Method for measuring nicotine content in cigar smoke and application

Abstract

The invention discloses a method for determining the nicotine content in cigar smoke and application. The method comprises the following steps: capturing smoke total particulate matters of a to-be-detected cigar by using a Cambridge filter; adding an internal standard solution and an alcohol solvent into the Cambridge filter, and performing ultrasonic extraction to obtain an extracting solution; and determining and analyzing the extracting solution by adopting a gas chromatography-flame ionization detection method to obtain the nicotine content in the cigar smoke. The method is easy to operate and high in accuracy, compared with a traditional determination method, the problem that it is difficult to detect cigar from a sample due to the fact that the detection limit is high is effectively solved, sensitivity is remarkably improved, and a scientific basis is provided for development and classification evaluation of high-quality cigar products.

Description

Technical Field

[0001] The present application relates to the technical field of tobacco detection, and in particular to a method for determining the nicotine content in cigar smoke and its application. Background Art

[0002] Alkaloids are substances in cigar smoke that directly affect the chemical properties and sensory characteristics of smoke. They primarily include nicotine, nornicotine, myosmine, dienicotine, anabasine, anatabine, cotinine, and 2,3'-bipyridine. Nicotine accounts for over 95% of the total alkaloid content in cigar tobacco leaves. The energizing and satisfying feeling of cigar smoking is closely related to the nicotine in the smoke. Nicotine in mainstream cigar smoke is primarily produced by direct transfer of nicotine from the tobacco leaves and by high-temperature pyrolysis of the leaves. Therefore, accurate measurement of nicotine in mainstream cigar smoke plays a crucial role in the smokeability of cigars.

[0003] Currently, the main methods for analyzing nicotine in tobacco smoke include gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography, ion chromatography, and supercritical fluid chromatography, with GC being the mainstream method. The YC / T465-2013 standard specifies the use of GC for the detection of total particulate matter in mainstream cigar smoke, but this method suffers from a high detection limit, making it difficult to detect in samples. Summary of the Invention

[0004] Based on this, it is necessary to provide a method for determining the nicotine content in cigar smoke and its application.

[0005] A first aspect of the present application provides a method for determining the nicotine content in cigar smoke, comprising the following steps:

[0006] Cambridge filter was used to capture the total particulate matter in the smoke of the cigar to be tested;

[0007] adding an internal standard solution and an alcohol solvent to the Cambridge filter disc, performing ultrasonic extraction, and obtaining an extract;

[0008] The extract is measured and analyzed by gas chromatography-flame ionization detection to obtain the nicotine content in cigar smoke.

[0009] In some embodiments, the internal standard in the internal standard solution includes one or more of carvone, quinoline, n-heptadecane, 2-methylquinoline, and n-octadecane; and the alcohol solvent includes isopropanol.

[0010] In some embodiments, the ultrasonic extraction time is 30 min to 70 min.

[0011] In some embodiments, the concentration of the internal standard solution is 7.6 mg / mL to 7.8 mg / mL.

[0012] In some embodiments, the volume ratio of the internal standard solution to the alcohol solvent is 1:(30~35).

[0013] In some embodiments, the diameter of the Cambridge filter is 50 mm to 55 mm.

[0014] In some embodiments, the Cambridge filter is used to capture the particulate matter in the mainstream smoke of the cigar to be tested and then smoked and captured by a smoking machine.

[0015] In some embodiments, the aspiration volume is 40 mL to 50 mL.

[0016] In some embodiments, the suction time is 1 s to 2 s.

[0017] In some embodiments, the puffing frequency is 40s to 45s.

[0018] In some embodiments, before collecting the particulate matter in the mainstream smoke of the cigar to be tested using the Cambridge filter, the method further includes: equilibrating the cigar to be tested at a temperature of 20° C. to 24° C. and a relative humidity of 60% to 64% for 70 hours to 74 hours.

[0019] In some embodiments, the assay comprises quantitatively analyzing the nicotine content in cigar smoke using an internal standard curve method.

