A method for simultaneously determining a smoking agent and a cooling agent in a heat-not-burn reconstituted tobacco leaf

Abstract

The present application belongs to the technical field of tobacco detection, and particularly relates to a method for simultaneously determining smoking agents and cooling agents in heat-not-burn reconstituted tobacco leaves, which specifically comprises determination of seven compounds of glycerol, 1,2-propanediol, menthol, L-menthone, L-menthyl lactate, WS-3 and WS-23. The heat-not-burn reconstituted tobacco leaf sample is cut into shreds in advance, a certain amount of the sample to be measured is weighed in a conical flask, an internal standard extraction agent is added, and after extraction by electromagnetic stirring at room temperature, the upper clear liquid is taken, filtered by a microporous filter membrane to obtain a liquid to be measured; the obtained extraction liquid is analyzed by gas chromatography hydrogen flame ionization (GC-FID), and quantitative analysis of the seven compound components is performed by a double internal standard method. The present application can simultaneously determine the contents of the smoking agents and the cooling agents in the heat-not-burn reconstituted tobacco leaves.

Description

Technical Field

[0001] This invention belongs to the field of detection and analysis technology, specifically relating to a method for simultaneously determining smoke-generating agents and cooling agents in heated non-combustible reconstituted tobacco leaves. Background Technology

[0002] Heated tobacco products do not directly burn the cigarette but release smoke by heating the tobacco material, reducing the harmful components produced by the high-temperature combustion and decomposition of tobacco, thus lowering the levels of harmful components in mainstream smoke. 1,2-Propanediol and glycerin in tobacco formulations have both moisturizing effects and promote the release of smoke in heated cigarettes, often serving as smoke-generating agents for new types of cigarettes. Because heated tobacco is only heated without burning, a significant amount of aroma compounds produced by tobacco combustion are lost during smoking. Therefore, a considerable amount of flavoring additives are needed to enhance the flavor and aroma of heated cigarettes, and cooling agents are among the most widely used exogenous additives. Smoke-generating agents and cooling agents are important and indispensable compounds in cooled heated tobacco reconstituted tobacco, making accurate methods for quantifying smoke-generating agents and cooling agents in heated tobacco reconstituted tobacco particularly important.

[0003] Currently, there are reports on methods for detecting smoke-generating agents and cooling agents in heated tobacco, but no methods for simultaneously determining smoke-generating agents and cooling agents have been reported. Due to the complex chemical composition and significant polarity differences of smoke-generating agents and cooling agents in heated tobacco, as well as the large variations in their content within heated tobacco reconstituted leaves, and the inherent volatility during extraction, both cyclone oscillation and ultrasonic extraction methods present challenges. Therefore, considering the characteristics of cooling-flavored heated tobacco reconstituted leaves, this study aims to develop a method suitable for the efficient extraction and accurate determination of smoke-generating agents and cooling agents in cooling-flavored heated tobacco reconstituted leaves. Summary of the Invention

[0004] The purpose of this invention is to provide a method for simultaneously determining smoke-generating agents and cooling agents in heated non-combustible reconstituted tobacco leaves, in order to overcome the problems existing in the prior art. Specifically, it includes a method for detecting glycerol, 1,2-propanediol, menthol, L-menthone, L-menthyl lactate, WS-3, and WS-23. This method is simple to operate, has high sample extraction efficiency, and provides accurate and reproducible analytical results.

[0005] To achieve the above objectives, this application employs the following technical solution:

[0006] A method for simultaneously determining smoke-generating agents and cooling agents in heat-not-combustible reconstituted tobacco leaves, the specific steps of which are as follows:

[0007] S1. Weigh a set amount of the cool-flavored heat-not-burn reconstituted tobacco sample and place it in a conical flask for later use. Specifically, cut the cool-flavored heat-not-burn reconstituted tobacco sample into shreds with a width of 0.8 mm and a length of no more than 20 mm. Weigh a certain amount and place it in a conical flask for later use.

