Method for testing transfer behavior of cooling agent in whole cigarette and method for analyzing content of cooling agent

Abstract

This invention belongs to the field of testing and analysis of cooling agents, specifically involving a method for testing the transfer behavior of cooling agents in whole cigarettes and a method for analyzing the content of cooling agents. The testing method includes the following steps: pulverizing the whole cigarette, adding methanol and an internal standard for extraction to obtain a cigarette sample solution; capturing the mainstream smoke of the corresponding whole cigarette, extracting it under the same conditions to obtain a smoke sample solution; performing gas chromatography-mass spectrometry analysis under the same conditions, wherein the cooling agent contains at least WS-3 and menthone glycerol ketal; and calculating the transfer rate of the cooling agent in the whole cigarette. This invention addresses the challenges of complex cigarette matrices and the difficulty in separating WS-3 and menthone glycerol ketal by using a highly polar chromatographic column with optimized temperature programming conditions to achieve baseline separation of novel compound cooling agents such as WS-3 and menthone glycerol ketal. All compounds exhibit sharp peaks with good symmetry, enabling accurate quantification and accurate evaluation of the transfer rate.

Description

Technical Field

[0001] This invention belongs to the field of testing and analysis of cooling agents, specifically involving a method for testing the transfer behavior of cooling agents in a whole cigarette and a method for analyzing the content of cooling agents. Background Technology

[0002] Cooling agents are one of the important additives in menthol cigarettes. Commonly used cooling agents in menthol cigarettes include D,L-menthol, L-menthone, menthyl acetate, menthyl lactate, isoprene, N-ethyl-p-menthyl-3-carboxamide (WS-3, CAS No. 9711-79-0), and N,2,3-trimethyl-2-isopropylbutyramide (WS-23, CAS No. 51115-67-4). These cooling agents can improve the sensory quality of cigarettes, enhance the harmony and smoothness of cigarette smoke, and reduce oral and throat irritation. Therefore, they are widely used in various types of cigarettes.

[0003] Different cooling agents have different melting and boiling points, and their transfer rates from the cigarette stick to the mainstream cigarette smoke also vary. The transfer rate of cooling agents directly affects the amount of cooling agents released into the mainstream cigarette smoke, thus affecting the sensory quality of the cigarette. Therefore, the determination of cooling agent content and the study of transfer rates are of great significance for cigarette quality control.

[0004] Chinese invention patent application CN 116539737 A, published on August 4, 2023, discloses a method for analyzing cooling agents in heated cigarettes using gas chromatography-quadrupole time-of-flight mass spectrometry (GC-QTOF MS). This method enables the determination of 16 target cooling agents in heated cigarettes, including isomenthyl alcohol, menthone, menthofuran, neomenthyl alcohol, D,L-menthyl alcohol, piperonone, WS-23, menthyl acetate, menthyl lactate, menthol, menthyl isovalerate, WS-3, WS-10, menthyl succinate, WS-5, and WS-12.

[0005] The aforementioned quadrupole / time-of-flight analyzer combines quadrupole mass selection with time-of-flight mass spectrometry (TOF-MS) high-resolution detection. The quadrupole facilitates ion screening and transport, while the time-of-flight analyzer enables high-resolution ion detection. Due to its high resolution, the TOF-MS analyzer can accurately identify specific compounds in complex mixture samples. However, GC-QTOF MS analysis requires establishing a Personal Compound Database (PCDL) containing retention times, elemental composition, and corresponding theoretically accurate mass numbers for qualitative analysis. This process is cumbersome, costly, and impractical for routine analysis of cigarette cooling agents.

[0006] Gas chromatography-quadrupole mass spectrometry (GC-MS) has advantages such as good selectivity, high resolution, high sensitivity, and short analysis time, making it the most widely used method for determining cooling agents. Wang Ziyan et al. (Tobacco Science & Technology, June 2020, Vol. 53, No. 6) used GC-MS to determine eight cooling agents in heated cigarette tobacco materials, including isoprene, D,L-menthol, L-menthone, menthyl acetate, menthyl lactate, WS-3, WS-5, and WS-23.

[0007] Wang Ziyan et al. (Tobacco Science and Technology, October 2020, Vol. 53, No. 10) also conducted a comparative study on the transfer rate of cooling agents in electrically heated cigarettes and traditional cigarettes. Their gas chromatography-mass spectrometry (GC-MS) determined the content of nine cooling agents (L-menthone, isomenthone, isomenthol, D,L-menthol, menthyl acetate, menthyl lactate, WS-3, WS-5 and WS-23) in tobacco shreds / reconstituted tobacco leaves of the two types of cigarettes (nine samples each) and their mainstream smoke release, and calculated the smoke transfer rate of seven cooling agents.

[0008] In menthol cigarettes, the use of multiple cooling agents in combination is becoming increasingly common. This combination can overcome the limitations of menthol alone. By complementing the different components in terms of evaporation rate and cooling intensity, it creates a three-dimensional sensory experience with distinct layers in the "first, middle and last inhalation stages", making the cooling sensation and the natural aroma of tobacco blend more smoothly and harmoniously.

[0009] With the continuous development of research on cooling agents, new combinations of cooling agents are constantly emerging. For example, the combination of WS-3 and menthone glycerol acetal (6-isopropyl-9-methyl-1,4-dioxospiro[4,5]decane-2-methanol) utilizes WS-3 to provide a strong and lasting cooling impact, and utilizes the mild and rounded characteristics of menthone glycerol acetal to modify the taste and add flavor. Combined with the similar thermal stability of the two, it has become an ideal combination for creating high-quality cooling cigarettes.

[0010] WS-3 and menthone glycerol ketal have similar polarities, which can easily lead to overlapping chromatographic peaks and poor peak shape, making baseline separation impossible. This makes it difficult to achieve effective separation and accurate quantification of WS-3 and menthone glycerol ketal when analyzing multiple cooling agents such as WS-3 and menthone glycerol ketal simultaneously.