[0020] In some embodiments, the detection limit of the internal standard curve method for nicotine content in cigar smoke is 0.0096 mg / mL, and the quantification limit is 0.032 mg / mL.

[0021] In some embodiments, the gas chromatography in the gas chromatography-flame ionization detection method satisfies one or more of the following conditions:

[0022] The chromatographic column was a TG-WAXMS capillary column with specifications of 30 m × 0.25 mm × 0.25 μm;

[0023] The carrier gas control mode is constant flow mode, and the carrier gas flow rate is 1.5 mL / min~2 mL / min;

[0024] The injection volume is 1 μL~2 μL;

[0025] The injection method was split injection with a split ratio of (8-12):1;

[0026] The injection port temperature is 250℃~300℃;

[0027] Heating program: initial temperature is 100℃~120℃, maintain for 3min~5min, increase the temperature to 200℃~250℃ at a rate of 10℃ / min~15℃ / min, and maintain for 2min~3min.

[0028] In some embodiments, the flame ionization in the gas chromatography-flame ionization detection method satisfies one or more of the following conditions:

[0029] The temperature of the flame ionization detector is 250℃~300℃;

[0030] Air flow rate is 250 mL / min~300 mL / min;

[0031] The hydrogen flow rate is 50 mL / min~80 mL / min.

[0032] The second aspect of the present application provides a method for analyzing or classifying the nicotine content in the mainstream smoke of different types of cigars, comprising using the method for determining the nicotine content in the mainstream smoke of cigars described in the first aspect of the present application to test and obtain the nicotine content in the mainstream smoke of different types of cigars.

[0033] The aforementioned method, based on optimized Cambridge filter pretreatment conditions, measures nicotine content in cigar smoke using gas chromatography-flame ionization detection. This method is simple to use and highly accurate. Compared to traditional methods, it addresses the problem of high detection limits that hinder sample detection, significantly improving sensitivity and providing a scientific basis for the development, classification, and evaluation of high-quality cigar products. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments and examples of this application and to provide a more complete understanding of the application and its beneficial effects, the following briefly introduces the drawings required for use in the description of the embodiments or examples. Obviously, the drawings described below are only some embodiments of this application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0035] Figure 1 This is a chromatogram of a nicotine standard solution in one embodiment of the present application;

[0036] Figure 2 The effect of extraction time on nicotine extraction efficiency in one embodiment of the present application;

[0037] Figure 3 This is the standard working curve in one embodiment of the present application;

[0038] Figure 4This is a chromatogram of a cigar mainstream smoke sample solution in one embodiment of the present application;

[0039] Figure 5 The nicotine content in the mainstream smoke of cigars at different locations in one embodiment of the present application;

[0040] Figure 6 Nicotine content in mainstream smoke of different grades of cigars in one embodiment of the present application;

[0041] Figure 7 The nicotine content in the mainstream smoke of cigars from different origins in one embodiment of the present application. DETAILED DESCRIPTION

[0042] To facilitate understanding of the present application, a more comprehensive description of the present application will be provided below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present application. However, the present application may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present application.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0044] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0045] In this application, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute a limitation on the scope of protection of this application.

[0046] As used herein, the terms "having," "containing," "including," and "comprising" are synonymous and are inclusive or open-ended, not excluding additional, unrecited members or features. Examples of members or features include materials or components, structures, elements, and instruments. Non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, and states.

[0047] In this application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0048] In this application, when referring to the unit of a data range, if the unit is only followed by the right endpoint, it means that the units of the left endpoint and the right endpoint are the same.

[0049] In this application, if a method flow involves multiple steps, unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in an order other than the order described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages do not necessarily need to be completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating, or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0050] In this application, exemplary descriptions such as "in some embodiments (or examples)" and "in one embodiment (or example)" may include but are not limited to the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0051] In this application, the terms "first" and "second" in "the first aspect" and "the second aspect" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor should they be understood as implicitly indicating the importance or quantity of the technical features indicated. Furthermore, "first" and "second" serve only as non-exhaustive enumeration and description and should be understood not to constitute a closed-ended limitation on quantity.