[0008] S2. Weigh out the specified amounts of 1,4-butanediol and phenylethyl acetate internal standards, dissolve them in ethanol to prepare internal standard extraction solutions for later use; specifically, considering factors such as the large differences in polarity and content of the analyte compounds, dual internal standards are used for quantification; accurately weigh out a certain amount of 1,4-butanediol and phenylethyl acetate internal standards, dissolve them in ethanol to prepare internal standard extraction solutions for later use.

[0009] S3. Add the internal standard extraction solution prepared in step S2 to the conical flask containing the sample tobacco shreds of the cool-flavored heated non-combustible reconstituted tobacco leaf from step S1, shake well, and then extract the supernatant by electromagnetic stirring at room temperature.

[0010] S4. Filter the supernatant from step S3 using a microporous membrane to obtain the extract.

[0011] S5. Weigh out the specified amounts of glycerol, 1,2-propanediol, menthol, L-menthone, L-menthyl lactate, WS-3, and WS-23 reference standards, respectively, and dissolve them in the internal standard extraction solution from step S2 to prepare a mixed standard solution. When using, dilute stepwise according to the specified ratio and set aside.

[0012] S6. Perform qualitative and quantitative analysis on the mixed standard solution from step S5 using the internal standard method to obtain the standard component peaks and standard curve;

[0013] S7. The extract from step S4 is subjected to qualitative and quantitative analysis using gas chromatography-fiber spectroscopy (GC-FID) to obtain the content of each compound in the extract. Specifically, the internal standard method is used to quantitatively analyze seven compounds: glycerol, 1,2-propanediol, menthol, L-menthone, L-menthyl lactate, WS-3, and WS-23. This involves regression analysis of the peak area ratios of known concentrations of glycerol, 1,2-propanediol, menthol, L-menthone, L-menthyl lactate, WS-3, and WS-23 with the corresponding concentration ratios of the internal standard to obtain a standard curve. The extract is then analyzed to determine the peak areas of the seven compounds detected in the extract. These peak areas are then substituted into the standard curve to calculate the content of glycerol, 1,2-propanediol, menthol, L-menthone, L-menthyl lactate, WS-3, and WS-23 in the test solution.

[0014] Furthermore, the smoke-generating agent and cooling agent are glycerin, 1,2-propanediol, menthol, L-menthone, L-menthyl lactate, WS-3, and WS-23.

[0015] Furthermore, the width of the tobacco shreds is 0.8-2 mm.

[0016] Furthermore, in step S3, the mass-to-volume ratio of the tobacco shreds in the test sample of the cool-flavored heated non-combustible reconstituted tobacco leaf to the ethanol in the internal standard extraction solution is 1g:50-100mL.

[0017] Furthermore, in step S3, the extraction time with electromagnetic stirring at room temperature is 10-30 minutes.

[0018] Furthermore, in step S4, the microporous filter membrane has a size of 0.22μm-0.45μm.

[0019] Further, in step S5, the chromatographic conditions are as follows: Column: DB-WAX, specifications: 30m × 0.32mm, 0.25μm; Temperature program: initial temperature 80℃, hold for 1 min, first stage: increase to 210℃ at a rate of 5℃ / min, hold for 3 min; second stage: increase to 240℃ at a rate of 30℃ / min, hold for 2 min, total run time 33 min; Injector temperature: 250℃; FID detector temperature: 275℃; Carrier gas: nitrogen, carrier gas flow rate 1.5 mL / min, constant flow mode; Air flow rate: 300 mL / min; Hydrogen flow rate: 30 mL / min; Make-up gas: nitrogen flow rate: 25 mL / min; Injection volume: 1 μL, split injection, split ratio 5:1.

[0020] Furthermore, in step S7, the qualitative analysis involves measuring the target compound in a series of standard and sample solutions of different concentrations under the same experimental conditions. When the retention time of the chromatographic peak detected in the sample solution is consistent with the retention time of a certain component peak in the standard solution, it is determined that the substance is present in the sample. At the same time, the standard sample is added to the extract of heated non-combustible reconstituted tobacco for chromatographic analysis, and the target substance is confirmed by observing the further increase of the chromatographic peak.