[0011] In addition, existing technologies mostly test and analyze cooling agents in single parts such as tobacco, filter (including flavor capsules), cigarette paper, or tipping paper. However, when actually smoking cigarettes with cooling agents added in multiple parts, the cooling agents in different parts of the cigarette will affect each other. Existing studies on the transfer behavior of cooling agents in single parts cannot reflect the actual transfer of cooling agents in the whole cigarette.

[0012] When analyzing a whole cigarette, the matrix of the whole cigarette is more diverse and complex than that of a single part. More than 5,000 components of cigarette matrix have been detected so far. Under the interference of complex matrix, it is more difficult to analyze the combination of novel cooling agents (such as WS-3 and menthone glycerol ketal) in the whole cigarette. Summary of the Invention

[0013] The purpose of this invention is to provide a method for testing the transfer behavior of cooling agents in whole cigarettes, solving the problem that existing gas chromatography-mass spectrometry analysis methods cannot accurately evaluate the transfer rate of novel compound cooling agents such as WS-3 and menthone glycerol ketal.

[0014] The second objective of this invention is to provide a method for analyzing the content of cooling agents in a whole cigarette, thereby solving the problem that existing gas chromatography-mass spectrometry analysis methods cannot achieve baseline separation and accurate quantification of novel compound cooling agents such as WS-3 and menthone glycerol ketal in a whole cigarette.

[0015] The third objective of this invention is to provide a method for analyzing the content of cooling agents in mainstream cigarette smoke, thereby solving the problem that existing gas chromatography-mass spectrometry analysis methods cannot achieve baseline separation and accurate quantification of novel compound cooling agents such as WS-3 and menthol glycerol ketal in mainstream cigarette smoke.

[0016] To achieve the above objectives, the technical solution of the method for testing the transfer behavior of cooling agents in whole cigarettes adopted in this invention is as follows:

[0017] The test method for the transfer behavior of cooling agents in a whole cigarette includes the following steps:

[0018] (1) After the whole cigarette is crushed, methanol and internal standard are added for extraction to obtain cigarette sample solution; the mainstream smoke of the corresponding whole cigarette is captured and extracted under the same conditions to obtain smoke sample solution.

[0019] (2) The cigarette sample solution and the smoke sample solution were subjected to gas chromatography-mass spectrometry analysis under the same conditions to quantify the cooling agent in the sample;

[0020] The cooling agent contains at least WS-3 and menthone glycerol ketal;

[0021] The gas chromatography-mass spectrometry analysis conditions were as follows: a highly polar column; a programmed temperature increase: an initial temperature of 55-65℃, followed by an increase at a rate of 10-20℃ / min to 240-260℃ and holding.

[0022] (3) Calculate the transfer rate of cooling agent in the whole cigarette based on the quantitative results of step (2).

[0023] This invention is an improved invention. In response to the difficulty in separating complex cigarette matrices and WS-3 and menthone glycerol ketal, a highly polar chromatographic column is selected and matched with optimized temperature program conditions to achieve baseline separation of novel compound cooling agents such as WS-3 and menthone glycerol ketal. The chromatographic peaks of all compounds are sharp and symmetrical, enabling accurate quantification and accurate evaluation of transfer rate.

[0024] Meanwhile, by comparing the relative standard deviations of the extraction effects of various extraction solvents on target cooling agents such as (-)-menthyl acetate, menthone glycerol ketal, and WS-23, methanol showed better extraction effect, which is beneficial for the accurate analysis of multiple cooling agents at the same time.

[0025] This method can provide an overall evaluation of the transfer behavior of novel compound cooling agents such as WS-3 and menthone glycerol ketal, which is of great significance for the research and development and optimization of corresponding menthol cigarette products.

[0026] Preferably, the cooling agent includes p-menthane-3,8-diol and WS-27. Both p-menthane-3,8-diol and WS-27 are novel cooling agents, and this method can simultaneously achieve accurate quantitative analysis of these novel cooling agents.

[0027] More preferably, the cooling agent includes one or more of (+)-isopreneol, L-menthone, isomenthone, D,L-menthone, WS-23, and (-)-menthyl acetate. The boiling points of (+)-isopreneol and L-menthone, isomenthone and D,L-menthone, and WS-23 and (-)-menthyl acetate are quite close, approximately 90°C, 216°C, and 230°C respectively, making baseline separation difficult. Furthermore, these three pairs of compounds are common cooling agents in menthol cigarettes. Simultaneous analysis of these cooling agent varieties further demonstrates the adaptability of this method, facilitating a systematic and comprehensive analysis of the cooling agent in the entire cigarette.

[0028] Preferably, the cooling agents comprise 13 types, namely L-menthone, isomenthone, (-)-menthyl acetate, (+)-isomenthyl alcohol, D,L-menthyl alcohol, piperonone, WS-23, WS-27, menthyl lactate, p-menthane-3,8-diol, WS-3, menthone glycerol ketal, and WS-5. These cooling agents include all four types specified in Appendix A of the national standard for electronic cigarettes (GB 41700): menthyl acetate, D,L-menthyl alcohol, WS-23, and WS-3 (WS-3 and WS-23 are currently the most widely used carboxamide cooling agents); they also contain many cooling agent intermediates (isomenthyl alcohol and menthone can be reduced to menthol, and isomenthyl alcohol is an indispensable intermediate in the synthesis of menthol); menthyl lactate is usually an important component of compound cooling agents. This method enables simultaneous analysis of the above 13 types of cooling agents, improving the efficiency of routine analysis of various common and novel cooling agents in cigarettes and reducing analysis costs.

[0029] More preferably, isomenthone, L-menthone, (-)-menthyl acetate, (+)-isomenthyl alcohol, and D,L-menthyl alcohol use acetophenone-D8 as internal standards, while the remaining compounds use 2',2-bipyridine-D8 as internal standards. Using this internal standard quantification method ensures high quantitative accuracy because the retention times of the internal standard compounds are close to those of the target analytes, resulting in similar quantification accuracy.