[0052] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values within the numerical interval is deemed to be continuous and includes the two numerical endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe a feature or characteristic, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. "Numerical interval" allows for a broad range of numerical interval types including percentage intervals, ratio intervals, and ratio intervals.

[0053] The gas chromatography method specified in the current YC / T465-2013 standard for detecting total particulate matter in mainstream cigar smoke has a high detection limit, making it difficult to detect from samples.

[0054] Based on this, the embodiments of the present application at least provide a method and application for determining the nicotine content in cigar smoke.

[0055] In a first aspect of the present application, a method for determining the nicotine content in cigar smoke is provided, comprising the following steps:

[0056] S100: Use Cambridge filter to capture the total particulate matter in the smoke of the cigar to be tested;

[0057] S200: Add internal standard solution and alcohol solvent to the Cambridge filter disc, perform ultrasonic extraction, and obtain the extract;

[0058] S300: The extract is analyzed using gas chromatography-flame ionization detection to obtain the nicotine content in cigar smoke.

[0059] In some embodiments, in step S200, the internal standard in the internal standard solution includes one or more of carvone, quinoline, n-heptadecane, 2-methylquinoline, and n-octadecane.

[0060] In some embodiments, in step S200, the alcohol solvent includes isopropyl alcohol.

[0061] In some embodiments, in step S200, the volume ratio of the internal standard solution to the alcohol solvent is 1:(30-35). Without limitation, the volume ratio of the internal standard solution to the alcohol solvent can be, but is not limited to, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, or a ratio or range between any two of the above ratios.

[0062] In some embodiments, in step S200, an internal standard solution is prepared by dissolving the internal standard in isopropanol, and the concentration of the internal standard solution is 7.6 mg / mL to 7.8 mg / mL. Without limitation, the concentration of the internal standard solution can be, but is not limited to, 7.6 mg / mL, 7.7 mg / mL, 7.8 mg / mL, or a value or range between any two of the foregoing values.

[0063] In some embodiments, in step S200, the ultrasonic extraction time is 30 min to 70 min. Non-limitingly, the ultrasonic extraction time can be, but is not limited to, 30 min, 40 min, 50 min, 60 min, 70 min, or a value or range between any two of the above values.

[0064] In some embodiments, in step S100, the diameter of the Cambridge filter disc is 50 mm to 55 mm. In a non-limiting manner, the diameter of the Cambridge filter disc can be, but is not limited to, 50 mm, 51 mm, 52 mm, 53 mm, 54 mm, 55 mm, or a value or range between any two of the above values.

[0065] In some embodiments, in step S100, the Cambridge filter is used to capture the particulate matter in the mainstream smoke of the cigar to be tested and then the particulate matter is captured by a smoking machine. It should be noted that the smoking machine is a conventional smoking machine in the art and can be commercially available.

[0066] In some embodiments, the aspiration volume is 40 mL to 50 mL. Without limitation, the aspiration volume can be, but is not limited to, 40 mL, 42 mL, 44 mL, 46 mL, 48 mL, 50 mL, or a value or range between any two of the above values.

[0067] In some embodiments, the puff time is 1 s to 2 s. Without limitation, the puff time can be, but is not limited to, 1 s, 1.5 s, 2 s, or a value or range between any two of the above values.

[0068] In some embodiments, the puffing frequency is 40s to 45s. Without limitation, the puffing frequency can be, but is not limited to, 41s, 42s, 43s, 44s, 45s, or a value or range between any two of the above values.

[0069] In some embodiments, prior to step S100, the method further includes equilibrating the cigar under test at a temperature of 20°C to 24°C and a relative humidity of 60% to 64% for 70 to 74 hours. Without limitation, the temperature may be, but is not limited to, 20°C, 22°C, 24°C, or any value or range between any two of the aforementioned values; the relative humidity may be, but is not limited to, 60%, 62%, 64%, or any value or range between any two of the aforementioned values; and the equilibration time may be, but is not limited to, 70 hours, 72 hours, 74 hours, or any value or range between any two of the aforementioned values.