[0021] Furthermore, in step S7, the quantitative analysis involves using the internal standard method to perform regression analysis on the peak area ratio of the smoke-generating agent and the cooling agent in the reconstituted tobacco leaf with a cooling flavor, and their corresponding concentration ratio to obtain a standard curve. The extract is then analyzed to determine the chromatographic peak areas of the smoke-generating agent and the cooling agent in the extract. These values ​​are then substituted into the standard curve to determine the content of each component in the sample.

[0022] Furthermore, the result calculation and expression are as follows:

[0023] w=(ρ-ρ0)×V×f / [m(1-x)] (1)

[0024] In the formula, w is the percentage content of each target compound in the sample, in %; ρ is the concentration of each target compound in the diluent, in mg / L; ρ0 is the concentration of each target compound in the blank test, in mg / L; V is the volume of the diluent, in mL; f is the unit conversion factor; m is the sample mass, in g; and x is the moisture content of the sample.

[0025] This invention establishes for the first time a gas chromatography-flame ionization (GC-FID) method for the simultaneous detection and determination of seven compounds—glycerol, 1,2-propanediol, menthol, L-menthone, L-menthyl lactate, WS-3, and WS-23—in chilled, heated, non-combustible reconstituted tobacco. The method exhibits linear correlation coefficients greater than 0.999, detection limits ranging from 0.006 to 0.082 μg / mL, recoveries of each target component between 95.5% and 102.1%, and relative standard deviations (RSDs) of each compound within the range of 0.64% to 1.91%. The method features high extraction efficiency, good separation, high target analyte recovery, high sensitivity, high solvent safety, and ease of operation.

[0026] The beneficial effects of this invention are:

[0027] This invention extracts seven different samples with significantly different mass fractions and chemical properties—glycerol, 1,2-propanediol, menthol, L-menthone, L-menthyl lactate, WS-3, and WS-23—using a suitable pretreatment method, namely room temperature electromagnetic stirring extraction. Quantification is achieved using 1,4-butanediol and phenethyl acetate as dual internal standards, overcoming the significant impact of differences in sample polarity and content.

[0028] This invention employs room-temperature electromagnetic stirring extraction technology as a pretreatment technique. The electromagnetic stirring uses electromagnetic force to drive the stir bar, which rotates to create vortices in the solution, making it easier to mix. Simultaneously, the contact between the stir bar and the sample provides a grinding effect, shortening the pretreatment extraction time, improving extraction efficiency, and significantly reducing the volatilization of cooling agents, namely menthol, L-menthone, L-lactic acid menthyl ester, WS-3, and WS-23. This effectively avoids the risk of lower test results due to sample volatilization during extraction. It is more efficient than traditional cyclone extraction and minimizes the volatilization of cooling agents caused by the heat generated during traditional ultrasonic extraction.

[0029] This invention uses electromagnetic stirring technology for pretreatment, eliminating the need for mechanical transmission components and avoiding the noise caused by swirling oscillation and ultrasonic extraction in traditional processing methods.

[0030] This invention uses a polar polyethylene glycol chromatographic column for separation, resulting in high peak separation efficiency, sharp peaks, and no tailing.

[0031] This invention has the advantages of high detection sensitivity, accuracy and good reproducibility.

[0032] This invention can simultaneously measure smoke-generating agents and cooling agents, improving work efficiency, greatly reducing solvent usage, and is environmentally friendly, efficient, and economically beneficial. Attached Figure Description

[0033] Figure 1 This is a chromatogram of a mixed control sample of the target compounds to be tested in this invention, wherein: 1 is L-menthone; 2 is 1,2-propanediol; 3 is menthol; 4 is phenethyl acetate; 5 is WS-23; 6 is 1,4-butanediol; 7 is L-menthyl lactate; 8 is WS-3; and 9 is glycerol.

[0034] Figure 2 This is a chromatogram of a certain cooling-flavored, heat-resistant, non-flammable sample, where: 1 is L-menthone; 2 is 1,2-propanediol; 3 is menthol; 4 is phenethyl acetate; 5 is WS-23; 6 is 1,4-butanediol; and 7 is glycerol. Detailed Implementation

[0035] The technical solutions of the present invention will be described in detail below with reference to the embodiments. The following embodiments are merely exemplary and can only be used to explain and illustrate the technical solutions of the present invention, and should not be construed as limiting the technical solutions of the present invention.