[0030] Preferably, the highly polar chromatographic column is HP-INNOWAX, the initial temperature is held for 1-2 minutes, and the temperature is raised to 240-260°C and held for 6-7 minutes. Under these preferred chromatographic conditions, the analysis time is less than 20 minutes, achieving good separation results in a short time and meeting the requirements of a rapid and efficient method.

[0031] Preferably, 2-5 cigarettes containing cigarette paper and filters are crushed. If the filters contain flavor capsules, these capsules are crushed. Then, 50-60 mL of methanol is added for extraction. This sampling method effectively avoids the adverse effects of sample inhomogeneity on testing. The internal standard is added in the form of an internal standard solution; 0.5-0.8 mL of internal standard solution is added for every 50 mL of methanol added. The solvent for the internal standard solution can be methanol, etc. The amount of internal standard solution added is controlled to maintain an internal standard concentration of 2.0-4.0 μg / mL in the sample solution.

[0032] More preferably, the extraction is performed using shaking extraction for 40-60 minutes. Although ultrasonic extraction is a common method to improve extraction efficiency, prolonged ultrasonication can cause the temperature of the sample solution to rise and may lead to thermal decomposition of the target analyte. Compared to ultrasonic extraction, shaking extraction avoids thermal decomposition of the target analyte and ensures extraction equilibrium within the aforementioned extraction time.

[0033] Preferably, the transfer rate is calculated according to the following formula:

[0034]

[0035] TR1 represents the transfer rate of each cooling agent from the cigarette to the mainstream smoke, in percentage.

[0036] C 烟气 and C 烟支 The concentrations (μg / mL) of each cooling agent in the mainstream smoke extract and cigarette extract are respectively.

[0037] V 烟气 and V 烟支 The volumes, in mL, are the mainstream smoke extract and the cigarette extract, respectively.

[0038] n 烟气 and n 烟支 These represent the number of cigarettes collected during mainstream smoke extraction and the number of cigarettes extracted, respectively.

[0039] The technical solution of the method for analyzing the content of cooling agent in a whole cigarette according to the present invention is as follows:

[0040] The analytical method for the content of cooling agent in a whole cigarette includes the following steps:

[0041] S1. After crushing the whole cigarette, methanol and internal standard were added for extraction to obtain the cigarette sample solution;

[0042] S2. Perform gas chromatography-mass spectrometry analysis on the cigarette sample solution to quantify the cooling agent in the sample;

[0043] The cooling agent contains at least WS-3 and menthone glycerol ketal;

[0044] The gas chromatography-mass spectrometry analysis conditions were as follows: a highly polar column; a programmed temperature increase of 55-65℃ initially, followed by a rate of 10-20℃ / min to 240-260℃ and holding.

[0045] The present invention provides a method for analyzing the content of cooling agents in whole cigarettes. Through optimized chromatographic columns and programmed temperature conditions, it achieves GC-MS analysis of novel compound cooling agents such as WS-3 and menthone glycerol ketal in cigarettes. This method solves the separation problem of substances with similar polarity and thermal stability, such as WS-3 and menthone glycerol ketal, and enables accurate quantification of the content of various compound cooling agents. Compared with GC-QTOF MS, it simplifies the analytical operation and reduces analytical costs.

[0046] Preferably, the cooling agent includes one or more of (+)-isopreneol, L-menthone, isomenthone, D,L-menthone, WS-23, (-)-menthyl acetate, piperone, p-menthane-3,8-diol, WS-27, menthyl lactate, and WS-5.

[0047] Preferably, the highly polar chromatographic column is HP-INNOWAX, the initial temperature is held for 1-2 minutes, and the temperature is raised to 240-260°C and held for 6-7 minutes.

[0048] Preferably, 2-5 cigarettes containing cigarette paper and filter are crushed. If the filter contains a flavor capsule, the flavor capsule is crushed and then 50-60 mL of methanol is added for extraction.

[0049] The technical solution of the method for analyzing the content of cooling agents in mainstream cigarette smoke of the present invention is as follows:

[0050] The analytical method for the content of cooling agents in mainstream cigarette smoke includes the following steps:

[0051] a) After smoking the cigarette, place the filter containing the total particulate matter of the mainstream smoke in a container, add methanol and internal standard for extraction, and obtain the smoke sample solution.

[0052] b) Perform gas chromatography-mass spectrometry analysis on the flue gas sample solution to quantify the cooling agent in the sample;

[0053] The cooling agent contains at least WS-3 and menthone glycerol ketal;

[0054] The gas chromatography-mass spectrometry analysis conditions were as follows: a highly polar column; a programmed temperature increase of 55-65℃ initially, followed by a rate of 10-20℃ / min to 240-260℃ and holding.

[0055] The method for analyzing the content of cooling agents in mainstream cigarette smoke of the present invention can effectively separate cooling agents such as WS-3 and menthol glycerol ketal from mainstream cigarette smoke, thereby improving the quantitative accuracy of the above-mentioned cooling agents in mainstream cigarette smoke.

[0056] Preferably, the cooling agent includes one or more of (+)-isopreneol, L-menthone, isomenthone, D,L-menthone, WS-23, (-)-menthyl acetate, piperone, p-menthane-3,8-diol, WS-27, menthyl lactate, and WS-5.

[0057] Preferably, the highly polar chromatographic column is HP-INNOWAX, the initial temperature is held for 1-2 minutes, and the temperature is raised to 240-260°C and held for 6-7 minutes.