[0070] In some embodiments, in step S300, the assay includes quantitatively analyzing the nicotine content in cigar smoke using an internal standard curve method.

[0071] For example, the detection limit of the quantitative analysis of nicotine content in cigar smoke using the internal standard curve method is 0.0096 mg / mL, and the quantification limit is 0.032 mg / mL.

[0072] In some embodiments, in step S300, the gas chromatography conditions in the gas chromatography-flame ionization detection method include one or more of the following: the chromatographic column is a TG-WAXMS capillary column with a specification of 30 m×0.25 mm×0.25 μm;

[0073] The carrier gas control mode is constant flow mode, and the carrier gas flow rate is 1.5 mL / min~2 mL / min;

[0074] The injection volume is 1 μL~2 μL;

[0075] The injection method was split injection with a split ratio of (8-12):1;

[0076] The injection port temperature is 250℃~300℃;

[0077] Heating program: initial temperature is 100℃~120℃, maintain for 3min~5min, increase the temperature to 200℃~250℃ at a rate of 10℃ / min~15℃ / min, and maintain for 2min~3min.

[0078] Exemplarily, the gas chromatography conditions include: the carrier gas control mode is a constant flow mode, the carrier gas flow rate is 1.5 mL / min; the injection volume is 1 μL; the split ratio is 10:1; the injection port temperature is 250°C; and the heating program is: the initial temperature is 100°C, maintained for 4 min, and then increased to 230°C at a rate of 15°C / min and maintained for 2.5 min.

[0079] In some embodiments, in step S300, the flame ionization conditions in the gas chromatography-flame ionization detection method include one or more of the following:

[0080] The temperature of the flame ionization detector is 250℃~300℃;

[0081] Air flow rate is 250 mL / min~300 mL / min;

[0082] The hydrogen flow rate is 50 mL / min~80 mL / min.

[0083] Illustratively, the flame ionization conditions include: a flame ionization detector temperature of 275° C.; an air flow rate of 250 mL / min; and a hydrogen flow rate of 60 mL / min.

[0084] In some embodiments, the same cigar sample to be tested may not produce normal peaks when tested using the method specified in the YC / T465-2013 standard.

[0085] The above-mentioned method, based on optimized Cambridge filter pretreatment conditions, measures nicotine content in cigar smoke using gas chromatography-flame ionization detection. This method is simple to use and highly accurate. Compared to traditional methods, it addresses the problem of high detection limits that make it difficult to detect nicotine in samples, significantly improving sensitivity and providing a scientific basis for the development, classification, and evaluation of high-quality cigar products.

[0086] In a second aspect of the present application, a method for analyzing or classifying the nicotine content in mainstream smoke of different types of cigars is provided, comprising using the above-mentioned method for determining the nicotine content in mainstream smoke of different types of cigars to test and obtain the nicotine content in mainstream smoke of different types of cigars.

[0087] Some examples are provided below.

[0088] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and may also be based on the experimental manuals or conventional conditions in this area, or on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0089] In the following examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operating accuracy are allowed.

[0090] Example 1

[0091] 1. Materials and Methods

[0092] 1.1 Instruments, reagents, and materials

[0093] Instruments: Ultrasonic cleaner (Tianjin Aotesense Instrument Co., Ltd.); analytical balance (Shanghai Ohaus Instrument Co., Ltd.); gas chromatograph (Agilent 7890A, equipped with a hydrogen flame detector, Agilent Technologies, USA).

[0094] Reagents: Nicotine standard (98.2%, Beijing North Weiye Institute of Metrology and Technology), isopropyl alcohol (chromatographic grade, Adamas), n-heptadecane (99%, Rohn's reagent).

[0095] Materials: Cigar samples came from four production areas in China, namely Sichuan, Yunnan, Hubei and Hainan, and were provided by Sichuan China Tobacco Great Wall Cigar Factory.

[0096] 1.2 Standard solution preparation

[0097] Internal standard solution: Accurately weigh 0.1929 g of n-heptadecane, dissolve it in isopropanol and dilute to volume in a 25 mL volumetric flask to obtain a 7.716 mg / mL internal standard solution. Refrigerate at 0-4°C.

