precise detection system for multiple categories of sugar compounds in tobacco

Abstract

This invention discloses a method for constructing a precise detection system for multiple types of carbohydrate compounds in tobacco, comprising the following steps: S1, tobacco sample pretreatment; S2, establishment of a one-dimensional liquid chromatography-evaporative light scattering detection method; S3, establishment of a center-cut two-dimensional liquid chromatography-electro-fogging detection method; S4, integration and validation of the detection system. This invention's method for constructing a precise detection system for multiple types of carbohydrate compounds in tobacco overcomes the limitations of existing standard methods that can only determine a few monosaccharides and disaccharides, and can simultaneously cover 26 carbohydrate components, including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, sugar alcohols, and sugar acids, meeting the needs of refined qualitative and quantitative analysis of tobacco and filling the technical gap in the inability to accurately determine low-abundance carbohydrates.

Description

Technical Field

[0001] This invention relates to the field of tobacco chemistry and analytical detection technology, specifically to a method for constructing a precise detection system for multiple types of carbohydrate compounds in tobacco. Background Technology

[0002] The flavor quality of tobacco products is a core indicator for cigarette formulation design, raw material evaluation, and processing technology control. Sugar compounds are key endogenous substances affecting the aroma, taste, irritation, and combustion characteristics of tobacco. Sugars account for 25%–50% of the dry weight of flue-cured tobacco leaves, encompassing various categories such as monosaccharides, disaccharides, trisaccharides, tetrasaccharides, sugar alcohols, and sugar acids. During combustion, through Maillard reactions, caramelization, and thermal decomposition, they generate aroma-producing components such as furfural, furanones, pyrazines, and pyridines, playing a crucial regulatory role in the sweetness, smoothness, and harmony of the smoke, directly impacting the sensory quality of cigarettes. Therefore, comprehensive, accurate, and high-throughput detection of various endogenous sugar compounds in tobacco is a vital technical support for raw material selection, leaf blend design, variety breeding, processing technology optimization, flavoring and additive addition, and product quality control. Currently, the tobacco industry generally adopts industry standard methods for sugar detection, mainly including YC / T159-2019 "Determination of Water-Soluble Sugars in Tobacco and Tobacco Products - Continuous Flow Method" and YC / T251-2008 "Determination of Glucose, Fructose and Sucrose in Tobacco and Tobacco Products - Ion Chromatography Method", etc.

[0003] However, the above methods are mainly suitable for routine detection of total sugars, reducing sugars, and a few common monosaccharides and disaccharides, and have the following drawbacks: 1. There are more than 20 endogenous sugar components in tobacco, but the current standard methods cover a serious lack of components, making it difficult to meet the growing demand for comprehensive quantitative and refined analysis. 2. Current methods have limited separation capabilities and are difficult to simultaneously determine structurally similar isomers. 3. Gas chromatography requires derivatization of sugars such as silanization and acylation, which is cumbersome and time-consuming. Derivatization efficiency is easily affected by matrix interference, making repeatability and stability difficult to guarantee, and it cannot directly and simultaneously determine sugars with sugar alcohols and sugar acids. 4. Although ion chromatography does not require derivatization and has high sensitivity, its detector selectivity is strong, making it difficult to simultaneously accommodate sugar alcohols, sugar acids, and oligosaccharides, and it has high operating costs and low throughput.

[0004] Therefore, there is a need to provide a method for constructing a precise detection system for multiple types of carbohydrate compounds in tobacco to meet the growing detection demands. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing a precise detection system for multiple types of sugar compounds in tobacco, in order to solve the shortcomings of existing detection technologies mentioned in the background art, such as limited component coverage, limited separation ability, and difficulty in simultaneously determining trace sugars and structurally similar isomers. These technologies cannot meet the requirements for precise analysis of more than 20 types of sugars, including ribose, mannose, galactose, rhamnose, arabinose, raffinose, stachyose, sugar alcohols, and sugar acids, with simultaneous qualitative and quantitative analysis. Furthermore, existing gas chromatography methods require derivatization of sugars through silanization, acylation, and other processes, which are cumbersome and time-consuming. The derivatization efficiency is easily affected by matrix interference, and repeatability and stability are difficult to guarantee. Moreover, it cannot simultaneously determine sugar alcohols and sugar acids. Although ion chromatography does not require derivatization and has high sensitivity, its detector selectivity is strong, making it difficult to simultaneously accommodate sugar alcohols and sugar acids. In addition, it has high operating costs and low detection throughput.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for constructing a precise detection system for multiple types of carbohydrate compounds in tobacco, comprising the following steps:

[0007] S1. Tobacco sample pretreatment: The tobacco leaves were dried at low temperature, crushed and sieved. Ethanol homogenization was combined with water bath reflux extraction. After centrifugation, the extracts were combined, concentrated and freeze-dried, reconstituted and filtered to obtain the sample solution to be tested.

[0008] S2. Establishment of a one-dimensional liquid chromatography-evaporative light scattering detection method: Using ultra-high performance liquid chromatography-evaporative light scattering detector, the detector temperature, column type and gradient elution program were optimized to establish a one-dimensional detection method suitable for the quantitative determination of multiple types of endogenous carbohydrate compounds in tobacco.

[0009] S3. Establishment of a center-cut two-dimensional liquid chromatography-electro-fogging detection method: A center-cut two-dimensional liquid chromatography system was built, and the combination of one-dimensional and two-dimensional chromatographic columns, mobile phase composition, alkaline regulator, buffer salt and column temperature conditions were optimized to establish a two-dimensional detection method suitable for the separation of trace sugars and isomers.

[0010] S4. Integration and Validation of Detection System: Methodological validation is performed using multiple categories of sugar standards to integrate qualified one-dimensional and two-dimensional detection methods into a unified detection system for qualitative and quantitative detection of multiple categories of sugar compounds in tobacco samples.

[0011] Preferably, in step S1, the tobacco sample pretreatment specifically involves: drying tobacco leaves at 40°C for 2 hours until they are easily crushed by hand, then pulverizing them and passing them through a 40-mesh sieve to obtain uniform tobacco powder; accurately weighing 0.1g of tobacco powder into a container, adding 1mL of 80% ethanol solution, and homogenizing for 10 minutes; transferring the homogenate into a 5mL ampoule, refluxing in an 80°C water bath for 3 hours, centrifuging at 12000 rpm for 10 minutes, and collecting the supernatant; repeatedly extracting the residue with 80% ethanol twice, combining the supernatants, centrifuging, concentrating, and lyophilizing, adding 500μL of ultrapure water to reconstitute, filtering through a 0.22μm microporous membrane, and storing in a chromatographic bottle at 4-8°C for later use. This precise pretreatment, employing low-temperature drying, homogenization-assisted low-temperature water bath reflux, multiple extractions, and concentration-lyophilization-reconstitution, improves sugar extraction efficiency and sample stability, reduces matrix interference, ensures the purity and component integrity of the test solution, and provides a reliable sample basis for subsequent accurate detection.

[0012] Preferably, in step S2, the temperature of the evaporation tube of the evaporative light scattering detector is optimized to 100°C, the carrier gas is nitrogen, and the carrier gas flow rate is 1.5–2.0 L / min; the chromatographic column is selected as GRACE Prevail Carbohydrate ES or ACQUITY UPLCBEH Amide; gradient elution is performed using acetonitrile-water as the mobile phase to achieve baseline separation of various sugar compounds in tobacco, optimize the detector temperature, chromatographic column and mobile phase system, improve the resolution, response intensity and peak shape symmetry of conventional sugars, and be compatible with gradient elution, thereby improving the quantitative accuracy and detection stability of one-dimensional methods for high-abundance sugars.

[0013] Preferably, in step S2, the gradient elution program is as follows: 0-5 min, acetonitrile volume fraction is maintained at 90%-95%; 5-20 min, acetonitrile volume fraction linearly decreases to 60%-70%; 20-30 min, acetonitrile volume fraction linearly decreases to 40%-50%; 30-35 min, acetonitrile volume fraction recovers to 90%-95%, completing column equilibration. The precise gradient program enables multi-component sugars to elute sequentially and achieve baseline separation, avoiding peak overlap and peak broadening, shortening analysis time, and improving the quantitative repeatability and detection efficiency of the one-dimensional method.