[0058] Preferably, 2-5 cigarettes are smoked. Before smoking cigarettes with flavor capsules, the flavor capsule is broken, and 50-60 mL of methanol is added for extraction. This sampling method, used for mainstream cigarette smoke, can improve sample homogeneity and achieve reproducibility and stability of test results. Attached Figure Description

[0059] Figure 1 The separation effect of 13 cooling agents and 2 internal standards in this invention on a DB-624UI chromatographic column is shown. The compounds corresponding to No. 1-15 are L-menthone, isomenthone, (-)-menthyl acetate, (+)-isomenthyl alcohol, D,L-menthyl alcohol, acetophenone-D8 (internal standard 1), piperone, WS-23, WS-27, menthyl lactate, p-menthane-3,8-diol, 2'2-bipyridine-D8 (internal standard 2), WS-3, menthone glycerol ketal and WS-5;

[0060] Figure 2 The separation effect of 13 cooling agents and 2 internal standards in this invention on a DB-5MS column is shown. The compounds corresponding to No. 1-15 are the same as those in the DB-5MS column. Figure 1 ;

[0061] Figure 3 The separation effect of 13 cooling agents and 2 internal standards in this invention on the HP-INNOWAX chromatographic column is shown. The compounds corresponding to No. 1-15 are the same as those in the previous invention. Figure 1 ;

[0062] Figure 4 The separation effect of 13 cooling agents and 2 internal standards in this invention under the first programmed temperature rise condition is shown. The compounds corresponding to No. 1-15 are the same. Figure 1 ;

[0063] Figure 5 This invention demonstrates the separation effect of 13 cooling agents and 2 internal standards under the second programmed temperature rise condition. Compounds 1-15 correspond to the same... Figure 1 ;

[0064] Figure 6 This invention demonstrates the separation effect of 13 cooling agents and 2 internal standards under the third programmed temperature rise condition. Compounds 1-15 correspond to the same... Figure 1 ;

[0065] Figure 7 The separation effect of 13 cooling agents and 2 internal standards in this invention under the fourth temperature program is shown. Compounds 1-15 correspond to the same... Figure 1 ;

[0066] Figure 8 The separation effect of 13 cooling agents and 2 internal standards in this invention under the fifth temperature program condition is shown. Compounds 1-15 correspond to the same... Figure 1 ;

[0067] Figure 9 This is the result of optimizing the extraction solution type using actual samples in this invention;

[0068] Figure 10 The results of the optimized cigarette count and extract volume ratio using actual samples in this invention;

[0069] Figure 11 The ultrasonic extraction time is the result of optimization using actual samples in this invention;

[0070] Figure 12 This is the result of the optimized oscillation extraction time using actual samples in this invention;

[0071] Figure 13 This is a typical chromatogram obtained from analyzing actual samples in this invention. Detailed Implementation

[0072] (I) Preferred embodiments of the method for testing the transfer behavior of cooling agents in whole cigarettes and the method for analyzing the content of cooling agents of the present invention

[0073] To enable routine analysis and quality optimization of cooling agents in cigarettes that use a variety of existing cooling agent blends, and to adapt to the analysis of novel cooling agent combinations, this invention provides a gas chromatography-mass spectrometry (GC-MS) method covering 13 cooling agents. This method can accurately determine and quantify multiple groups of cooling agents with similar polarity and thermal stability, such as WS-3 with menthone glycerol ketal, (+)-isomentheptyl alcohol and L-menthone, isomenthone and D,L-menthol, WS-23 and (-)-menthyl acetate, as well as novel cooling agents such as menthane-3,8-diol and WS-27. This allows for comprehensive analysis of the content testing and transfer behavior analysis of various cooling agent combinations throughout the entire cigarette.

[0074] The difficulty in simultaneously analyzing the above 13 cooling agents lies in the presence of easily pyrolytic substances (such as isomenthone and isomenthol); the significant differences in polarity among the target cooling agents, making it impossible to simultaneously guarantee a high extraction rate; and the presence of combinations of cooling agents that are difficult to separate, such as WS-3 and menthone glycerol ketal.

[0075] Under the aforementioned constraints, this invention, through comparison of seven extraction solvents, determined that methanol exhibits the best extraction rate and is the optimal solvent for achieving near-perfect extraction of various target cooling agents. By comparing three chromatographic columns and matching at least five temperature programs, effective separation and accurate quantification of each target cooling agent were achieved. The optimization of the solvent extraction and chromatographic-mass spectrometry analysis conditions fully considered the influence of pyrolysis temperature, ultimately determining the key control conditions.

[0076] Compared with existing GC-MS methods, this method can simultaneously analyze a wider range of cooling agents, including combinations of cooling agents that are difficult to separate, such as WS-3 and menthone glycerol ketal. The recoveries of each target cooling agent are concentrated between 90.4% and 105.5%, demonstrating better accuracy than existing technologies and achieving unexpected technical results. Based on these results, a comprehensive analysis of various mainstream cooling agent combinations in cigarettes can be achieved, providing efficient and economical basic data for quality monitoring and style optimization of menthol cigarettes.

[0077] For GC-MS analysis, the chromatographic column was HP-INNOWAX; the temperature program was as follows: initial temperature 55~65℃, hold for 1~2 min, increase to 240~260℃ at a rate of 10~20℃ / min and hold for 6~7 min.

[0078] The splitless injection mode was used with an injection volume of 1.0 μL and a solvent cut-off time of 5.5 min. High-purity He was used as the carrier gas at a flow rate of 1.0 mL / min. The temperatures of the injection port, ion source, and transfer line were 250℃, 230℃, and 250℃, respectively. The mass spectrometer ionization source was an EI source with an ionization voltage of 70 eV. The scanning mode was selected ion scanning.

[0079] The preferred embodiments of the present invention will be illustrated below with reference to examples.