[0098] Nicotine standard stock solution: Weigh 0.0947 g of nicotine standard, dissolve it in isopropanol and dilute to volume in a 25 mL volumetric flask to obtain a 3.788 mg / mL nicotine standard stock solution. Refrigerate at 0-4°C.

[0099] Nicotine standard solution: Gradient pipette the nicotine standard stock solution into six 10 mL volumetric flasks, add 0.42 mL of internal standard solution to each flask, dilute with isopropanol, shake well to obtain nicotine standard solutions with different concentration gradients, and store refrigerated at 0-4°C.

[0100] 1.3 Experimental methods and conditions

[0101] 1.3.1 Capture of aerosol particulate matter

[0102] According to YC / T 462-2013 and YC / T 461-2013, the cigar samples to be tested were equilibrated in an environment of (22±1)°C and (60±2)% relative humidity for 72 h. The puff duration, puff frequency, and puff volume were set to 1.5 s, 40 s, and 45 mL, respectively. A Ø55 mm Cambridge filter was used to capture the total particulate matter in the mainstream smoke of one cigar. After puffing, the Cambridge filter was stored in a sealed bag.

[0103] 1.3.2 Sample preparation for nicotine analysis in aerosol

[0104] Place a Cambridge filter disc in a 50 mL conical flask, add 0.3 mL of internal standard solution on the Cambridge filter disc, then add 10 mL of isopropanol, sonicate for 60 min, take 1 mL of the extract into an injection bottle, and perform GC-FID (gas chromatography-flame ionization detection) analysis.

[0105] 1.4 Gas chromatography analysis conditions

[0106] The experimental conditions are as follows, referring to YC / T465-2013 and the literature (Determination of nicotine in heat-not-burn reconstituted tobacco leaves by gas chromatography [J]. Chemical Analysis and Metering, 2022, 31(10):40): chromatographic column: TG-WAXMS capillary column (30 m×0.25 mm×0.25 μm) (Shanghai Xiyan Scientific Instrument Co., Ltd.); heating program: initial temperature is 100 ℃, maintained for 4 min, heated to 230 ℃ at a rate of 15 ℃ / min, maintained for 2.5 min; injection port temperature: 250 ℃; FID detector temperature: 275 ℃; carrier gas: nitrogen, flow rate is 1.5 mL / min, constant flow mode; air flow rate: 250 mL / min; hydrogen flow rate: 60 mL / min; injection volume: 1 μL; injection mode: split injection, split ratio is 10:1.

[0107] 1.5 Optimization of sample processing methods

[0108] 1.5.1 Extraction method selection

[0109] Different extraction methods have different extraction efficiencies. Under the conditions of 1.3.2, this example investigated the effects of different extraction methods (ultrasound and oscillation) on the extraction efficiency of the same cigar smoke sample. The ultrasound and oscillation times were each 60 minutes, and the results were analyzed and compared.

[0110] 1.5.2 Extraction time selection

[0111] Different extraction times result in different extraction efficiencies. Under the conditions described in 1.3.2, this example investigates the effects of different extraction times (30, 40, 50, 60, and 70 min) on the extraction efficiency of the same cigar smoke sample, and analyzes and compares the results.

[0112] 1.6 Sample measurement and data analysis

[0113] The nicotine content of multiple cigar smoke Cambridge filters was determined using an optimized sample pretreatment method, and the differences in nicotine content between cigar smoke from different parts, grades, and production areas were briefly analyzed.

[0114] 2 Results and Discussion

[0115] 2.1 Selection of internal standard

[0116] The internal standard should be similar to the analyte in chemical properties. Nicotine is often determined by using carvone, quinoline, n-heptadecane, 2-methylquinoline, n-octadecane, etc. as internal standards. This example uses n-heptadecane as the internal standard. The chromatogram of the nicotine standard solution is as follows: Figure 1 shown.