[0014] Preferably, in step S3, during the establishment of the center-cut two-dimensional liquid chromatography-electro-fogging detection method, the system consists of dual pumps, an autosampler, an electro-fogging detector, a dual column oven, and switching valves. The column oven is equipped with a 2-position / 6-port column switching valve and a 6-position / 7-port column switching valve. The one-dimensional chromatographic column is an Acquity BEH Amide column, and the two-dimensional chromatographic column is a Hypercarb™ PGC porous graphitized carbon column. The center-cut two-dimensional system combined with the amide column + porous graphitized carbon column improves the separation capacity, solves the problem of difficult separation of isomers, and achieves accurate resolution of trace sugars in complex matrices.

[0015] Preferably, in step S3, the mobile phase is an acetonitrile-water system, with 0.005% ammonia added as an alkaline regulator and 5mM ammonium acetate added as a volatile buffer salt. The pH of the mobile phase is adjusted to 10.5-10.9. The column temperature is set to 60℃, the one-dimensional flow rate is 0.2-0.3 mL / min, and the two-dimensional flow rate is 0.1-0.2 mL / min. The use of a low-concentration ammonia and ammonium acetate system and a high-temperature column temperature eliminates the bimodal peaks of reducing sugars, suppresses baseline drift, improves peak tailing, enhances the response stability and method robustness of the electro-fogging detector (CAD), and is suitable for long-term high-throughput detection.

[0016] Preferably, in step S4, during the integration and validation of the detection system, 26 compounds are selected as targets, including erythritol, ribose, xylose, arabinose, rhamnose, glucosamine, fructose, mannose, glucose, galactose, tagatose, inositol, sorbitol, glucuronic acid, galacturonic acid, raffinose, sucrose, cellobiose, trehalose, maltose, melibiose, isomaltose, maltotriose, mesotriose, glucopyranosyl sucrose, and stachyose. Single and mixed standard solutions are prepared, using these 26 sugars as targets to cover monosaccharides to tetrasaccharides, sugar alcohols, sugar acids, and sugar derivatives, thereby expanding the detection range and meeting the needs for refined and comprehensive characterization of tobacco sugars.

[0017] Preferably, in step S4, the linear range, limit of detection, limit of quantitation, spiked recovery rate, and precision of the one-dimensional liquid chromatography-evaporative light scattering detection method and the two-dimensional liquid chromatography-electro-fogging detection method are verified respectively to confirm that both methods meet the requirements for sugar detection in tobacco matrix. The systematic methodological verification ensures that the linearity, limit of detection, limit of quantitation, recovery rate, and precision all meet the requirements for tobacco detection, thereby improving the reliability of the method and the credibility of the data, and meeting the needs of standard testing and results transformation.

[0018] Preferably, the integrated detection system simultaneously covers monosaccharides, disaccharides, trisaccharides, tetrasaccharides, sugar alcohols, sugar derivatives, and sugar acids. It can stably detect ribose, mannose, galactose, glucuronic acid, and galacturonic acid, which are difficult to detect by evaporative light scattering detectors. It can stably detect ELSD-resistant components such as ribose, mannose, galactose, glucuronic acid, and galacturonic acid, filling the gap in existing technologies that cannot detect weakly responsive and trace sugars.

[0019] Preferably, the constructed precision detection system is suitable for high-throughput qualitative and quantitative analysis of multiple types of carbohydrate compounds in tobacco leaves and cigarette tobacco shreds of different varieties, production areas, and processing techniques. A single analysis takes 35–40 minutes and is suitable for high-throughput batch detection of multiple types of tobacco samples. It supports raw material evaluation, process optimization and quality control, and enhances the practicality and promotion value of the detection system.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] Breaking through the limitations of existing standard methods that can only determine a few monosaccharides and disaccharides, it can simultaneously cover 26 types of sugar components, including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, sugar alcohols, sugar derivatives, and sugar acids, thus meeting the needs of refined qualitative and quantitative analysis of tobacco.

[0022] It enables the stable detection of ELSD-resistant or weakly responsive components such as ribose, mannose, galactose, glucuronic acid, and galacturonic acid, filling the technological gap in the accurate determination of trace and weakly responsive sugars.

