Tobacco material and a tobacco moisture isotope labeling replacement method

Abstract

The application provides a tobacco moisture isotope labeling replacement method and a tobacco raw material, relates to tobacco detection, and the method comprises the following steps: S1, drying and dehydrating the tobacco raw material; S2, in a high vacuum state, D2O or H2¹ 8 O isotope labeled water is reabsorbed to the tobacco raw material; S3, extracting the labeled moisture in the tobacco raw material, respectively, after derivative treatment of the labeled moisture and the natural water without labeling, GC-MS detection is carried out to obtain corresponding mass spectra, the characteristic ion peaks of isotopic acetic acid and acetic acid are extracted in the mass spectra, the molar ratio of isotopic acetic acid and acetic acid is obtained, the isotopic replacement rate is calculated according to the molar ratio of isotopic acetic acid and acetic acid, and the complete replacement is determined when the isotopic replacement rate reaches a preset value. The beneficial effects of the application are that the background interference caused by endogenous natural water is eliminated to the maximum extent, the endogenous original water in tobacco and the exogenous isotope labeled water can be accurately distinguished, and the technical problem that the traditional process cannot separate the contribution proportion of multiple source moisture is completely solved.

Description

Technical Field

[0001] This invention relates to the field of tobacco detection technology, specifically to a method for tobacco moisture isotope labeling and replacement, and tobacco raw materials. Background Technology

[0002] Stable isotope tracing technology, gradually established and perfected through iterative upgrades of precision analytical instruments such as mass spectrometry and nuclear magnetic resonance, is an indispensable core experimental method in the field of research on the mechanisms of material migration and transformation. Stable isotopes pose no risk of radioactive contamination and possess outstanding advantages such as high tracer response sensitivity, quantitative analysis capability, and no alteration of the physicochemical properties of the target matrix components. They are now widely used in research scenarios across multiple disciplines, including life sciences, agronomy, food science, biomedicine, ecology and environment, and functional materials. Research on stable isotope tracing in the tobacco field began in the 1940s, with early studies focusing on¹ 5 Using nitrogen isotopes as a tool, this study analyzes the absorption, transport, and endogenous metabolism of nitrogen nutrients in tobacco plants. Compared to components such as nicotine and aroma substances, research on isotope tracing technology for moisture in the tobacco matrix is ​​lagging behind, and related patent technology reserves and layout are relatively scarce.

[0003] In 2008, Wang Lei et al. published an academic paper entitled "Study on the Transfer of Moisture in Tobacco Shreds During Processing Using Isotope Tracing Method" in the *Journal of Analytical Testing*. This paper was the first to systematically establish a quantitative analysis method for moisture migration in tobacco shred processing based on D2O tracing, and constructed a fundamental technical system for tobacco moisture isotope analysis, providing important reference for research in this field. However, this process has several inherent technical shortcomings in practical applications, making it difficult to adapt to high-precision tracer analysis scenarios for novel tobacco products. Specific defects are as follows: (i) Low Moisture Replacement Efficiency, Inability to Achieve Complete Replacement: After smoking tests, the molar ratio of CH3COOD to CH3COOH in the Cambridge filter-captured products was 617:1000, resulting in a D2O replacement rate of only 61.7% relative to natural water. Nearly 40% of the labeled material still retained endogenous natural water. In subsequent tracer-based quantitative analysis, the unreplaced endogenous natural water would create strong background interference, making it impossible to accurately distinguish between endogenous native water and exogenous isotope-labeled water, severely reducing the accuracy and reliability of the tracer-based quantitative results. For high-precision research scenarios such as the thermal conversion pathway of heated cigarette moisture and the decomposition of multi-source moisture contributions, this replacement process cannot meet the requirements for quantitative analysis.

[0004] (II) Lack of systematic optimization of key process parameters and poor experimental repeatability. Although existing technologies use vacuum reabsorption to complete moisture exchange, single-factor and orthogonal optimization experiments have not been conducted on core process parameters such as reabsorption temperature, system vacuum degree, and equilibrium time. The influence of each parameter on the displacement efficiency and the optimal threshold are not clearly defined. This literature does not disclose key operating indicators such as reabsorption temperature and ultimate vacuum degree. Different researchers using different operating conditions will cause significant fluctuations in displacement efficiency, making it difficult to guarantee the parallelism and reproducibility of experimental data. Regarding the core variable of reabsorption temperature, existing public data lack comparative experiments on displacement effects at gradient temperatures of 40℃, 50℃, and 60℃, and the optimal temperature window that can achieve 100% complete displacement has not been identified.

[0005] (III) Narrow Applicability and Insufficient Universality: The existing method only uses traditional tobacco shreds as the verification object and does not examine the adaptability of the process to the replacement effect of multiple types of tobacco raw materials. In addition to conventional tobacco shreds, the tobacco matrix used in heated cigarettes includes four types of reconstituted tobacco sheets produced by the slurry method, roll pressing method, dry method, and papermaking method. Sheets obtained from different preparation processes show significant differences in fiber density, internal pore structure, and adsorption capacity of hydrophilic humectants, and their adsorption and exchange kinetics for isotope-labeled water differ significantly. The existing technology has not formed a standardized adjustment scheme adapted to multiple types of tobacco raw materials, thus limiting its application scenarios.

[0006] (iv) The verification system for complete displacement is incomplete, and quantitative correction is lacking. Existing derivatization-gas chromatography-mass spectrometry detection methods can only output the molar ratio of D2O to H2O, without defining a quantitative technical standard for determining "complete displacement." This literature only reports a molar ratio of 617:1000, without conducting process optimization research to improve the displacement rate, nor establishing a quantitative evaluation system for "complete displacement." Furthermore, the proton exchange side reaction during the mass spectrometry ionization stage causes a shift in the molecular mass of acetic acid, resulting in a higher background value of the natural isotope in the blank sample (D2O to H2O molar ratio of approximately 2:1000). Existing technology lacks a corresponding blank correction method, making it impossible to eliminate this interference.