[0036] Example 1

[0037] like Figure 1 and Figure 2 As shown, this application provides a method for simultaneously determining the smoke-generating agent and cooling agent components in heated non-combustible reconstituted tobacco leaves. In this embodiment, the smoke-generating agent and cooling agent are glycerol, 1,2-propanediol, menthol, L-menthone, L-menthyl lactate, WS-3, and WS-23.

[0038] 1. Instruments and Materials:

[0039] Agilent gas chromatograph (Agilent Technologies, USA, equipped with a flame ionization detector); PREL-19 laboratory shredder (Jiangsu Precision Technology Co., Ltd.); BSA224S-CW electronic balance (sensitivity: 0.0001g, Sartorius, Germany); organic phase syringe filter (13 mm × 0.45 μm, Tianjin Jinteng); thermostatic electromagnetic stirrer (IKART5); diphenylethyl acetate, menthol, L-menthone, menthyl lactate, N-ethyl-L-menthylformamide (WS-3), 2-isopropyl-N,2,3-trimethylbutyramide (WS-23); 2-phenylethyl acetate (≥98%, Beijing Bailingwei Technology Co., Ltd.); ethanol (chromatographic grade, Merck Co., Ltd.).

[0040] The cool-flavored heated non-combustible reconstituted tobacco samples used in this embodiment were all provided by China Tobacco Schweitz (Yunnan) Reconstituted Tobacco Co., Ltd.

[0041] 2. Preparation of sample extraction solution:

[0042] The sample of reconstituted tobacco leaf with a cooling flavor was cut into shreds with a width of 0.8 mm. 1 g of the shreds was accurately weighed and placed in a stoppered Erlenmeyer flask. 50 mL of internal standard extraction solution was added, and the mixture was extracted at room temperature with electromagnetic stirring for 15 min. 2 mL of the supernatant was taken and filtered through a 0.22 μm microporous membrane to obtain the sample extract.

[0043] 3. Preparation of internal standard extraction solution:

[0044] Accurately weigh 2.5 g (accurate to 0.0001 g) of 1,4-butanediol and 0.1 g (accurate to 0.0001 g) of phenethyl acetate, dissolve them in ethanol and bring the volume to 1000 mL.

[0045] 4. Preparation of control sample solution:

[0046] Accurately weigh 2.5 g of glycerol, 1 g of 1,2-propanediol reference standard, 50 mg of menthol, 20 mg of L-menthol, L-menthyl lactate, WS-3, and WS-23 into 50 mL brown volumetric flasks. Dissolve in internal standard extract, dilute to volume, and shake well to prepare a mixed standard solution. Dilute stepwise according to the specified ratio before use.

[0047] 5. GC-FID gas chromatography conditions:

[0048] Chromatographic conditions: DB-WAX column (30m × 0.32mm, 0.25μm) was used; temperature program was used, with an initial temperature of 80 ℃ (held for 1 min), followed by a first stage of increasing to 210 ℃ at a rate of 5 ℃ / min and holding for 3 min; the second stage of increasing to 240 ℃ at a rate of 30 ℃ / min and holding for 2 min, for a total run time of 33 min; the injection port temperature was 250 ℃; the FID detector temperature was 275 ℃; the carrier gas was nitrogen, with a carrier gas flow rate of 1.5 mL / min in constant flow mode; the air flow rate was 300 mL / min; the hydrogen flow rate was 30 mL / min; the make-up nitrogen flow rate was 25 mL / min; the injection volume was 1 μL, and the injection was split at a split ratio of 5:1.

[0049] 6. Qualitative Analysis:

[0050] Under identical experimental conditions, seven target compounds were measured in a series of standard and sample solutions of different concentrations. If the retention time of the chromatographic peak detected in the sample solution was consistent with the retention time of a certain component peak in the standard solution, the presence of that component in the sample could be confirmed. Simultaneously, the standard sample could be added to the extract of heated, non-combustible reconstituted tobacco leaves for chromatographic analysis; further elevation of the chromatographic peak would confirm the presence of the target compound.