[0023] The method employs a combination of one-dimensional liquid chromatography and center-cut full two-dimensional liquid chromatography, along with complementary separation using amide columns and porous graphitized carbon columns, effectively resolving the issues of overlapping isomers and bimodal peaks due to optical rotational isomerism of reducing sugars, resulting in significant improvements in resolution and peak shape.

[0024] An integrated pretreatment process is employed, which includes low-temperature drying, ethanol homogenization followed by water bath reflux, multiple extractions and merging, concentration, freeze-drying, and reconstitution. This process ensures stable extraction efficiency, low matrix interference, and avoids component degradation and loss. Attached Figure Description

[0025] Figure 1 This is an overall flowchart of a method for constructing a precise detection system for multiple types of carbohydrate compounds in tobacco according to the present invention;

[0026] Figure 2 This is a technical roadmap for constructing a precise detection system for multiple types of carbohydrate compounds in tobacco according to the present invention;

[0027] Figure 3 This is a partial mixed standard chromatogram under drift tube conditions of 80°C and 100°C, for the method of constructing a precise detection system for multiple types of carbohydrate compounds in tobacco according to the present invention.

[0028] Figure 4 The figure shows the experimental results of column optimization for the construction of a precise detection system for multiple types of carbohydrate compounds in tobacco according to the present invention.

[0029] Figure 5 This is a single-standard stacked chromatogram of the GracePrevail sugar column portion of the present invention, which is a method for constructing a precise detection system for multiple types of sugar compounds in tobacco.

[0030] Figure 6 This is a partial mixed standard chromatogram under different gradient conditions for the method of constructing a precise detection system for multiple types of carbohydrate compounds in tobacco according to the present invention;

[0031] Figure 7This is a Prevail sugar column chromatogram of nine monosaccharides as a standard for constructing a precise detection system for multiple types of sugar compounds in tobacco according to the present invention.

[0032] Figure 8 This is a Prevail sugar column mixed standard chromatogram of a method for constructing a precise detection system for multiple types of sugar compounds in tobacco according to the present invention;

[0033] Figure 9 This is a galactose chromatogram under acidic or alkaline mobile phases for a method of constructing a precise detection system for multiple types of carbohydrate compounds in tobacco according to the present invention.

[0034] Figure 10 This is a blank chromatogram before and after rinsing, representing a method for constructing a precise detection system for multiple types of carbohydrate compounds in tobacco according to the present invention. Detailed Implementation

[0035] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0036] Please see Figure 1 -10. This invention provides a method for constructing a precise detection system for multiple types of carbohydrate compounds in tobacco. To make the objectives, technical solutions, and beneficial technical effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described in this specification are merely for explaining the invention and are not intended to limit the invention.

[0037] A method for constructing a precise detection system for multiple types of carbohydrate compounds in tobacco

[0038] S1. Tobacco Sample Pretreatment

[0039] The tobacco leaves were dried at low temperature, crushed, and sieved. Ethanol homogenization combined with water bath reflux extraction was used. The sample solution was prepared by centrifugation, combining the extracts, concentrating and freeze-drying, reconstituted, and filtered.

[0040] During the operation, the tobacco leaves were dried at 40℃ for 2 hours until they could be easily crushed by hand. After crushing, they were passed through a 40-mesh sieve to obtain uniform tobacco powder. 0.1g of tobacco powder was accurately weighed, 1mL of 80% ethanol solution was added, and 3mm stainless steel beads were added. The mixture was homogenized for 10 minutes. The homogenate was transferred to a 5mL ampoule and extracted by reflux in an 80℃ water bath for 3 hours. The supernatant was collected by centrifugation at 12000rpm for 10 minutes. The residue was extracted twice with 80% ethanol. The supernatants were combined, centrifuged, concentrated, and freeze-dried. The mixture was reconstituted with 500μL of ultrapure water, filtered through a 0.22μm microporous membrane, and stored in a chromatographic bottle at 4-8℃ for later use.