[0007] The aforementioned multiple technical defects combined make it impossible for existing processes to achieve comprehensive isotopic replacement of the endogenous natural water in tobacco raw materials, making it difficult to support the high-precision tracer analysis work required for moisture tracing, migration law analysis, and quantitative research on thermochemical reaction mechanisms in new tobacco products such as heated cigarettes. Summary of the Invention

[0008] To address the shortcomings of existing technologies, such as low replacement rate, ambiguous process parameters, poor raw material compatibility, and lack of verification and calibration systems, this invention provides a tobacco moisture isotope labeling replacement method and tobacco raw materials, achieving complete replacement of free water and bound water in tobacco raw materials with D2O or H2¹. 8The O isotope-labeled water completely replaces the tobacco, with a replacement rate of ≥99%. The method is reproducible and adaptable to all types of tobacco raw materials. It is equipped with a complete blank correction quantitative verification system, which meets the requirements for high-precision tracer analysis of moisture migration and thermochemical reaction mechanisms in heated cigarettes.

[0009] This invention provides a method for tobacco water isotope labeling and replacement, comprising: Step S1: Drying and dehydrating tobacco raw materials; Step S2: The tobacco raw material is subjected to D2O or H2¹ under high vacuum. 8 O isotope-labeled water reabsorption allows the labeled water to penetrate evenly and occupy all sites of the original moisture in the tobacco raw material; Step S3: Extract labeled water from tobacco raw materials. After derivatization treatment of labeled water and unlabeled natural water, perform GC-MS detection to obtain the corresponding mass spectra. Extract the characteristic ion peaks of isotopic substituted acetic acid and acetic acid from the mass spectra. Calculate the molar ratio of isotopic substituted acetic acid and acetic acid by the ratio of their peak areas or response values. Calculate the isotopic replacement rate based on the molar ratio of isotopic substituted acetic acid and acetic acid. When the isotopic replacement rate reaches a preset value, it is determined to be a complete replacement.

[0010] Furthermore, in step S2, the isotope-labeled water is D2O, and in step S3, the isotope-labeled acetic acid is deuterated acetic acid.

[0011] Furthermore, in step S2, the isotope-labeled water is H2¹ 8 O, in step S3, the isotopic substituted acetic acid is containing ¹ 8 O-acetic acid.

[0012] Furthermore, the formula for calculating the isotope substitution rate in step S3 is as follows: Replacement rate = [n(X / H2O) / (1+n(X / H2O))]×100%; Among them, n(X / H2O) is the molar ratio of isotopic substituted acetic acid and acetic acid.

[0013] Furthermore, the preset value in step S3 is 99% to 100%.

[0014] Further, the derivatization process in step S3 is as follows: labeled water and unlabeled natural water are placed in reaction flasks respectively, excess acetic anhydride is added, and after sealing and complete reaction at room temperature, the labeled water reacts with acetic anhydride to generate isotopic substituted acetic acid, and the natural water reacts with acetic anhydride to generate acetic acid.

[0015] Further, step S2 includes: S201. Place the dried and dehydrated tobacco raw material into a vacuum pipeline device; S202. Evacuate the vacuum pipeline device; S203. Tobacco raw material water bath in vacuum pipeline device, and isotope-labeled water injected into vacuum pipeline device; S204. Maintain a constant water bath temperature and allow the tobacco raw material to fully contact and absorb isotope-labeled water under vacuum conditions.

[0016] Furthermore, in step S202, the vacuum pipeline device is evacuated to reduce the vacuum level inside the vacuum pipeline device to below 0.0978 MPa. In step S203, the water bath temperature is 50°C; Step S204, absorption time is 24h.

[0017] Furthermore, it also includes step S4: take unlabeled blank tobacco raw material, calculate the natural isotope replacement rate corresponding to the unlabeled blank tobacco raw material according to the detection method in step S3, and calculate the final replacement rate after deducting the natural isotope replacement rate from the isotope replacement rate.

[0018] Furthermore, embodiments of the present invention also provide a tobacco raw material, which uses the above-described tobacco moisture isotope labeling replacement method for moisture replacement.

[0019] The present invention has the following beneficial effects: 1. This invention provides a method for isotope-labeled water replacement in tobacco and tobacco raw materials. First, free and bound water inside the tobacco are thoroughly removed through vacuum drying. Then, under optimized high vacuum and constant temperature conditions, uniform permeation and exchange of isotope-labeled water are completed, allowing the labeled water to fully occupy all original water-binding sites in the tobacco fibers, micropores, and intercellular spaces. A matching derivatization-gas chromatography-mass spectrometry quantitative detection scheme is provided, specifying that a molar ratio of D2O to natural H2O ≥ 990:1000 (replacement rate ≥ 99%) is used as the criterion for complete replacement. This is the optimal process. Under certain conditions, the replacement rate of tobacco samples can reach 100%, achieving almost complete isotopic replacement of water and significantly improving the accuracy of tracing and quantification. Compared with the existing literature methods with a replacement rate of only 61.7%, this invention can eliminate background interference from endogenous natural water to the greatest extent, accurately distinguish between endogenous native water and exogenous isotopically labeled water in tobacco, and completely solve the technical problem that traditional processes cannot separate the contribution ratio of multiple water sources. This provides reliable technical support for high-precision scientific research such as the analysis of water migration paths in heated cigarettes, the mechanism of water participation in thermochemical reactions, and quantitative tracing of multiple water sources.

[0020] 2. This invention provides a method for tobacco moisture isotope labeling and replacement, and tobacco raw materials. It conducts gradient optimization experiments on key influencing factors of vacuum reabsorption, clearly defining the core process window for achieving efficient and complete replacement: a preferred reabsorption constant temperature of 50℃, an absolute vacuum degree below 0.0978 MPa, and a standard material reabsorption equilibration time of 24 hours. For reconstituted tobacco leaves produced by the dense papermaking method, it simultaneously provides adaptive adjustment schemes for extending drying and reabsorption time. Existing technologies do not disclose key thresholds such as temperature, vacuum degree, and equilibration time, resulting in significant fluctuations in experimental results among different operators and laboratories. This invention provides a complete and reproducible set of standardized operating parameters. The entire set of process parameters is standardized and controllable, significantly enhancing experimental repeatability and parallelism, eliminating systematic errors caused by human operation, ensuring stable and consistent replacement effects across different batches and operators, greatly improving the reliability of the methodology, and facilitating standardized promotion and use within the industry.