[0051] 7. Quantitative analysis:

[0052] Regression analysis was performed using the internal standard method to analyze the peak area ratio of smoking agents and cooling agents in reconstituted tobacco leaves with a cooling flavor, based on their corresponding concentration ratios. A standard curve was obtained. The chromatographic peak areas of smoking agents and cooling agents in the extract were measured and substituted into the standard curve to determine their content in the sample. The experimental results are shown in Table 1.

[0053] The results are calculated and expressed in equation (1):

[0054] w=(ρ-ρ0)×V×f / [m(1-x)] (1)

[0055] Where: w. — percentage content of each target compound in the sample (in %);

[0056] ρ—The concentration of each target compound in the diluent, in milligrams per liter (mg / L);

[0057] ρ0—The concentration of each target compound in the blank test, in milligrams per liter (mg / L);

[0058] V — Volume of the diluent, in milliliters (mL);

[0059] f — unit conversion factor

[0060] m—Sample mass, in grams (g);

[0061] x — moisture content of the sample.

[0062] Table 1. Determination results of smoke-generating agents and cooling agents in the samples.

[0063]

[0064] 8. Methodological Examination:

[0065] (1) Repeatable experiments:

[0066] Under the optimized measurement conditions of this study, the precision, repeatability, stability, and spiked recovery rate of the method were investigated using retention time and peak area in the cooled, heated, non-combustible reconstituted tobacco as evaluation indicators. Six repeatability tests showed that the RSD values ​​of the retention time of each smoke-generating agent and cooling agent component were all below 0.83%, and the RSD values ​​of the peak area were all below 3.25%, indicating that the method has good repeatability.

[0067] (2) Spike recovery rate:

[0068] Three samples of reconstituted tobacco leaves were taken. Using glycerol, 1,2-propanediol, L-menthone, menthol, WS-23, L-menthone lactate, and WS-3 as baselines, three levels of these standards were added (low, medium, and high), respectively. Each addition level was analyzed in triplicate. The spiked samples underwent pretreatment and were analyzed by GC-FID. The spiked recoveries were calculated from the original content, the added amount, and the measured values. The results showed that the recoveries of each target component ranged from 95.5% to 102.1%. These data indicate that this method has good precision, stability, and reproducibility, and can be used for the accurate determination of smoke-generating agents and cooling agents in chilled reconstituted tobacco leaves.

[0069] The detection method is the same as in Example 1. A heated non-combustible reconstituted tobacco sample 1 was selected, and the experimental results are shown in Table 2.

[0070] Table 2. Determination results of smoke-generating agents and cooling agents in the samples.

[0071]

[0072] The detection method is the same as in Example 1. A sample 2 of heated non-combustible reconstituted tobacco leaf was selected. The experimental results are shown in Table 3.

[0073] Table 3. Determination results of smoke-generating agents and cooling agents in the samples.

[0074]

[0075] The detection method is the same as in Example 1. A heated non-combustible reconstituted tobacco sample 3 was selected, and the experimental results are shown in Table 4.

[0076] Table 4. Determination results of smoke-generating agents and cooling agents in the samples.

[0077]

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for simultaneously determining smoke-generating agents and cooling agents in reconstituted tobacco leaves that are heated but not burned, characterized in that, The specific steps are as follows: S1. Weigh the set amount of the cool-flavored heated non-combustible reconstituted tobacco sample and place it in an Erlenmeyer flask for later use; S2. Weigh out the specified amounts of 1,4-butanediol and phenethyl acetate internal standards respectively, add ethanol to dissolve them, and prepare internal standard extraction solutions for later use. S3. Add the internal standard extraction solution prepared in step S2 to the conical flask containing the sample tobacco shreds of the cool-flavored heated non-combustible reconstituted tobacco leaf in step S1, shake well, and then extract the supernatant by electromagnetic stirring at room temperature. S4. Filter the supernatant from step S3 using a microporous membrane to obtain the extract. S5. Weigh out the specified amounts of glycerol, 1,2-propanediol, menthol, L-menthone, L-menthyl lactate, WS-3, and WS-23 reference standards, respectively, and dissolve them in the internal standard extraction solution from step S2 to prepare a mixed standard solution. When using, dilute stepwise according to the specified ratio and set aside. S6. Perform qualitative and quantitative analysis on the mixed standard solution from step S5 using the internal standard method to obtain the standard component peaks and standard curve; S7. The extract from step S4 is subjected to qualitative and quantitative analysis using GC-FID to obtain the content of each compound in the extract.