[0080] Example 1

[0081] The method for testing the transfer behavior of cooling agent in a whole cigarette in this embodiment adopts the following steps:

[0082] The first step was the preparation of a series of standard solutions: using methanol as the solvent and 13 cooling agent standards as solutes—L-menthone, isomenthone, (-)-menthyl acetate, (+)-isomenthyl alcohol, D,L-menthyl alcohol, piperonone, WS-23, WS-27, menthyl lactate, p-menthane-3,8-diol, WS-3, 6-isopropyl-9-methyl-1,4-dioxospiro[4,5]decane-2-methanol (i.e., menthone glycerol ketal) and WS-5—as well as acetophenone-D8 and 2'2-bipyridine-D8 as internal standards, a series of standard solutions with the following concentrations were prepared: 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, and 20.0. The concentrations of piperonitrile and WS-5 were 0.45, 0.90, 2.25, 4.50, 9.00, 22.50, 45.00 and 90.00 μg / mL, and the internal standard concentration in all standard series solutions was 2.0 μg / mL.

[0083] The second step is the preparation of the sample solution: The whole cigarette is pulverized and extracted with methanol and an internal standard to obtain a cigarette sample solution; the mainstream smoke from the corresponding whole cigarette is captured and extracted under the same conditions to obtain a smoke sample solution. Specifically:

[0084] Extraction of cooling agent in cigarettes: Cut two cigarettes containing cigarette paper and filter into small segments of 0.5-1.0 cm. If the filter contains a flavor capsule, crush the capsule. Add 0.50 mL of internal standard solution (solvent is methanol, internal standard concentration is 200 μg / mL) and 50 mL of methanol. Place the mixture on a vortex mixer and extract at a rate of 2000 rpm for 40 min. After standing, collect the supernatant, which is the cigarette sample solution, for later use.

[0085] Collection and extraction of cooling agents in mainstream cigarette smoke: Cigarettes were smoked 5 times each time using a linear smoking machine according to the conditions specified in GB / T 19609 and ISO 4387. Before smoking cigarettes with flavor capsules, the flavor capsules were broken. The filter containing the total particulate matter of the mainstream smoke was placed in a container, and 0.50 mL of internal standard solution (as above) and 50 mL of methanol were added. The mixture was placed in a vortex mixer and extracted at a rate of 2000 rpm for 40 min. After standing, the supernatant was collected as the smoke sample solution for later use.

[0086] The third step involved gas chromatography-mass spectrometry (GC-MS) analysis of the standard series solutions and sample solutions under the same conditions: GC-MS conditions were as follows: HP-INNOWAX column, 30 m × 0.25 mm × 0.25 μm; temperature program: initial temperature 60℃, held for 1.0 min, increased to 240℃ at a rate of 20℃ / min and held for 6.0 min; splitless injection mode, injection volume 1.0 μL, solvent cut-off time 5.5 min; high-purity He as carrier gas, flow rate 1.0 mL / min; injection port, ion source, and transfer line temperatures 250℃, 230℃, and 250℃, respectively; EI source; ionization voltage 70 eV; selected ion scan mode. The retention times and monitored ion mass-charge ratios of the 13 target analytes and 2 internal standards under the above conditions are shown in Table 1 below.

[0087] Table 1. Retention times of target analytes and internal standards, and monitoring ions in the method of this invention.

[0088]

[0089] Step 4: Characterization of cooling agent transfer behavior:

[0090] (1) Quantification of cooling agents in the sample: The ratio of the peak area of ​​each cooling agent in the standard series solution to the peak area of ​​the internal standard is used as the ordinate (isomenthone, L-menthone, (-)-menthyl acetate, (+)-isomenthyl alcohol and D,L-menthyl alcohol are internally referenced with acetophenone-D8, and the other compounds are internally referenced with 2'2-bipyridine-D8), and the concentration of each cooling agent in the standard series solution is used as the abscissa. The working curve is shown in Table 2. Based on the ratio of the peak area of ​​each cooling agent in the sample solution to the peak area of ​​the internal standard, the content of each cooling agent in the sample is obtained according to the working curve.

[0091] The concentrations corresponding to the signal-to-noise ratio (S / N) of the target analyte at 3 and 10 are defined as the limits of detection and the limits of quantitation. The linear range, working curve, limit of quantitation, and limit of detection of the target analyte in this invention are shown in Table 2.

[0092] Table 2. Linear range, working curve, limit of quantitation, and limit of detection of the target analyte in this invention.

[0093]

[0094] To examine the repeatability of this method, samples with low, medium, and high concentrations were injected, and five samples prepared within one day were measured to calculate the intraday relative standard deviation at different concentrations. Samples prepared over three consecutive days were extracted to calculate the interday relative standard deviation at different concentrations.

[0095] The results are shown in Table 3. The intra-day and inter-day precision of the target analyte at different concentrations were less than 6.6% and 7.3%, respectively. This indicates that the sensitivity and repeatability of the method can meet the requirements for detecting the content of cooling agents.

[0096] Table 3. Repeatability data of the target analyte in this invention

[0097]

[0098] (2) Calculation of the transfer rate of cooling agent in cigarettes: Substitute the contents of the cigarette extract and the mainstream cigarette smoke into the following formula to calculate the transfer rate of cooling agent in the whole cigarette.

[0099]

[0100] TR1 represents the transfer rate of each cooling agent from the cigarette to the mainstream smoke, in percentage.

[0101] C 烟气 and C 烟支 The concentrations (μg / mL) of each cooling agent in the mainstream smoke extract and cigarette extract are respectively.

[0102] V 烟气 and V 烟支The volumes, in mL, are the mainstream smoke extract and the cigarette extract, respectively.

[0103] n 烟气 and n 烟支 These represent the number of cigarettes collected during mainstream smoke extraction and the number of cigarettes extracted, respectively.

[0104] Example 2

[0105] The method for analyzing the content of cooling agent in whole cigarettes in this embodiment involves pulverizing the whole cigarette, adding methanol and an internal standard for extraction to obtain a cigarette sample solution, and then performing gas chromatography-mass spectrometry analysis on the cigarette sample solution to quantify the cooling agent in the sample. The specific implementation details are the same as the analysis process of the cigarette sample solution in Example 1.