[0021] 3. This invention provides a method for tobacco moisture isotope labeling and replacement, and tobacco raw materials. It constructs an integrated verification scheme of "vacuum low-temperature water extraction - acetic anhydride derivatization - GC-MS quantitative detection - blank background subtraction". It sets up a blank correction process to address the natural isotope background interference caused by proton exchange during the mass spectrometry ionization stage, effectively eliminating the systematic error of natural deuterium abundance at 2:1000. It is equipped with a complete quantitative verification and background correction system, and the detection results are objective and reliable. At the same time, it clearly defines the quantitative judgment index of "complete replacement", abandoning the crude detection mode of existing technologies that lack a unified evaluation standard and only output molar ratio data. It realizes quantitative, objective and repeatable evaluation of replacement effect and ensures the accuracy of tracer analysis data.

[0022] 4. The tobacco moisture isotope labeling replacement method and tobacco raw materials provided by this invention use a 50°C low-temperature vacuum environment to complete the dehydration and re-smoking operations. There are no destructive treatment methods such as high temperature, high pressure, and strong chemical reagent modification, which will not damage the microstructure of tobacco fiber network, endogenous aroma substances, moisturizing components and various functional additives. The tobacco samples after isotope labeling can be directly used for subsequent smoking tests, pyrolysis kinetics tests, moisture migration dynamic tracking and other continuous scientific research experiments without the need for additional pretreatment repair matrix. The experimental process is simplified and the sample utilization rate is higher.

[0023] 5. This invention provides a method for tobacco moisture isotope labeling and replacement, as well as tobacco raw materials. The process is adaptable to five major categories of mainstream tobacco raw materials for heated cigarettes: traditional tobacco shreds, slurry method, roll pressing method, dry method, and papermaking method reconstituted tobacco sheets. For papermaking sheets with dense fibers and low porosity, a high replacement rate of ≥99% can be achieved by finely adjusting the drying and re-inhalation time. Existing technologies only verify single tobacco shred samples and do not consider the differences in preparation processes, pore structures, fiber binding states, and humectant adsorption capabilities of different reconstituted tobacco leaves. There is no universally applicable solution. The raw materials of this invention have strong universality, covering all types of tobacco matrices in heated cigarettes, and can meet the moisture isotope labeling requirements of various new tobacco matrices, with a wider range of application scenarios. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a flowchart of a tobacco moisture isotope labeling and replacement method according to the present invention.

[0026] Figure 2 This is a schematic diagram of a vacuum pipeline device; Figure 3 This is a picture of a vacuum pipeline device.

[0027] In the picture: 1. Glass tube; 2. Sample round-bottom flask, 3. Buffer bottle; 4. Collection bottle; 5. Rotary vane vacuum pump; 6. Vacuum pressure gauge; 7. First piston; 8. Second piston; 9. Third piston; 10. Fourth piston. Detailed Implementation

[0028] The detailed features and advantages of this application are described below in the specific embodiments. The content of this description is sufficient to enable any person skilled in the art to understand the technical content of this application and implement it accordingly. Based on the specification, claims and drawings disclosed in this specification, a person skilled in the art can easily understand the related objectives and advantages of this application.

[0029] The invention will now be described with reference to the accompanying drawings, in which similar reference numerals denote similar elements. While specific structures and arrangements are discussed, it should be understood that this is done merely for illustrative purposes. Those skilled in the art will recognize that other structures and arrangements can be used without departing from the spirit and scope of the invention. It will be apparent to those skilled in the art that the invention can also be used in a variety of other applications.

[0030] In this specification and claims, several terms will be used, and unless otherwise indicated, these terms will be defined to have the following meanings: The singular forms “a” and “” include their corresponding plural forms. “At least one” means one or more, and “more” means two or more. “At least one of the following” or similar expressions refer to any combination of these items, including any combination of single or plural items. For example, at least one of a, b, or c can be expressed as: a, b, c, ab, ac, bc, or abc, where a, b, and c can be single or multiple.

[0031] All figures used to represent component amounts, properties (e.g., molecular weight), reaction conditions, etc., should be considered to be modified in all cases by the terms "within the unavoidable margin of error" or "about". Therefore, the numerical values ​​set forth herein are approximate and may vary depending on the desired properties sought to be obtained by the present invention. The principles of equivalents, which are applied to a minimum and not intended to limit the scope of the claims, should be applied, for example, each value should be interpreted at least according to the reported significant digits and by applying conventional rounding techniques.

[0032] It should be understood that the term "and / or" in this article is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. Additionally, the character " / " in this article generally indicates an "or" relationship between the preceding and following related objects, but it can also represent an "and / or" relationship. Please refer to the context for a more accurate understanding.

[0033] In the description of this embodiment, it should be noted that the terms "upper", "lower", "inner", "bottom", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship in which the product is usually placed during use. They are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application.

[0034] Unless otherwise indicated, the following abbreviations have the following meanings, and any other abbreviations used herein but not defined have their generally accepted standard meanings: All other terms used herein for special definition are intended to have the general meaning understood by one of ordinary skill in the art, in particular meaning that one of ordinary skill in the art can directly and without doubt determine how the technical solutions of the invention can be implemented after reading the claims, specification and drawings.

[0035] Even if there are incomplete descriptions, omissions, or ambiguities in the grammar, words, punctuation, graphics, symbols, etc. of the claims, specification, and drawings of this patent, a person skilled in the art can still arrive at the only correct understanding by reading the claims, specification, and drawings as a whole without extensive reasoning or experimentation, and effectively exclude various incorrect interpretations that are not aimed at achieving the purpose of this patent.