[0117] 2.2 Sample processing method selection

[0118] 2.2.1 Extraction method

[0119] To determine the optimal extraction method, this example investigated the effects of different extraction methods on the same cigar smoke sample. The experimental results are shown in Table 1. Ultrasonic extraction was significantly more efficient than oscillation extraction for nicotine extraction from Cambridge filter discs, so ultrasonic extraction was selected as the sample extraction method.

[0120] Table 1 Effect of extraction method on nicotine extraction efficiency

[0121]

[0122] 2.2.2 Extraction time

[0123] In order to determine the optimal extraction time, this example investigated the effects of different extraction times on the extraction of the same cigar smoke sample. The experimental results are as follows: Figure 2 As shown in the figure, as the extraction time increases, the nicotine detection results gradually increase. When the extraction time reaches 60 minutes, the nicotine detection results decrease with time. Therefore, the ultrasonic time is selected as 60 minutes.

[0124] 2.3 Method Evaluation

[0125] 2.3.1 Standard curve, detection limit, and quantification limit

[0126] Under the conditions of 1.4, 6 nicotine standard solutions of different concentrations were analyzed by GC-FID. The internal standard method was used for quantification. The ratio of the peak area of nicotine to the internal standard was used as the ordinate, and the ratio of the concentration of nicotine to the internal standard was used as the abscissa to plot the result. Figure 3 The standard working curve was y = 0.6805x + 0.0736, with a correlation coefficient of 0.9995. The linear relationship of the nicotine standard curve was good, meeting the requirements of quantitative analysis.

[0127] The lowest concentration of nicotine standard solution in the standard curve was measured eight times. The nicotine concentration was determined using the standard curve, and the standard deviation of the test results was calculated. 3 times and 10 times the standard deviation were used as the method detection limit (LOD) and quantification limit (LOQ). The detection limit of nicotine was 0.0096 mg / mL, and the quantification limit was 0.032 mg / mL.

[0128] 2.3.2 Method recovery and precision

[0129] The accuracy of the method was evaluated using recovery measurements. Nicotine standard solutions were spiked onto Cambridge filters capturing cigar smoke at low, medium, and high spike levels. Precision was also used to assess the reproducibility of the method. Eight replicates of the same cigar sample were measured under the same conditions. The results are shown in Tables 2 and 3. Recoveries ranged from 96.6% to 101.0%, with a relative standard deviation of 2.5%, demonstrating that the method was accurate and reliable, meeting quantitative requirements.

[0130] Table 2 Nicotine recovery rate in total particulate matter in mainstream cigar smoke

[0131]

[0132] Table 3 Precision of nicotine in total particulate matter in mainstream cigar smoke (n=8)

[0133]

[0134] 2.4 Sample measurement and data analysis

[0135] According to the optimized method, the nicotine content of 28 cigar smoke Cambridge filters (11 in Yunnan, 5 in Hainan, 6 in Hubei, and 6 in Sichuan) was determined. The chromatogram of the cigar mainstream smoke sample solution is shown in Figure 2. Figure 4 shown.

[0136] 2.4.1 Analysis of Nicotine Differences in Cigar Smoke from Different Parts

[0137] The average value of the nicotine test results in the upper, middle and lower cigar smoke is calculated, e.g. Figure 5 As shown in the figure, the nicotine content in the Cambridge filter of cigar smoke made from upper tobacco leaves is higher than that in samples made from middle tobacco leaves, and the nicotine content in the Cambridge filter of cigar smoke made from middle tobacco leaves is higher than that in samples made from lower tobacco leaves. In the literature (Effects of ecological conditions and germplasm factors on the content of secondary metabolites in flue-cured tobacco leaves [J]. Journal of Northwest Agriculture and Forestry University (Natural Science Edition), 2013, 41(12): 80-85, 92), tobacco leaves from different production areas in Guizhou were studied and it was found that light has a significant effect on the content of secondary metabolites (such as nicotine) in tobacco leaves. Since the light conditions of cigar plants are greater in the upper part than in the middle part, and greater in the middle part than in the lower part, the nicotine content in the upper cigar leaves is higher than that in the middle cigar leaves, and the nicotine content in the middle cigar leaves is higher than that in the lower cigar leaves. During the curing process of cigar tobacco leaves, glutamine synthetase and nitrate reductase work together to promote nitrogen metabolism in plants, playing a key role in the strength of nitrogen metabolism. Nitrate reductase and glutamine synthetase are positively correlated with nicotine content. At the same time, the activities of nitrate reductase and glutamine synthetase increase with the increase of the position, indicating that the nicotine content gradually increases with the increase of the position.