[0106] Example 3

[0107] The method for analyzing the content of cooling agents in mainstream cigarette smoke in this embodiment involves placing a filter containing the total particulate matter of mainstream smoke in a container after smoking the cigarette, adding methanol and an internal standard for extraction to obtain a smoke sample solution, and then performing gas chromatography-mass spectrometry analysis on the smoke sample solution to quantify the cooling agents in the sample. The specific implementation details are the same as the analysis process of the smoke sample solution in Example 1.

[0108] (ii) Comparative Example

[0109] Comparative Example 1: Selection of Gas Chromatography Column

[0110] To achieve ideal separation results, this study investigated the separation performance of three different coated and polar columns using a mixed standard solution. Specific information on the columns and temperature programs are shown in Table 4. The total ion chromatograms of the preliminary separation of the mixed standard solution on the three columns are shown below. Figure 1 .

[0111] Table 4. Column Specifications

[0112]

[0113] Depend on Figure 1 It can be seen that the response value of menthone glycerol acetal (compound 14) is small, and the chromatographic peak has a tail.

[0114] Furthermore, compared to the other two columns, the target analytes exhibited longer overall retention times on the DB-624UI column: all 12 target analytes and 2 internal standard compounds were eluted within 25 minutes, with the last compound (WS-5) retaining for over 34 minutes. In contrast, all compounds on the other two columns were eluted within 20 minutes. This is likely due to the longer column length and thicker membrane of the DB-624UI, resulting in stronger retention of the target compounds.

[0115] Depend on Figure 2 It can be seen that the relative response value of menthone glycerol acetal (compound 14) is improved compared with the DB-624UI column, but it leads to poor separation from WS-3 (compound 13). At the same time, the relative response value of 2'2-bipyridine-D8 (internal standard 2, compound 12) is found to be significantly reduced, which may bring uncertainty to the subsequent quantification.

[0116] Meanwhile, since the boiling points of (+)-isoprene and L-menthone, isoprene and D,L-menthone, and WS-23 and (-)-menthyl acetate are relatively close, with boiling points of approximately 90℃, 216℃ and 230℃ respectively, these three pairs of compounds could not be baseline separated on the DB-5MS column.

[0117] Depend on Figure 3 It can be seen that all compounds can be well separated on the HP-INNOWAX column, and the analysis time is less than 20 minutes, which meets the requirements of a rapid and efficient method. Therefore, subsequent experiments were carried out on the HP-INNOWAX column.

[0118] Comparative Example 2: Optimization of Program Temperature Rise Conditions

[0119] To achieve effective separation of all target compounds in a shorter time, the temperature program was investigated based on the previously optimized column, including optimization of the initial column temperature and heating rate, as detailed in Table 5.

[0120] Table 5. Heating Program

[0121]

[0122] Generally, a higher initial column temperature can reduce chromatographic separation time; however, in this invention, the applicant unexpectedly discovered that, as Figure 4 As shown, when the initial temperature is 100℃, the chromatographic peaks of the seven compounds with the earliest retention times exhibit poor peak shape and cannot be separated; for example... Figure 5 As shown, when the initial temperature is reduced to 80℃, the number of compounds with poor peak shape decreases to 4; Figure 6 As shown, when the initial temperature was further reduced to 60℃, the chromatographic peaks of all compounds were sharp and symmetrical. This is likely because compounds with earlier retention times undergo low-temperature condensation at the column head, reducing diffusion at high temperatures and thus resulting in better separation. To achieve good separation within a shorter time, the initial temperature of the chromatographic column was set to 60℃.

[0123] Appropriately increasing the heating rate can also reduce the overall chromatographic analysis time. Therefore, with the initial column temperature set at 60℃, different heating rates were investigated. The results are as follows: Figures 6-8 As shown, with the continuous increase in heating rate, the elution time of all compounds decreased from 22 minutes to 14 minutes. Considering that the separation degree of (-)-menthyl acetate and (+)-isomenthyl alcohol, 2',2-bipyridine-D8 and WS-3 decreased with the continuous increase in heating rate, baseline separation of the two groups of compounds could be basically achieved at a heating rate of 20 °C / min, and all compounds could be eluted in a relatively short time (14 minutes). Therefore, a faster heating rate was not considered, and the temperature program rate was set to 20 °C / min.

[0124] Comparative Example 3: Optimization of Cigarette Extract Types

[0125] Since the research object is the whole cigarette, it is not possible to selectively add a spike to specific parts of the cigarette (e.g., only tobacco, filter, flavor capsule, cigarette paper, tipping paper) when examining the sample pretreatment conditions. Moreover, adding a spike cannot guarantee the homogeneity of the sample. Therefore, in the actual experiment, unspiked commercial cigarettes containing the target analyte were used as the research object.

[0126] The 13 cooling agents in this invention have certain differences in properties (polarity, solubility, etc.). In order to ensure that all compounds have good extraction effects, a certain domestic cigarette was used as the research object, and the extraction effects of seven solvents with different polarities (methanol, ethanol, isopropanol, ethyl acetate, acetone, dichloromethane, and cyclohexane) on the cooling agents in the actual sample were investigated.

[0127] Crush the flavor capsules from two cigarettes, cut the whole cigarette into small pieces of 0.5-1.0 cm, fix the extraction volume at 50 mL, shake and extract for 15 minutes, collect the supernatant after standing, and analyze it by gas chromatography-mass spectrometry. The results are as follows. Figure 9 As shown.

[0128] Seven cooling agents were detected in the cigarette. The relative standard deviations (RSDs) of most compounds under different extraction solvents ranged from 7.2% to 10.9%, indicating little difference in extraction efficiency between different solvents. For the compounds with significantly different RSDs (RSDs) under different extraction solvents, namely (-)-menthyl acetate, menthone glycerol ketal, and WS-23 (RSDs ranging from 17.9% to 33.8%), methanol showed better extraction efficiency. Therefore, methanol was ultimately selected as the extraction solvent.