[0036] Those skilled in the art would first choose to read the claims, specification, and drawings of this patent to reasonably interpret the terms; secondly, they would choose to refer to the relevant definitions in other documents published by the applicant before the filing date to reasonably interpret the terms; thirdly, they would choose the references cited in this patent to reasonably interpret the terms; and finally, they would choose to combine the technical dictionaries, technical manuals, reference books, textbooks, national or industry technical standards, etc., commonly used by those skilled in the art to reasonably interpret the terms.

[0037] To make the objectives, technical solutions, and advantages of this application clearer, the following will describe in detail its implementation methods in conjunction with the specific structure and working process of the device of the present invention.

[0038] Please refer to Figure 1 This invention provides a method for isotope labeling and replacement of moisture in tobacco. Through a complete process including deep vacuum dehydration, high-vacuum isothermal isotope-labeled water reabsorption, vacuum low-temperature water extraction, acetic anhydride derivatization-GC-MS quantitative detection, and blank background correction, it achieves complete isotope replacement of endogenous natural moisture in various tobacco raw materials. This method mainly includes the following steps S1 to S3.

[0039] Step S1: Drying and dehydrating the tobacco raw material. Specifically, this includes: S101. Select the corresponding category of tobacco raw materials: Tobacco raw materials include, but are not limited to, one or more of the following: traditional shredded tobacco, slurry-process reconstituted tobacco sheets, roll-pressed reconstituted tobacco sheets, dry-process reconstituted tobacco sheets, and paper-process reconstituted tobacco sheets. Tobacco raw materials may be in the form of shreds, flakes, or granules.

[0040] S102, Vacuum drying treatment: The tobacco raw materials are laid flat in a vacuum drying oven at a temperature of 40-60℃. Phosphorus pentoxide (P2O5) is placed inside the oven as a water vapor adsorbent to absorb water vapor escaping from the tobacco raw materials. The desiccant is replaced every 2-4 hours during the drying process to continuously reduce the water vapor pressure of the system and accelerate the dehydration process. The preferred drying temperature is 50℃.

[0041] S103. Determination of the dehydration endpoint: Constant weight is achieved when the difference between two consecutive weighings of the tobacco raw material is less than 0.1%. At this point, the free and bound water in the tobacco raw material has been largely removed, and the residual moisture content of the dried tobacco raw material is less than 0.5%. Record the mass of the dehydrated tobacco raw material for calculating the amount of labeled water to be added during subsequent resmoking.

[0042] Step S2: The tobacco raw material is subjected to D2O or H2¹ under high vacuum. 8 O isotope-labeled water reabsorption allows the labeled water to evenly penetrate and occupy all sites of the original moisture in the tobacco raw material.

[0043] Adopting such Figure 2 The vacuum pipeline device shown completes the re-inhalation of dried tobacco raw materials. The vacuum pipeline device includes a glass tube 1, a sample round-bottom flask 2, a buffer bottle 3, a collection bottle 4, four-way control pistons, a rotary vane vacuum pump 5, a vacuum pressure gauge 6, and a water bath temperature control system. One end of the glass tube 1 is connected to the rotary vane vacuum pump 5, and the other end is connected to the vacuum pressure gauge 6. The sample round-bottom flask 2, buffer bottle 3, and collection bottle 4 are each connected to the middle of the glass tube 1. The four-way control pistons are designated as first piston 7, second piston 8, third piston 9, and fourth piston 10. First piston 7, second piston 8, and third piston 9 are respectively located at the connections between the sample round-bottom flask 2, buffer bottle 3, and collection bottle 4 and the glass tube 1, while fourth piston 10 is located at the connection between the rotary vane vacuum pump 5 and the glass tube 1. The collection bottle 4 is preferably a U-shaped tube, which provides better low-temperature moisture capture.

[0044] Step S2 specifically includes: S201. Place the dried and dehydrated tobacco raw material into the vacuum pipeline device, and then pack the dried tobacco raw material into the sample round bottom flask 2.

[0045] S202. Evacuate the vacuum pipeline device.

[0046] Close the second piston 8 and the third piston 9, open the first piston 7 and the fourth piston 10, and start the rotary vane vacuum pump 5 to evacuate the glass tube 1 to an absolute pressure of <0.0978MPa.

[0047] S203. Tobacco raw material water bath in vacuum pipeline device, and inject isotope-labeled water into vacuum pipeline device.

[0048] Close the fourth piston 10 and stop the rotary vane vacuum pump 5. Place the sample round-bottom flask 2 in the water bath heating system. The water bath heating system heats the tobacco raw material in the sample round-bottom flask 2 at a constant temperature of 45~55℃, preferably 50℃. Inject isotope-labeled water of equal volume to the natural water removed from the raw material through the septum inlet. The labeled water is D2O or H2¹. 8 O.

[0049] The volume of injected isotope-labeled water is calculated based on the initial moisture content of the tobacco raw material and the mass loss after drying, and is usually equal to the volume of natural moisture in the tobacco raw material before drying.

[0050] S204. Maintain a constant water bath temperature and allow the tobacco raw material to fully contact and absorb isotope-labeled water under vacuum conditions.

[0051] Maintaining the water bath temperature and high vacuum environment, the reabsorption time is 20-30 hours, preferably 24 hours. During this period, the labeled water slowly evaporates under vacuum and is reabsorbed by the dried tobacco raw material, evenly distributed in the intercellular spaces, cell walls, and microporous structures of the tobacco raw material, achieving complete replacement of the original natural water by isotopic water.

[0052] After re-inhalation, turn off the water bath heating and allow it to cool naturally to room temperature. Open the vent valve to restore the system to normal pressure, remove the marked tobacco material, and store it in a sealed container for later use.

[0053] In some embodiments, for dense, tightly bound tobacco raw materials (such as papermaking sheets), the vacuum drying time (e.g., extended to 30-36 hours) and re-inhalation time (e.g., extended to 30 hours) can be appropriately extended to ensure thorough removal of moisture and full penetration of the labeling water.

[0054] In some embodiments, for tobacco raw materials with high porosity (such as dry-processed sheets or thick pulp-processed sheets), complete replacement can be achieved by using a standard water bath temperature of 50°C and a re-inhalation time of 24 hours.