[0041] It should be noted that the low-temperature drying (40℃) used in this embodiment aims to minimize the degradation of reducing sugars or Maillard reactions caused by high temperatures; the homogenization-assisted water bath reflux combines the advantages of mechanical disruption and thermal extraction, significantly improving cell wall rupture efficiency and ensuring the full release of intracellular polysaccharides and free sugars; the three extractions combined with the supernatant followed by concentration, freeze-drying and reconstitution process effectively removes impurities such as ethanol solvent and pigments, enriches trace components, reduces interference from complex matrices, and ensures the purity and component integrity of the test solution, providing a highly stable sample basis for subsequent dual-strategy detection.

[0042] S2. Establishment of a one-dimensional liquid chromatography-evaporative light scattering detection method.

[0043] A one-dimensional detection method suitable for the quantitative determination of common sugars (high abundance) in tobacco was established by using ultra-high performance liquid chromatography-evaporative light scattering detector (UPLC-ELSD), optimizing detector temperature, column type and gradient elution program.

[0044] During operation, the temperature of the evaporation tube of the evaporative light scattering detector was optimized to 100℃, the carrier gas was nitrogen, and the carrier gas flow rate was 1.5-2.0 L / min; the chromatographic column used was GRACE Prevail Carbohydrate ES or ACQUITY UPLC BEH Amide; gradient elution was performed using acetonitrile-water as the mobile phase.

[0045] The gradient elution program is as follows: 0–5 min, acetonitrile volume fraction is maintained at 90%–95%; 5–20 min, acetonitrile volume fraction linearly decreases to 60%–70%; 20–30 min, acetonitrile volume fraction linearly decreases to 40%–50%; 30–35 min, acetonitrile volume fraction recovers to 90%–95%, and column equilibration is completed.

[0046] It should be noted that in this embodiment, an amide column was selected to utilize its hydrophilic interaction chromatography (HILIC) mechanism. The high proportion of acetonitrile environment promotes the retention and separation of sugars on the packing surface. The evaporation tube temperature of 100°C and the nitrogen flow rate of 1.5-2.0 L / min optimized the balance between nebulization and volatilization, significantly improving the response intensity and peak symmetry of ELSD for sugars without UV absorption. The precise gradient elution program uses the change of acetonitrile ratio to control the elution intensity, so that monosaccharides to tetrasaccharides with large polarity differences can be eluted sequentially and the baseline separated, avoiding peak overlap and peak broadening. While ensuring the resolution, the analysis time is compressed to within 35 minutes, improving quantitative repeatability and detection efficiency.

[0047] S3. Establishment of a center-cut, full two-dimensional liquid chromatography-electro-spray detection method.

[0048] A centrally cut two-dimensional liquid chromatography system was constructed, and the combination of one-dimensional and two-dimensional chromatographic columns, mobile phase composition, alkaline regulator, buffer salt and column temperature conditions were optimized to establish a two-dimensional detection method suitable for the separation of trace sugars and isomers.

[0049] During operation, the system consists of dual pumps, an autosampler, an electro-fogging detector (CAD), dual column ovens, and switching valves. The column ovens are equipped with 2-position / 6-port and 6-position / 7-port column switching valves. The one-dimensional chromatographic column is an Acquity BEH Amide column, and the two-dimensional chromatographic column is a Hypercarb™ PGC porous graphitized carbon column. The mobile phase is an acetonitrile-water system, with 0.005% ammonia added as an alkaline adjuster and 5mM ammonium acetate added as a volatile buffer salt. The pH of the mobile phase is adjusted to 10.5–10.9. The column temperature is set to 60℃, the one-dimensional flow rate is 0.2–0.3 mL / min, and the two-dimensional flow rate is 0.1–0.2 mL / min.

[0050] It should be noted that in this embodiment, a center-cutting mode of amide column (one-dimensional) and porous graphitized carbon column (two-dimensional) is adopted. The unique planar recognition capability of PGC column is utilized, and an alkaline system (ammonia water and ammonium acetate) is introduced and the pH is adjusted to above 10.5. This effectively suppresses the cyclic-chain tautomerism of the terminal aldehyde group of reducing sugar, eliminates the bimodal phenomenon common in reducing sugar, and improves peak tailing. The high column temperature of 60℃ reduces the viscosity of the mobile phase, further improving the response stability and system pressure resistance of the CAD detector, and adapting to the needs of long-term high-throughput detection.