[0129] Comparative Example 4: Optimization of the tobacco extract feed-to-liquid ratio (number of cigarettes / volume of extract)

[0130] To determine the appropriate number of cigarettes and the volume ratio of the extract, based on previous optimizations, different numbers of cigarettes were extracted with 50 mL of methanol as the extractant using shaking for 15 minutes. After standing, the supernatant was collected and analyzed by gas chromatography-mass spectrometry. The results are as follows: Figure 10 As shown.

[0131] It can be observed that the relative standard deviations of all compounds under different extraction solvents range from 5.4% to 9.3%, indicating that the test results do not differ significantly with different numbers of cigarettes. Considering the sample volume and minimizing the impact of sample inhomogeneity on the test results, the cigarette extraction solution ratio (number of cigarettes / volume of extraction solution) was fixed at 50 mL of extraction solution for 2 cigarettes.

[0132] Comparative Example 5: Optimization of Extraction Time and Extraction Method

[0133] Two cigarettes were extracted with 50 mL of methanol as the extraction solvent by ultrasonication or shaking for different times. After standing, the supernatant was collected and analyzed by gas chromatography-mass spectrometry. The results are as follows: Figure 11 and Figure 12 As shown.

[0134] It can be observed that both extraction methods show the same results: except for WS-23, all other compounds can achieve good extraction results within 20 min. For WS-23, extraction equilibrium is reached at 40 min, therefore the extraction time is set at 40 min.

[0135] In terms of extraction method selection, prolonged ultrasound can cause the temperature of the sample solution to rise, which is not conducive to the stability of the target analyte. In order to avoid frequent replacement of water in the ultrasound generator during the extraction process to ensure a constant temperature, the extraction method is set as vortex oscillation.

[0136] Comparative Example 6 Recovery Test

[0137] To verify the accuracy of the established method, a spiked experiment was conducted using a domestically produced cigarette brand. The spiked concentrations were 2, 20, and 100 times the lowest linear range of each target analyte. After spiked analysis, extraction and subsequent gas chromatography-mass spectrometry (GC-MS) analysis were performed. The ratio of the obtained peak areas was substituted into the standard working curve of the target analyte to calculate the measured concentration, and the relative recovery rate was obtained by comparing it with the actual spiked amount.

[0138] As shown in Table 6, the relative recoveries of the target analytes at the three concentrations in the samples ranged from 90.4% to 105.5%, indicating that the method has good accuracy and can meet the accuracy requirements for the analysis of cooling agents in routine samples.

[0139] Table 6. Recovery rate data (%) of the target analyte in this invention

[0140]

[0141] Analysis of Cooling Agent Transfer in Comparative Example 7 Cigarettes

[0142] The method of this invention was used to analyze six types of cigarettes and their corresponding mainstream cigarette smoke. The results of the detected compounds are shown in Table 7.

[0143] Table 7. Results of analysis of cigarette cigarettes and mainstream cigarette smoke in this invention (μg / mL, “ND” indicates not detected)

[0144]

[0145] The results show that a total of 8 compounds were detected in all cigarette samples, namely isomenthone, L-menthone, (-)-menthyl acetate, D,L-menthol, WS-23, piperone, menthone glycerol acetal, and WS-3.

[0146] Among them, sample F contained seven types of cooling agents, making it the sample with the highest variety and content of cooling agents. Its typical chromatogram is shown below. Figure 13 As shown, under optimized conditions, the matrix does not interfere with the quantification of the target analyte, indicating good selectivity of this method. Sample A was the second most frequently detected, containing four different cooling agents, making it the sample with the second highest variety and content of cooling agents. D,L-menthol was detected in all cigarette samples, making it the compound with the highest detection rate, followed by WS-23, which was detected in five cigarette samples. Substituting these results into the transfer rate formula, the transfer values ​​of different cooling agents from cigarettes to the mainstream smoke can be obtained, as shown in Table 8.

[0147] Table 8. Transfer rate of cooling agent from cigarette cigarettes to mainstream smoke (%, “ / ” indicates that the cooling agent was not added to the cigarette)

[0148]

[0149] The results show that due to differences in cigarette design parameters and the physicochemical properties of the cooling agents themselves, the transfer rates of different cooling agents within the same sample or the same cooling agent in different samples varied. Specifically, all cooling agents in sample F transferred to the mainstream smoke to varying degrees, and D,L-menthol in all cigarette samples also transferred to the mainstream smoke to varying degrees. Excluding cooling agents that did not transfer to the mainstream smoke, the transfer rates of cooling agents to the mainstream smoke in different cigarette samples ranged from 1.01% to 18.14%.

[0150] As can be seen from the description, this method can detect 13 target substances, covering single-component cooling agents containing menthol, single-component cooling agents without menthol, and artificial cooling agents. The number of target analytes is significantly greater than that under existing analytical conditions. Furthermore, it achieves effective separation and quantification of several difficult-to-separate cooling agent combinations, such as WS-3 and menthone glycerol ketal. Simultaneously, this method can measure the transfer rate of cooling agents from the entire cigarette to the mainstream smoke, avoiding the mutual influence between different components of the cigarette (such as tobacco, filter, flavor capsule, cigarette paper, and tipping paper). The relevant methods and evaluation data can provide technical guidance for cigarette research and development, formula maintenance, and quality evaluation.

[0151] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. 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 testing the transfer behavior of cooling agents in a whole cigarette, characterized in that, Includes the following steps: (1) After the whole cigarette is crushed, methanol and internal standard are added for extraction to obtain cigarette sample solution; The mainstream smoke of the entire cigarette was captured and extracted under the same conditions to obtain a smoke sample solution. (2) The cigarette sample solution and the smoke sample solution were subjected to gas chromatography-mass spectrometry analysis under the same conditions to quantify the cooling agent in the sample; The cooling agent contains at least WS-3 and menthone glycerol ketal; The gas chromatography-mass spectrometry analysis conditions were as follows: a highly polar column; a programmed temperature increase: an initial temperature of 55-65℃, followed by an increase at a rate of 10-20℃ / min to 240-260℃ and holding. (3) Calculate the transfer rate of cooling agent in the whole cigarette based on the quantitative results of step (2).