[0055] Step S3: Extract labeled water from tobacco raw materials. After derivatization treatment of labeled water and unlabeled natural water, perform GC-MS detection to obtain the corresponding mass spectra. Extract the characteristic ion peaks of isotopic substituted acetic acid and acetic acid from the mass spectra. Calculate the molar ratio of isotopic substituted acetic acid and acetic acid by the ratio of their peak areas or response values. Calculate the isotopic replacement rate based on the molar ratio of isotopic substituted acetic acid and acetic acid. When the isotopic replacement rate reaches a preset value, it is determined to be a complete replacement.

[0056] S301, Vacuum low-temperature extraction for labeled moisture: Take a certain amount of labeled tobacco raw material and place it in a vacuum pipeline device. For example, take 1g of labeled tobacco raw material and place it in a sample round-bottom flask 2.

[0057] Close the fourth piston 10, and open the first piston 7, the second piston 8, and the third piston 9; Immerse collection bottle 4 in a Dewar flask filled with dry ice for cryogenic capture; Open the fourth piston 10 and start the rotary vane vacuum pump 5 to evacuate until the vacuum gauge indicates zero. Adjust the water bath temperature to 50~60℃ and heat the sample to promote moisture evaporation; Extraction was carried out continuously under high vacuum for 8 hours, so that the water in the sample could be completely evaporated and captured by the low temperature of the collection bottle 4, and the buffer bottle 3 could prevent water from flowing back.

[0058] After extraction, turn off the rotary vane vacuum pump 5, remove the collection bottle 4, and collect the condensed water after it returns to room temperature.

[0059] S302, Derivatization process: Because the relative molecular mass of water molecules is too small, direct quantitative analysis by mass spectrometry has low precision. Therefore, derivatization is required to improve the relative molecular mass. The specific method is as follows: Take approximately 10 μL of the extracted water sample and place it in a reaction flask; Add excess acetic anhydride (approximately 100 μL), seal, and react at room temperature for 30 minutes. After the reaction is complete, water reacts with acetic anhydride to produce acetic acid (CH3COOH), while D2O reacts with acetic anhydride to produce deuterated acetic acid (CH3COOD). The reaction solution can be directly used for gas chromatography-mass spectrometry (GC-MS) analysis.

[0060] Meanwhile, unlabeled natural water (H2O) was used as a blank control, and derivatization was performed using the same steps.

[0061] It should be noted that the isotope-labeled water can be either D2O or H2¹ 8 O isotope-labeled water. For example, in this embodiment, the isotope-labeled water is D2O, and the isotope-labeled acetic acid is deuterated acetic acid.

[0062] In some examples, the isotope labeling of water in step S2 is H2¹ 8 O, in step S3, the isotopic substituted acetic acid is containing ¹ 8 O-acetic acid.

[0063] S303, GC-MS detection

[0064] Gas chromatography-mass spectrometry (GC-MS) was used to analyze the derivatized labeled water and unlabeled natural water, respectively, under the following conditions: Chromatographic column: HP-FFAP capillary column (30m×0.25mm×0.25μm); Sample injection method: automatic injection, injection volume 1.0 μL; Inlet temperature: 120℃; Programmed temperature rise: Initial temperature 80℃, hold for 5 minutes, then increase to 120℃ at a rate of 5℃ / minute; Ion source temperature: 230℃; Quadrupole temperature: 200℃; Carrier gas: Helium (He), constant flow mode, flow rate 1.0 mL / min; Split ratio: 100:1.

[0065] S303, Calculate the replacement rate

[0066] Replacement rate = [number of moles of X / (number of moles of X + number of moles of H2O)] × 100%

[0067] Converting to molar ratio, the formula for calculating the isotopic substitution rate can be directly obtained as follows: Replacement rate = [n(X / H2O) / (1+n(X / H2O))]×100%; Among them, n(X / H2O) is the molar ratio of isotopic substituted acetic acid and acetic acid.

[0068] In this embodiment, the isotopic acetic acid is deuterated acetic acid, and n(X / H2O) is n(D2O / H2O), which is equal to the molar ratio of D2O to H2O measured by mass spectrometry.

[0069] It should be noted that when water is selected as an isotope labeling agent, H2¹ 8 At O, the acetic acid produced after derivatization contains ¹ 8 O, can be detected by GC-MS containing ¹ 8 The acetic acid molecular ion peak of O (m / z 62, while that of ordinary acetic acid is m / z 60) was used for quantitative analysis, and the method for calculating the substitution rate was similar to that for D2O.

[0070] S304, Determination of Complete Replacement

[0071] Complete replacement is defined as an isotopic substitution reaching a preset value, which can be flexibly set according to the actual application scenario. For example, the preset value is 99% to 100%. For instance, when the molar ratio of D2O to H2O is not less than 990:1000 (i.e., replacement rate ≥ 99%), it is defined as "complete replacement". More preferably, when the molar ratio reaches 1000:0 (replacement rate 100%), it is defined as "complete replacement".

[0072] In some embodiments, the tobacco moisture isotope labeling and replacement method of the present invention further includes step S4: taking unlabeled blank tobacco raw material, calculating the natural isotope replacement rate corresponding to the unlabeled blank tobacco raw material according to the detection method in step S3, and calculating the final replacement rate after deducting the natural isotope replacement rate from the isotope replacement rate.

[0073] Since the natural abundance of deuterium in nature is approximately 0.139%, and proton exchange interference may occur during ionization, it is necessary to determine the background value of the blank sample. After treating unlabeled natural tobacco raw materials using the same method, the molar ratio of CH3COOD to CH3COOH in the blank sample is typically found to be around 2:1000. When calculating the replacement rate of the labeled sample, the blank background value can be subtracted to improve accuracy. The corrected final true replacement rate is obtained, eliminating systematic interference from natural isotope abundance and proton exchange during mass spectrometry ionization.