[0138] 2.4.2 Analysis of Nicotine Differences in Cigar Smoke of Different Grades

[0139] The average value of the nicotine determination results in the smoke of first-grade, second-grade and third-grade cigars is calculated, e.g. Figure 6 As shown, the nicotine content in Cambridge filter smoke from cigars made from Grade II tobacco leaves is higher than that from Grade III tobacco leaves, and the nicotine content in Cambridge filter smoke from Grade III tobacco leaves is higher than that from Grade I tobacco leaves. Cigar tobacco leaves are primarily graded based on maturity, oil content, and uniformity (according to the YC / T 588-2021 standard). Studies have shown that nicotine content increases with increasing maturity. Cigars have a high nicotine content and are highly pungent. To achieve the ideal tobacco quality, high-quality cigars typically require two or more fermentation processes. This is because nicotine degradation during fermentation by enzymes, microorganisms, and oxidation reduces nicotine content. Grade I cigars undergo more rigorous fermentation and aging processes, resulting in a fully blended nicotine smoke with a mellow, rich, and less pungent flavor. Grade II cigars undergo a relatively simple fermentation and aging process, but the nicotine is not fully blended, resulting in a certain pungent flavor. Grade III cigars use lower-quality tobacco leaves and are processed more simply, resulting in unstable nicotine content.

[0140] 2.4.3 Analysis of Nicotine Differences in Cigar Smoke from Different Origins

[0141] The average value of the nicotine determination results in cigar smoke from Sichuan, Hainan, Yunnan, and Hubei is calculated. Figure 7 As shown, the nicotine content in Cambridge filter smoke from Yunnan cigars is higher than that from Hubei cigars, the nicotine content in Cambridge filter smoke from Hubei cigars is higher than that from Hainan cigars, and the nicotine content in Cambridge filter smoke from Hainan cigars is higher than that from Sichuan cigars. Climate conditions (light, temperature, precipitation) and soil conditions significantly influence the nicotine content in tobacco leaves. Cigars are best grown in tropical or subtropical monsoon climates with abundant sunshine, suitable temperatures, and precipitation. Yunnan has a subtropical monsoon climate and a plateau mountain climate, with long sunshine hours, large temperature differences between day and night, and moderate precipitation. The soil is mostly red soil (rich in nitrogen), which is conducive to the synthesis and accumulation of nicotine; Sichuan has a subtropical monsoon climate, with sufficient sunshine and moderate precipitation. The soil is mostly purple soil or red soil (rich in organic matter), which is more suitable for the accumulation of nicotine; Hubei has a subtropical humid climate, with abundant precipitation and fertile soil (mostly alluvial soil), which is more suitable for the accumulation of nicotine; Hainan has a tropical marine monsoon climate, with sufficient sunshine but excessive precipitation, and the soil is mostly sandy soil (poor water retention and low nutrient content), which is not conducive to the accumulation of nicotine.

[0142] 3 Conclusion

[0143] To investigate the relationship between nicotine content differences in mainstream cigar smoke and its location, grade, and origin, several examples have established a method for simultaneous nicotine determination in cigar smoke using ultrasonic extraction and GC-FID. This method is simple to use and highly accurate. This method overcomes the high detection limit encountered in the application of the YC / T465-2013 standard, improving the sensitivity of detecting target substances in samples. The established method was also used to determine the nicotine content of 28 Cambridge filter cigars. Analysis of the correlation between nicotine content differences and location, grade, and origin revealed that nicotine content gradually increased with increasing location; nicotine content in second-grade cigar smoke was higher than that in first-grade cigar smoke, while nicotine content in third-grade cigar smoke fluctuated significantly; and nicotine content varied across different regions. This method can be used to analyze and categorize nicotine content in mainstream smoke from different cigar types.