[0051] S4. Integration and Validation of the Testing System

[0052] Methodological validation was conducted using multiple categories of carbohydrate standards, integrating qualified one-dimensional and two-dimensional detection methods into a unified detection system for the qualitative and quantitative detection of multiple categories of carbohydrate compounds in tobacco samples.

[0053] During the operation, 26 compounds, including erythritol, ribose, xylose, arabinose, rhamnose, glucosamine, fructose, mannose, glucose, galactose, tagatose, inositol, sorbitol, glucuronic acid, galacturonic acid, raffinose, sucrose, cellobiose, trehalose, maltose, melibiose, isomaltose, maltotriose, melitriose, glucopyranosylsucrose, and stachyose, were selected as target compounds, and single-standard and mixed-standard solutions were prepared. The linear range, limit of detection, limit of quantitation, spiked recovery rate, and precision of the one-dimensional liquid chromatography-evaporative light scattering detection method and the fully two-dimensional liquid chromatography-electro-spray detection method were validated. The integrated detection system simultaneously covers monosaccharides, disaccharides, trisaccharides, tetrasaccharides, sugar alcohols, sugar derivatives, and sugar acids, and can stably detect ribose, mannose, galactose, glucuronic acid, and galacturonic acid, which are difficult to detect with evaporative light scattering detectors. The constructed precision detection system has a single analysis cycle of 35-40 minutes and is suitable for high-throughput qualitative and quantitative analysis of multiple types of carbohydrate compounds in tobacco leaves and cigarette tobacco shreds of different varieties, production areas, and processing techniques.

[0054] It should be noted that, through systematic validation of 26 targets, a comprehensive database covering macro- and trace amounts, neutral and acidic substances, and monosaccharides and oligosaccharides was constructed in this embodiment. By leveraging the complementary advantages of ELSD for macro-sugar quantification (with broad linearity) and CAD for trace amounts, the method fills the technical gap that a single detector cannot simultaneously determine strong-response components and weak-response components (such as uronic acid). Validation shows that this method meets the analytical requirements of the complex tobacco matrix in terms of linearity, detection limit, recovery rate, and precision. A single analysis only takes 35-40 minutes, demonstrating its practicality and promotional value.

[0055] Example:

[0056] Take representative samples of flue-cured tobacco leaves, spread them evenly on a tray, and dry them in a 40℃ constant temperature oven for 2 hours until the leaves are dry and easily crumbled by hand. Remove the dried leaves and grind them thoroughly in a high-speed grinder. After grinding, sieve them through a 40-mesh standard sieve to remove incompletely ground tobacco stems and large particles, obtaining a uniformly sized and stable tobacco powder sample. Accurately weigh 0.1g of tobacco powder using a 0.01% analytical balance and transfer it to a clean centrifuge tube. Add 1mL of 80% ethanol aqueous solution to the centrifuge tube, along with a 3mm diameter stainless steel grinding bead. Fix the centrifuge tube on a high-speed homogenizer, set the homogenization time to 10 minutes, and start the homogenizer to fully disperse the tobacco powder and break down cell walls, improving the extraction efficiency of carbohydrate compounds. After homogenization, transfer all the homogenate to 5mL ampoules and seal them to prevent solvent evaporation during extraction. Then, place the ampoules in an 80℃ constant temperature water bath and continuously reflux for 3 hours. After extraction, remove the ampoule and allow it to cool to room temperature. Transfer the liquid inside to a centrifuge tube and centrifuge at 12,000 rpm for 10 minutes. After centrifugation, carefully aspirate the clear supernatant and collect it separately for later use. Add an equal volume of 80% ethanol solution to the remaining residue and repeat the reflux extraction and centrifugation steps twice. Combine all the supernatants obtained from the three extractions to ensure complete extraction of the carbohydrate components. Transfer the combined extract to a lyophilization bottle and freeze-dry it in a vacuum concentration freeze dryer at low temperature. After freeze-drying, add 500 μL of ultrapure water to the bottle and vortex thoroughly to completely reconstitute the lyophilized product. Filter the reconstituted solution using a 0.22 μm aqueous microporous membrane to remove small particulate impurities. Transfer the filtrate to a chromatographic injection bottle and store it at 4°C to 8°C for later use to prevent degradation of the carbohydrate components.