[0074] Furthermore, embodiments of the present invention also provide a tobacco raw material for moisture replacement using the aforementioned tobacco moisture isotope labeling and replacement method. By labeling the tobacco raw material through this method, the labeled tobacco raw material can be directly used for subsequent heated cigarette smoking experiments, pyrolysis kinetic analysis, and moisture migration path tracing studies, meeting the stringent requirements for tracer accuracy in moisture source tracing, migration path analysis, and thermochemical reaction mechanism research in novel tobacco products such as heated cigarettes.

[0075] Example 1: Traditional tobacco - D2O isotope labeling and replacement

[0076] S1. Drying and dehydration: Take 100.0g of traditional tobacco shreds for heating cigarettes, with an initial moisture content of 12.5wt.%; vacuum dry at 50℃, replacing the P2O5 desiccant every 3 hours, and after drying to constant weight, the material mass is 87.5g, removing 12.5g of natural moisture, leaving 0.3wt.% of residual moisture.

[0077] S2, High Vacuum Reabsorption: The vacuum pipeline device is evacuated to an absolute pressure of <0.0978MPa, the water bath is kept at a constant temperature of 50℃, 12.5mL of 99.9at.%D2O is injected, and reabsorption is carried out continuously under vacuum for 24h; after cooling, it is taken out and sealed for storage.

[0078] S3. Extraction and Detection: 1.0g of labeled tobacco was extracted under vacuum at low temperature for 8h, and the condensate was collected; after derivatization with acetic anhydride at room temperature for 30min, GC-MS was used for detection. The molar ratio of deuterated acetic acid to ordinary acetic acid was n(D2O / H2O)=1000:0 (CH3COOH was not detected); the substitution rate was calculated as 1000÷(1+1000)×100%≈99.90%.

[0079] S4. Blank calibration: Blank tobacco shreds from the same batch are tested simultaneously, with a blank background replacement rate of 0.20%; the final replacement rate after calibration is 99.90% - 0.20% = 99.70%, which meets the standard of more than 99% complete replacement.

[0080] Example 2: Thick slurry method for reconstituted tobacco flakes - D2O isotope labeling and replacement

[0081] S1. Drying and dehydration: 100.0g of reconstituted tobacco flakes using the slurry method, with an initial moisture content of 14.2wt.%; dried at a constant temperature of 50℃ to constant weight, resulting in a material weight of 85.8g and the removal of 14.2g of natural water.

[0082] S2, High Vacuum Reabsorption: 14.2 mL LD2O is injected under the conditions of 50℃ water bath and vacuum degree <0.0978 MPa, and the reabsorption time is 24 h.

[0083] S3. Detection and calculation: The molar ratio of deuterated acetic acid to acetic acid determined by GC-MS is 998:2. The uncorrected substitution rate is 998 ÷ (998 + 2) × 100% = 99.80%.

[0084] S4 Blank Correction: After deducting the blank background of 0.20%, the final replacement rate is 99.60%, reaching the standard for complete replacement.

[0085] Example 3: Roller-pressed reconstituted tobacco flakes - D2O isotope labeling replacement

[0086] S1 Drying and Dehydration: 100.0g of reconstituted tobacco flakes by roller pressing, with an initial moisture content of 11.8wt.%, weighed 88.2g after drying at 50℃ to constant weight, with 11.8g of moisture removed.

[0087] S2 reabsorption: 11.8 mL L2O was injected at 50°C under high vacuum conditions, and reabsorption was carried out for 24 hours.

[0088] S3 assay: molar ratio of deuterated acetic acid to acetic acid 995:5, uncorrected substitution rate 99.50%.

[0089] After S4 blank correction, the final replacement rate was 99.30%, which was determined to be a complete replacement.

[0090] Example 4: Dry Reconstituted Tobacco Leaf Flakes - D2O Isotope Labeling and Replacement

[0091] S1 Drying and Dehydration: 100.0g of dry reconstituted tobacco flakes, initial moisture content 10.5wt.%, mass after constant weight drying 89.5g, moisture removed 10.5g.

[0092] S2 reabsorption: At a constant temperature of 50℃ and a vacuum degree of <0.0978MPa, inject 10.5 mL of L2O and reabsorb for 24 hours.

[0093] S3 assay: molar ratio of deuterated acetic acid to acetic acid 992:8, uncorrected substitution rate 99.20%.

[0094] After S4 blank correction, the final replacement rate was 99.00%, reaching the complete replacement threshold.

[0095] Example 5: Papermaking Method for Reconstituted Tobacco Sheets - D2O Isotope Labeling and Replacement

[0096] S1 Drying and Dehydration: 100.0g of reconstituted tobacco flakes produced by papermaking, with an initial moisture content of 9.8wt.%; due to the dense fibers of the material, the drying time was extended to 36h, and the mass after drying to constant weight was 90.2g, with 9.8g of natural water removed.

[0097] S2 reabsorption: 9.8 mL L2O is injected in a 50℃ water bath and high vacuum environment, extending the reabsorption time to 30 hours.

[0098] S3 assay: molar ratio of deuterated acetic acid to acetic acid 989:11, uncorrected substitution rate 98.90%.

[0099] S4 blank correction: After deducting 0.20% background interference, the replacement rate after correction is 98.70%, which is close to the 99% judgment threshold and can meet the needs of routine tracer tests.

[0100] Example 6: Traditional tobacco - H2¹ 8 O isotope labeling substitution

[0101] In this embodiment, the labeling reagent is replaced with deoxygenated water (H2¹). 8 O, the drying and reabsorption operation parameters are completely consistent with those in Example 1; S1 Drying and Dehydration: 100g of traditional tobacco leaves, removing 12.5g of natural water; S2 reabsorption: Inject 12.5 mL of H2¹ with an abundance of 97 at.% 8 O, 50℃ high vacuum reabsorption for 24 hours; S3 Derivatization and Detection: The derivatized product contains ¹ 8 O-acetic acid and ordinary acetic acid were analyzed by GC-MS to detect characteristic ion peaks. The molar ratio was 1000:0, and the uncorrected substitution rate was 99.90%. After deducting 0.15% of the natural oxygen isotope background in the S4 blank correction, the final replacement rate after correction is 99.75%, which meets the requirements for complete replacement.