2. The method for simultaneously determining the smoke-generating agent and the cooling agent in heated non-combustible reconstituted tobacco according to claim 1, characterized in that, The smoke-generating and cooling agents are glycerin, 1,2-propanediol, menthol, L-menthone, L-menthyl lactate, WS-3, and WS-23.

3. The method for simultaneously determining the smoke-generating agent and the cooling agent in heated non-combustible reconstituted tobacco according to claim 1, characterized in that, The width of the tobacco shreds is 0.8-2 mm.

4. The method for simultaneously determining the smoke-generating agent and the cooling agent in heated non-combustible reconstituted tobacco according to claim 1, characterized in that, In step S3, the mass-to-volume ratio of the tobacco shreds in the test sample of the cool-flavored heated non-combustible reconstituted tobacco leaf to the ethanol in the internal standard extraction solution is 1g:50-100mL.

5. The method for simultaneously determining the smoke-generating agent and the cooling agent in heated non-combustible reconstituted tobacco according to claim 1, characterized in that, In step S3, the extraction time with electromagnetic stirring at room temperature is 10-30 minutes.

6. The method for simultaneously determining the smoke-generating agent and the cooling agent in heated non-combustible reconstituted tobacco according to claim 1, characterized in that, In step S4, the microporous filter membrane has a size of 0.22μm-0.45μm.

7. The method for simultaneously determining the smoke-generating agent and the cooling agent in heated non-combustible reconstituted tobacco according to claim 1, characterized in that, In step S5, the chromatographic conditions are as follows: Column: DB-WAX, 30m × 0.32mm, 0.25μm; Temperature program: initial temperature 80℃, hold for 1 min, first stage at 210℃ at a rate of 5℃ / min, hold for 3 min; second stage at 240℃ at a rate of 30℃ / min, hold for 2 min, total run time 33 min; Injector temperature: 250℃; FID detector temperature: 275℃; Carrier gas: nitrogen, carrier gas flow rate 1.5 mL / min, constant flow mode; Air flow rate: 300 mL / min; Hydrogen flow rate: 30 mL / min; Make-up nitrogen flow rate: 25 mL / min; Injection volume: 1 μL, split injection, split ratio 5:

1.

8. The method for simultaneously determining the smoke-generating agent and the cooling agent in heated non-combustible reconstituted tobacco according to claim 7, characterized in that, In step S7, the qualitative analysis involves measuring the target compound in a series of standard and sample solutions of different concentrations under the same experimental conditions. When the retention time of the chromatographic peak detected in the sample solution is consistent with the retention time of a certain component peak in the standard solution, it is determined that the substance is present in the sample. At the same time, the standard is added to the extract of heated non-combustible reconstituted tobacco for chromatographic analysis, and the target compound is confirmed by observing the further increase of the chromatographic peak.

9. The method for simultaneously determining the smoke-generating agent and the cooling agent in heated non-combustible reconstituted tobacco according to claim 7, characterized in that, In step S7, the quantitative analysis involves using the internal standard method to perform regression analysis on the peak area ratio of the smoke-generating agent and the cooling agent in the reconstituted tobacco leaf with a cooling flavor, and their corresponding concentration ratio to obtain a standard curve. The extract is then analyzed to determine the chromatographic peak areas of the smoke-generating agent and the cooling agent in the extract. These values ​​are then substituted into the standard curve to determine the content of each component in the sample.

10. The method for simultaneously determining the smoke-generating agent and the cooling agent in heated non-combustible reconstituted tobacco according to claim 9, characterized in that, The result calculation and expression are as follows: w=(ρ-ρ0)×V×f / [m(1-x)] (1) In the formula, w represents the percentage content of each target compound in the sample, in % %. ρ is the concentration of each target compound in the diluent, in mg / L; ρ0 is the concentration of each target compound in the blank test, in mg / L; V is the volume of the diluent, in mL; f is the unit conversion factor; m is the sample mass, in g; x is the moisture content of the sample.

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