[0144] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0145] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art could make several modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the patent in this application shall be based on the appended claims, and the specification and drawings may be used to interpret the claims.

Claims

1. A method for determining the nicotine content in cigar smoke, characterized in that: The following steps are involved: Cambridge filter was used to capture the total particulate matter in the smoke of the cigar to be tested; adding an internal standard solution and an alcohol solvent to the Cambridge filter disc, performing ultrasonic extraction, and obtaining an extract; The extract is measured and analyzed by gas chromatography-flame ionization detection to obtain the nicotine content in cigar smoke.

2. The method for determining the nicotine content in cigar smoke according to claim 1, wherein: The internal standard in the internal standard solution includes one or more of carvone, quinoline, n-heptadecane, 2-methylquinoline and n-octadecane; and the alcohol solvent includes isopropyl alcohol.

3. The method for determining the nicotine content in cigar smoke according to claim 2, wherein: The ultrasonic extraction satisfies one or more of the following conditions: The ultrasonic extraction time is 30 min to 70 min; The concentration of the internal standard solution is 7.6 mg / mL to 7.8 mg / mL; The volume ratio of the internal standard solution to the alcohol solvent is 1:(30~35).

4. The method for determining the nicotine content in cigar smoke according to claim 1, wherein: The diameter of the Cambridge filter is 50 mm to 55 mm.

5. The method for determining the nicotine content in cigar smoke according to claim 4, wherein: Cambridge filter was used to capture the particulate matter in the mainstream smoke of the cigar to be tested and then smoked and captured by a smoking machine; Optionally, the suction satisfies one or more of the following conditions: The aspiration volume is 40mL~50mL; The suction time is 1s~2s; The suction frequency is 40s~45s.

6. The method for determining the nicotine content in cigar smoke according to claim 1, wherein: Before using the Cambridge filter to collect the mainstream smoke particulate matter of the cigar to be tested, the method further includes: equilibrating the cigar to be tested at a temperature of 20°C to 24°C and a relative humidity of 60% to 64% for 70 hours to 74 hours.

7. The method for determining the nicotine content in cigar smoke according to any one of claims 1 to 6, wherein: The determination and analysis includes quantitatively analyzing the nicotine content in cigar smoke using an internal standard curve method; Optionally, the detection limit of the internal standard curve method for nicotine content in cigar smoke is 0.0096 mg / mL, and the quantification limit is 0.032 mg / mL.

8. The method for determining the nicotine content in cigar smoke according to any one of claims 1 to 6, wherein: The gas chromatography in the gas chromatography-flame ionization detection method satisfies one or more of the following conditions: The chromatographic column was a TG-WAXMS capillary column with specifications of 30 m × 0.25 mm × 0.25 μm; The carrier gas control mode is constant flow mode, and the carrier gas flow rate is 1.5 mL / min~2 mL / min; The injection volume is 1 μL~2 μL; The injection method was split injection with a split ratio of (8-12):1; The injection port temperature is 250℃~300℃; Heating program: initial temperature is 100℃~120℃, maintain for 3min~5min, increase the temperature to 200℃~250℃ at a rate of 10℃ / min~15℃ / min, and maintain for 2min~3min.

9. The method for determining the nicotine content in cigar smoke according to any one of claims 1 to 6, wherein: The flame ionization in the gas chromatography-flame ionization detection method satisfies one or more of the following conditions: The temperature of the flame ionization detector is 250℃~300℃; Air flow rate is 250 mL / min~300 mL / min; The hydrogen flow rate is 50 mL / min~80 mL / min.

10. A method for analyzing or classifying the nicotine content in mainstream smoke of different types of cigars, characterized in that: The method comprises using the method for determining the nicotine content in cigar smoke according to any one of claims 1 to 9 to test and obtain the nicotine content in the mainstream smoke of different types of cigars.

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