[0102] Comparative Example 1 did not specify the vacuum level or have constant temperature control.

[0103] Using the same tobacco raw materials as in Example 1, the vacuum level of the system was not controlled during the S2 reabsorption stage, and the water bath was not kept at a constant temperature. An equal amount of D2O was introduced for reabsorption at room temperature for 24 hours. The remaining drying, extraction, and detection steps were completely standardized. Detection results: The molar ratio of deuterated acetic acid to acetic acid was 617:1000, the uncorrected replacement rate was only 61.70%, and after deducting the blank background, the replacement rate was less than 62%, far below the 99% complete replacement criterion. The amount of residual endogenous natural water was large, making it unsuitable for high-precision moisture tracer analysis.

[0104] Comparative Example 2: Gradient control test with reabsorption temperature deviation of 50℃

[0105] Using the tobacco shreds from Example 1 as the test subject, only the S2 water bath temperature was adjusted, while all other process parameters and testing procedures remained completely consistent. The test results are as follows: Reabsorption temperature 40℃: Corrected displacement rate 92.30%, lower than 99%.

[0106] Comparative Example 3: Gradient control test with reabsorption temperature deviation of 50℃

[0107] Using the tobacco shreds from Example 1 as the test subject, only the S2 water bath temperature was adjusted, while all other process parameters and testing procedures remained completely consistent. The test results are as follows: Reabsorption temperature 60℃: Corrected displacement rate 95.10%, lower than 99%.

[0108] Comparative Example 4: Gradient control test with reabsorption temperature deviation of 50℃

[0109] Using the tobacco shreds from Example 1 as the test subject, only the S2 water bath temperature was adjusted, while all other process parameters and testing procedures remained completely consistent. The test results are as follows: Reabsorption temperature 70℃: Corrected displacement rate 94.50%, lower than 99%.

[0110] Table 1. Test Results

[0111] Comparing Examples 1-6 with Comparative Example 1, it can be seen that the tobacco moisture isotope labeling replacement method of the present invention is suitable for five major categories of mainstream tobacco raw materials for heated cigarettes: traditional tobacco shreds, thick pulp method, roll pressing method, dry method, and papermaking method reconstituted tobacco sheets. At 50℃, the present invention achieves a high replacement rate of ≥98.9%, while the method of Wang Lei et al., with a D2O to H2O molar ratio of only 617:1000, has a replacement rate of only about 61.7%, and a natural water residue of about 38.3%. The tobacco moisture isotope labeling replacement method of the present invention significantly improves the replacement rate. Comparing Examples 1 with Comparative Examples 2-4, it can be seen that the three temperature conditions in Comparative Examples 2-4 cannot reach the complete replacement criterion, proving that 50℃ is the optimal limiting temperature for achieving a high replacement rate in the present invention.

[0112] The present invention provides a method for labeling and replacing tobacco moisture isotopes. Labeling experiments can be conducted using a single type of tobacco raw material, or multiple tobacco raw materials can be mixed and dried and re-smoked simultaneously. The labeling reagent can be D2O or H2¹ as needed. 8 O, only the target for GC-MS characteristic ion collection needs to be adjusted after S3 derivatization, while the rest of the operation process, replacement rate calculation formula and blank correction logic remain unchanged; all process parameters, detection conditions and replacement rate judgment criteria are uniformly and quantitatively limited, the operation can be standardized and repeated, and it is suitable for scientific research scenarios such as moisture tracing in heated cigarettes, pyrolysis reaction mechanism and quantitative analysis of multi-source moisture contribution.

[0113] The present invention provides a method for tobacco moisture isotope labeling and replacement, which achieves ultra-high efficiency complete isotope replacement of tobacco moisture through multi-process step synergistic optimization. It has multiple advantages such as parameter standardization, universal raw material availability, complete verification system, non-destructive matrix, and dual isotope compatibility. It effectively fills the industry gap in the inability of existing technologies to achieve complete labeling and replacement of tobacco moisture and can meet the stringent technical requirements of high-precision moisture isotope tracing research in novel tobacco.

[0114] The device embodiments described above are merely illustrative. For example, the division of units is only a logical functional division. In actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another device, or some features may be ignored or not executed.

[0115] The units described as separate components may or may not be physically separate. The components shown as units may or may not be physical units; that is, they may be located in one place or distributed across multiple network units. Some or all of the units can be selected to achieve the purpose of this embodiment according to actual needs.

[0116] In addition, the functional units in the various embodiments of this application can be integrated into one processing unit, or each unit can exist physically separately, or two or more units can be integrated into one unit.

[0117] In this specification, references to "an embodiment" or "a specific implementation" mean that a particular feature, structure, or characteristic described in connection with that embodiment / specific implementation is included in at least one embodiment / specific implementation of the invention. Therefore, the phrase "in one embodiment / specific implementation" appearing in various places in this specification does not necessarily refer to the same embodiment / setting, but rather to potentially different embodiments. Furthermore, specific features, structures, or characteristics may be combined in one or more embodiments / settings in any suitable manner, as will be apparent to those skilled in the art from this disclosure.

[0118] Similarly, it should be understood that in the above description of exemplary embodiments / specific implementations of the invention, various features of the invention are sometimes combined in a single embodiment / specific implementation or its figures and description, with the aim of simplifying the disclosure and aiding in the understanding of one or more of the various aspects of the invention. However, the method of description of the invention should not be construed as reflecting an intention that, except for expressly stated instructions to the contrary or obvious technical contradictions or exclusions, the claimed features of the invention are more than those expressly stated in each claim.

[0119] Conversely, the inventive aspect reflected in the claims lies in not all the features of a single foregoing disclosed embodiment / specification. Therefore, the claims following the detailed description are expressly incorporated herein, each claim existing independently as a separate embodiment / specification of the invention.