[0057] After pretreatment, initiate the one-dimensional liquid chromatography-evaporative light scattering (ELS) detection process. Correctly install the GRACE PrevailCarbohydrate ES column or ACQUITY UPLC BEH Amide column into the chromatographic system, connect the EAS detector, set the detector evaporation tube temperature to 100℃, use high-purity nitrogen as the carrier gas, control the carrier gas flow rate within the range of 1.5 L / min to 2.0 L / min, set the column temperature to 30℃, and the injection volume to 10 μL. Use an acetonitrile-water mobile phase. Edit the gradient elution program: maintain the acetonitrile volume fraction at 90% to 95% for 0 to 5 minutes, linearly decrease the acetonitrile volume fraction to 60% to 70% for 5 to 20 minutes, linearly decrease the acetonitrile volume fraction to 40% to 50% for 20 to 30 minutes, and rapidly recover the acetonitrile volume fraction to 90% to 95% for 30 to 35 minutes to fully equilibrate the column for the next sample detection. After the method is edited, the blank solvent is injected at least twice to observe whether the baseline is stable and free of interference from stray peaks. Then, single-standard solutions and mixed standard solutions are injected sequentially, and corresponding chromatograms are acquired to determine the retention times of each component and establish a standard working curve. The pretreated sample is placed into the autosampler, the detection program is started, and data acquisition is awaited. Peak integration and data recording are performed for high-abundance common sugar components such as fructose, glucose, sucrose, and maltose.

[0058] Subsequently, a center-cut two-dimensional liquid chromatography-electro-atomization detection was performed. An Acquity BEHAmide column was used for the one-dimensional chromatography, and a Hypercarb™ PGC porous graphitized carbon column was used for the two-dimensional chromatography. An acetonitrile-water mobile phase was prepared, with 0.005% ammonia added as a basicity adjuster and 5 mM ammonium acetate added as a volatile buffer. The pH of the mobile phase was adjusted to between 10.5 and 10.9 using ammonia. The column temperature was set to 60℃, the flow rate for one-dimensional chromatography was 0.2 mL / min to 0.3 mL / min, and the flow rate for two-dimensional chromatography was 0.1 mL / min to 0.2 mL / min. The electro-atomization detector was turned on, and after the signal stabilized, a blank solution was injected to check baseline drift and noise levels. The editing center employs a cutting valve switching program and a gradient elution program to cut components that are not completely separated in one dimension into two dimensions for secondary separation. This eliminates the bimodal phenomenon caused by the optical isomerism of reducing sugars and optimizes the peak shape and resolution of weakly responding components such as ribose, mannose, galactose, glucuronic acid, and galacturonic acid. After confirming the separation effect by injecting a mixed standard solution, the retention time of each component is determined, and a standard working curve is established. Then, the sample to be tested is added to start the detection, completing the separation and signal acquisition of trace sugars and isomer sugars.

[0059] Finally, the detection system was integrated and data processed. Based on the retention time of the standards, the 26 carbohydrate components in the sample were qualitatively identified one by one. The repeatability, spiked recovery rate, linear coverage, limit of detection, and limit of quantitation of the experimental data were checked and verified to confirm that all indicators met the requirements for tobacco matrix detection. The standard curve method was used to quantify the components in the sample whose peak areas were higher than the limit of quantitation, namely: conventional carbohydrates, trace carbohydrates, and weakly responsive carbohydrates. The detection data of each group were combined to generate a complete quantitative result report of multiple carbohydrate compounds in tobacco, thus completing the detection of multiple carbohydrate compounds in tobacco.