2. The method for testing the transfer behavior of cooling agent in a whole cigarette as described in claim 1, characterized in that, The cooling agent includes p-menthane-3,8-diol and WS-27.

3. The method for testing the transfer behavior of cooling agent in a whole cigarette as described in claim 2, characterized in that, The cooling agent includes one or more of (+)-isopreneol, L-menthone, isopreneol, D,L-menthone, WS-23, and (-)-menthyl acetate.

4. The method for testing the transfer behavior of cooling agent in a whole cigarette as described in claim 1, characterized in that, The cooling agents comprise 13 types, namely L-menthone, isomenthone, (-)-menthyl acetate, (+)-isomenthyl alcohol, D,L-menthyl alcohol, piperone, WS-23, WS-27, menthyl lactate, p-menthane-3,8-diol, WS-3, menthone glycerol ketal, and WS-5.

5. The method for testing the transfer behavior of cooling agent in a whole cigarette as described in claim 4, characterized in that, Isomenthone, L-menthone, (-)-menthyl acetate, (+)-isomenthyl alcohol, and D,L-menthyl alcohol were treated with acetophenone-D8 as internal standard, while the remaining compounds were treated with 2'2-bipyridine-D8 as internal standard.

6. The method for testing the transfer behavior of cooling agent in a whole cigarette as described in claim 1, characterized in that, The highly polar chromatographic column is HP-INNOWAX, and the initial temperature is maintained for 1-2 minutes, followed by heating to 240-260°C and maintaining the temperature for 6-7 minutes.

7. The method for testing the transfer behavior of cooling agent in a whole cigarette as described in claim 1, characterized in that, Crush 2-5 cigarettes containing cigarette paper and filter. If the filter contains a flavor capsule, crush the capsule and then extract with 50-60 mL of methanol.

8. The method for testing the transfer behavior of cooling agent in a whole cigarette as described in claim 7, characterized in that, The extraction was performed using shaking extraction for 40-60 minutes.

9. The method for testing the transfer behavior of cooling agent in a whole cigarette as described in any one of claims 1 to 8, characterized in that, The transfer rate is calculated using the following formula: TR1 represents the transfer rate of each cooling agent from the cigarette to the mainstream smoke, in percentage. C 烟气 and C 烟支 The concentrations (μg / mL) of each cooling agent in the mainstream smoke extract and cigarette extract are respectively. V 烟气 and V 烟支 The volumes, in mL, are the mainstream smoke extract and the cigarette extract, respectively. n 烟气 and n 烟支 These represent the number of cigarettes collected during mainstream smoke extraction and the number of cigarettes extracted, respectively.

10. A method for analyzing the content of cooling agents in a whole cigarette, characterized in that, Includes the following steps: S1. After crushing the whole cigarette, methanol and internal standard were added for extraction to obtain the cigarette sample solution; S2. Perform gas chromatography-mass spectrometry analysis on the cigarette sample solution to quantify the cooling agent in the sample; The cooling agent contains at least WS-3 and menthone glycerol ketal; The gas chromatography-mass spectrometry analysis conditions were as follows: a highly polar column; a programmed temperature increase of 55-65℃ initially, followed by a rate of 10-20℃ / min to 240-260℃ and holding.

11. The method for analyzing the content of cooling agent in a whole cigarette as described in claim 10, characterized in that, The cooling agent includes one or more of (+)-isopreneol, L-menthone, isomenthone, D,L-menthone, WS-23, (-)-menthyl acetate, piperone, p-menthane-3,8-diol, WS-27, menthyl lactate, and WS-5.

12. The method for analyzing the content of cooling agent in a whole cigarette as described in claim 10, characterized in that, The highly polar chromatographic column is HP-INNOWAX, and the initial temperature is maintained for 1-2 minutes, followed by heating to 240-260°C and maintaining the temperature for 6-7 minutes.

13. The method for analyzing the content of cooling agent in a whole cigarette as described in any one of claims 10 to 12, characterized in that, Crush 2-5 cigarettes containing cigarette paper and filter. If the filter contains a flavor capsule, crush the capsule and then extract with 50-60 mL of methanol.

14. A method for analyzing the content of cooling agents in mainstream cigarette smoke, characterized in that, Includes the following steps: a) After smoking the cigarette, place the filter containing the total particulate matter of the mainstream smoke in a container, add methanol and internal standard for extraction, and obtain the smoke sample solution. b) Perform gas chromatography-mass spectrometry analysis on the flue gas sample solution to quantify the cooling agent in the sample; The cooling agent contains at least WS-3 and menthone glycerol ketal; The gas chromatography-mass spectrometry analysis conditions were as follows: a highly polar column; a programmed temperature increase of 55-65℃ initially, followed by a rate of 10-20℃ / min to 240-260℃ and holding.

15. The method for analyzing the content of cooling agents in mainstream cigarette smoke as described in claim 14, characterized in that, The cooling agent includes one or more of (+)-isopreneol, L-menthone, isomenthone, D,L-menthone, WS-23, (-)-menthyl acetate, piperone, p-menthane-3,8-diol, WS-27, menthyl lactate, and WS-5.

16. The method for analyzing the content of cooling agents in mainstream cigarette smoke as described in claim 14, characterized in that, The highly polar chromatographic column is HP-INNOWAX, and the initial temperature is maintained for 1-2 minutes, followed by heating to 240-260°C and maintaining the temperature for 6-7 minutes.

17. The method for analyzing the content of cooling agent in mainstream cigarette smoke as described in any one of claims 14 to 16, characterized in that, Smoke 2-5 cigarettes. Before smoking a cigarette with a flavor capsule, break the capsule and add 50-60mL of methanol for extraction.

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