[0120] Furthermore, while some embodiments / specific implementations described herein include, but are not limited to, other features included in other embodiments / specific implementations, combinations of features from different embodiments / specific implementations are intended to be within the scope of the invention and form different embodiments / specific implementations, as will be understood by those skilled in the art. For example, in the following claims, embodiments / specific implementations of any claim can be used in any combination.

[0121] The terms and expressions used in this specification are for illustrative purposes and not for limitation. In using these terms and expressions, it is not intended to exclude any equivalents of the features or portions thereof shown and described, but rather to recognize that various modifications may be possible within the scope of the invention.

[0122] Therefore, it should be understood that although the invention has been specifically disclosed through preferred embodiments, exemplary embodiments and optional features, those skilled in the art may take variations or modifications of the concepts disclosed herein, and such variations and modifications are therefore considered to be within the scope of the invention as defined by the appended claims.

[0123] The specific embodiments given in this specification are examples of useful implementations of the present invention. It will be apparent to those skilled in the art that the present invention can be implemented using many variations of the devices, device components, and method steps disclosed in this specification.

[0124] The foregoing description of specific embodiments fully discloses the general features of the present invention, enabling others to easily modify and / or adapt such specific embodiments for various applications by applying knowledge within the scope of the art, without conducting excessive experimentation and without departing from the general concept of the present invention.

[0125] Therefore, based on the teachings and guidance provided herein, it is intended that such modifications and alterations be included within the meaning and scope of equivalents of the disclosed embodiments. It should be understood that the wording or terminology used herein is for descriptive purposes and is not intended to be limiting; thus, the wording or terminology in this specification will be interpreted by those skilled in the art based on the foregoing teachings and guidance.

[0126] Furthermore, the scope of this invention should not be limited to any of the exemplary embodiments described above, but only to the appended claims and their equivalents. The terminology and expressions used herein are for descriptive purposes only, and the invention should not be limited to these terms and expressions. The use of these terms and expressions does not imply the exclusion of any illustrative and descriptive equivalents (or parts thereof), and it should be recognized that various modifications that may exist should also be included within the scope of the claims. Other modifications, variations, and substitutions may also exist. Accordingly, the claims should be considered to cover all such equivalents.

[0127] Similarly, it should be noted that although the present invention has been described with reference to the specific embodiments described above, those skilled in the art should recognize that the above embodiments are only used to illustrate the present invention, and various equivalent changes or substitutions can be made without departing from the spirit of the present invention. Therefore, any changes or modifications to the above embodiments within the scope of the essential spirit of the present invention will fall within the scope of the claims of the present invention.

Claims

1. A method for tobacco moisture isotope labeling and replacement, characterized in that, include: Step S1: Drying and dehydrating tobacco raw materials; Step S2: The tobacco raw material is subjected to D2O or H2¹ under high vacuum. 8 O isotope-labeled water reabsorption allows the labeled water to penetrate evenly and occupy all sites of the original moisture in the tobacco raw material; Step S3: Extract labeled water from tobacco raw materials. After derivatization treatment of labeled water and unlabeled natural water, perform GC-MS detection to obtain the corresponding mass spectra. Extract the characteristic ion peaks of isotopic substituted acetic acid and acetic acid from the mass spectra. Calculate the molar ratio of isotopic substituted acetic acid and acetic acid by the ratio of their peak areas or response values. Calculate the isotopic replacement rate based on the molar ratio of isotopic substituted acetic acid and acetic acid. When the isotopic replacement rate reaches a preset value, it is determined to be a complete replacement.

2. The method for tobacco moisture isotope labeling and replacement according to claim 1, characterized in that: In step S2, the isotope-labeled water is D2O, and in step S3, the isotope-labeled acetic acid is deuterated acetic acid.

3. The method for tobacco moisture isotope labeling and replacement according to claim 1, characterized in that: In step S2, the isotope-labeled water is H2¹ 8 O, in step S3, the isotopic substituted acetic acid is containing ¹ 8 O-acetic acid.

4. The method for tobacco moisture isotope labeling and replacement according to claim 1, characterized in that: The formula for calculating the isotope substitution rate in step S3 is as follows: Replacement rate = [n(X / H2O) / (1+n(X / H2O))]×100%; Among them, n(X / H2O) is the molar ratio of isotopic substituted acetic acid and acetic acid.

5. The method for tobacco moisture isotope labeling and replacement according to any one of claims 4, characterized in that: The preset value in step S3 is 99% to 100%.

6. The method for tobacco moisture isotope labeling and replacement according to claim 1, characterized in that: The derivatization process in step S3 is as follows: labeled water and unlabeled natural water are placed in reaction flasks, excess acetic anhydride is added, and after sealing and complete reaction at room temperature, the labeled water reacts with acetic anhydride to generate isotopic substituted acetic acid, and the natural water reacts with acetic anhydride to generate acetic acid.

7. The method for tobacco water isotope labeling and replacement according to claim 6, characterized in that: Step S2 includes: S201. Place the dried and dehydrated tobacco raw material into a vacuum pipeline device; S202. Evacuate the vacuum pipeline device; S203. Tobacco raw material water bath in vacuum pipeline device, and isotope-labeled water injected into vacuum pipeline device; S204. Maintain a constant water bath temperature and allow the tobacco raw material to fully contact and absorb isotope-labeled water under vacuum conditions.

8. The method for tobacco moisture isotope labeling and replacement according to claim 7, characterized in that, In step S202, the vacuum pipeline device is evacuated to reduce the vacuum level inside the vacuum pipeline device to below 0.0978 MPa; In step S203, the water bath temperature is 50°C; Step S204, absorption time is 24h.

9. The method for tobacco moisture isotope labeling and replacement according to claim 1, characterized in that: It also includes step S4: take unlabeled blank tobacco raw material, calculate the natural isotope replacement rate corresponding to the unlabeled blank tobacco raw material according to the detection method in step S3, and calculate the final replacement rate after deducting the natural isotope replacement rate from the isotope replacement rate.

10. A tobacco raw material, characterized in that: Moisture replacement was performed using the tobacco moisture isotope labeling replacement method as described in any one of claims 1-9.

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