[0060] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a precise detection system for multiple types of carbohydrate compounds in tobacco, characterized in that, Includes the following steps: S1. Sample pretreatment: Pretreatment of tobacco samples to prepare the sample solution to be tested; S2. Construction of a one-dimensional liquid chromatography-evaporative light scattering detection module: Using an amide column, the parameters of the evaporative light scattering detector and the acetonitrile-water gradient elution program were optimized to establish a quantitative detection method suitable for macrocarbohydrates in tobacco. S3. Construction of a center-cut two-dimensional liquid chromatography-electro-fogging detection module: A center-cut two-dimensional system based on amide column and porous graphitized carbon column was built. Alkaline regulators and volatile buffer salts were introduced to optimize the separation environment and establish a method suitable for the separation and detection of trace sugars, sugar acids and isomers in tobacco. S4. System Integration and Validation: The two modules are validated using multiple types of carbohydrate standards. The validated modules are then integrated into a unified and accurate detection system to achieve qualitative and quantitative detection of multiple types of carbohydrate compounds in tobacco samples.

2. The construction method according to claim 1, characterized in that, The S1 step specifically includes: The tobacco leaves were dried at 40℃ until they could be easily crushed by hand, and then pulverized through a 40-mesh sieve. 0.1g of tobacco powder was accurately weighed and added to a system containing 80% ethanol solution and stainless steel beads for homogenization and crushing. The homogenate was extracted by reflux in an 80℃ water bath, centrifuged, and the supernatant was collected. The extraction was repeated twice and the supernatants were combined. The combined solution was concentrated, freeze-dried, and then reconstituted with ultrapure water. After filtration through a 0.22μm filter membrane, it was stored at 4~8℃ for later use.

3. The construction method according to claim 1, characterized in that, In step S2, the temperature of the evaporation tube of the evaporative light scattering detector is set to 100°C, the carrier gas is nitrogen, and the flow rate is 1.5~2.0 L / min; the chromatographic column is selected from GRACE PrevailCarbohydrate ES column or ACQUITY UPLC BEH Amide column.

4. The construction method according to claim 3, characterized in that, The gradient elution procedure in step S2 is as follows: From 0 to 5 minutes, the volume fraction of acetonitrile remains at 90% to 95%. Within 5–20 minutes, the volume fraction of acetonitrile decreased linearly from 90%–95% to 60%–70%. Over 20–30 minutes, the volume fraction of acetonitrile decreased linearly from 60%–70% to 40%–50%. After 30-35 minutes, the acetonitrile volume fraction recovered to 90%-95% and column equilibration was performed.

5. The construction method according to claim 1, characterized in that, In step S3, the two-dimensional system consists of dual pumps, an autosampler, an electro-fogging detector, a dual column oven, and a switching valve; the one-dimensional chromatographic column is an Acquity BEH Amide column, and the two-dimensional chromatographic column is a Hypercarb™ PGC porous graphitized carbon column; the center-cut flow path is controlled by a 2-position / 6-port valve and a 6-position / 7-port valve.

6. The construction method according to claim 5, characterized in that, In step S3, the mobile phase is an acetonitrile-water system, with 0.005% ammonia added as an alkaline regulator and 5mM ammonium acetate added as a volatile buffer salt. The pH of the mobile phase is controlled to be 10.5-10.

9. The column temperature is set to 60℃, the one-dimensional flow rate is 0.2-0.3 mL / min, and the two-dimensional flow rate is 0.1-0.2 mL / min.

7. The construction method according to claim 1, characterized in that, In step S4, the selected target standards cover monosaccharides, disaccharides, trisaccharides, tetrasaccharides, sugar alcohols, sugar derivatives, and sugar acids, totaling 26 types, including erythritol, ribose, xylose, arabinose, rhamnose, glucosamine, fructose, mannose, glucose, galactose, tagatose, inositol, sorbitol, glucuronic acid, galacturonic acid, raffinose, sucrose, cellobiose, trehalose, maltose, melibiose, isomaltose, maltotriose, pinetriose, glucopyranosyl sucrose, and stachyose.

8. The construction method according to claim 1, characterized in that, After integration, the system can detect trace components that are difficult to detect with conventional evaporative light scattering detectors, such as glucuronic acid and galacturonic acid, and the single analysis cycle is 35-40 minutes.

9. The construction method according to claim 1, characterized in that, The method is applicable to tobacco leaf and cigarette tobacco samples of different varieties, production areas and processing techniques, and can achieve high-throughput qualitative and quantitative analysis.

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