A detection method for volatile components of tobacco leaves during the fermentation process of cigars

Abstract

The present invention discloses a method for detecting volatile components in tobacco leaves during the fermentation process of cigars, comprising the following steps: preparation of samples, pretreatment of samples, headspace solid-phase microextraction, and GC-MS detection. This method derivatizes tobacco leaves with a silanization reagent, hexamethyldisilazane, and then can quickly remove hexamethyldisilazane by water washing. While improving the accuracy of tobacco leaf detection, it can also avoid the interference of the silanization reagent on the detection. This method has a wide detection range, is simple, convenient, fast, and efficient in operation.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis of volatile components in tobacco leaves, and particularly relates to a method for detecting volatile components in tobacco leaves during the fermentation process of cigar tobacco. Background Art

[0002] Cigarette combustion is a complex chemical system. In addition to the gaseous substances generated by the complete combustion and cracking of organic substances (such as low-molecular-weight hydrocarbon compounds such as carbon monoxide, water, and methane), due to insufficient oxygen supply in some areas, many complex chemical changes occur under anoxic conditions. Volatile substances in tobacco leaves (such as semi-volatile nitrogen-containing heterocyclic compounds with 5-membered and 6-membered rings) will also volatilize into the tobacco stream. Terpenoids, sugars, amino acids, cellulose, and many other components in tobacco produce volatile and semi-volatile gases through thermal decomposition, thermal synthesis, dry distillation, polymerization, condensation, free radical reactions, etc. The components of the flue gas generated after cigarette combustion are very complex, including carboxylic acids, lipids, aldehydes, ketones, nicotine, alkanes, etc., and there are more than 4,000 specific chemical substances. Among them, harmful substances will affect the sleep quality and fertility of smokers, leading to health problems such as chronic bronchitis and osteoporosis, and even triggering major diseases such as coronary heart disease and cancer.

[0003] Especially for the widely popular cigar tobacco leaves, since smoking cigars is somewhat different from smoking cigarettes, typical cigar smokers do not inhale the cigar smoke into the lungs, but let the smoke linger in the mouth. Therefore, the incidence rates of emphysema and lung cancer among cigar smokers are lower than those of cigarette smokers but higher than those of non-smokers. Currently, many people in the market have misunderstandings about cigars, believing that smoking cigars is harmless to health or causes less harm than smoking cigarettes. In fact, the nicotine absorbed from smoking cigars is higher than that from smoking cigarettes, and cigar smokers are more likely to develop oral cancer, tongue cancer, or throat cancer than non-smokers. However, there is still controversy about the increased cancer risk of smoking cigars. And the impact of occasional cigar smoking on health has not yet been concluded. Therefore, there is an urgent need for a method that can accurately detect the volatile components of cigar tobacco leaves. In the prior art, the main method for detecting flue gas components is the GC-MS method, but most of them have problems such as complex operation and small detection range. Due to the complex matrix of cigar tobacco leaf samples, especially the presence of high-concentration nicotine, it will affect the gas chromatography behavior of tobacco leaf components. Moreover, the above sample pretreatment methods not only have great defects in terms of solvents, sample usage, and extraction time, but also it is difficult to ensure the original fragrance characteristics of tobacco leaves during the treatment process, easily causing the loss of some volatile aroma components, and unable to avoid problems such as limited chromatographic peak capacity and insufficient resolution caused by impurities such as nicotine, resulting in a reduction in the accuracy of the detection results of aroma components, pesticide residues, and other harmful substances in tobacco leaves.

[0004] Chinese Patent [CN108680694A] discloses a method for determining volatile chemical components in flue gas by infrared mirror reflection furnace-headspace gas chromatography-mass spectrometry, which includes the following steps: (A) Placing tobacco leaf samples and glass fiber filter discs in a constant temperature and humidity environment for 48 h of equilibration; (B) Weighing not less than 0.50 g of the tobacco leaf samples obtained in step A, placing them in a quartz tube, and heating them with infrared rays in a temperature-programmed manner. During heating, air is introduced into the quartz tube to simulate the tobacco leaf combustion process. After a certain period of time, the infrared heating is stopped and nitrogen is introduced into the quartz tube to simulate the tobacco leaf smoldering process. The chemical components in the smoke are trapped with a glass fiber filter disc, and the steps of simulating combustion and simulating smoldering are repeatedly executed several times; (C) Placing the glass fiber filter disc into a headspace vial and performing headspace gas chromatography-mass spectrometry analysis. This method enables the tobacco cut filler usage to reach or even exceed the tobacco leaf raw material of 1 cigarette, ensuring the accurate analysis of trace volatile chemical components in tobacco leaf flue gas. However, this method uses an infrared mirror reflection furnace to simulate the flue gas combustion process. This method is difficult to exclude the interference of other impurity components in the tobacco leaf, with low detection accuracy and unable to accurately judge the content of each component in the tobacco leaf. Summary of the Invention

[0005] To solve the above problems, the present invention provides a method for detecting volatile components in tobacco leaves during the fermentation process of cigars, specifically including steps such as the preparation of cigar tobacco leaf samples, sample pretreatment, headspace solid-phase microextraction, GC-MS detection, etc. This method derivatizes tobacco leaves with the silanization reagent hexamethyldisilazane, and then hexamethyldisilazane can be quickly removed by water washing. While improving the detection accuracy of tobacco leaves, it can also avoid the interference of the silanization reagent on the detection. This method has a wide detection range, is simple, convenient, fast, and efficient in operation.

[0006] The technical solution provided by the present invention is as follows: A method for detecting volatile components in tobacco leaves during the fermentation process of cigars, characterized by including the following steps:

[0007] S1. Preparation of the sample: Removing the tobacco veins from the collected cigar tobacco leaf samples, then cutting the leaves into pieces or shreds, and placing the tobacco leaves or tobacco shreds in an oven for drying;

[0008] S2. Sample pretreatment: Crushing and sieving the dried tobacco leaf samples in step S1 above, putting the sieved powder into a derivatization bottle, adding the silanization reagent hexamethyldisilazane, performing water bath at 70 °C for 60 min of derivatization, then washing with water, filtering, and drying to obtain the derivatized powder;

[0009] S3. Headspace solid-phase microextraction: Taking out the powder derivatized in step S2 above and putting it into a 20 mL headspace injection vial, adding saturated NaCl solution and internal standard solution, then using a manual SPME injector to insert a 70 μm CAR / PDM extraction head into the headspace vial, heating for extraction, and desorbing to obtain the sample to be detected;

[0010] S4. GC-MS detection: Put the sample to be measured obtained in the above step S3 into the GC-MS detection system for detection and analysis.

[0011] Furthermore, in the above step S1, put the tobacco leaves or cut tobacco into an oven and dry at 35 - 40 °C for 3 - 4 h.

[0012] Furthermore, in the above step S2, the crushing time of the tobacco leaf sample is 1 - 2 min, and after crushing, it is sieved through a 40 - mesh sieve.

[0013] Furthermore, in the above step S2, the mass ratio of the sieved powder to hexamethyldisilazane is (0.4 - 1.2):3.

[0014] Furthermore, in the above step S3, the internal standard solution is ethyl caprate with a concentration of 50 μg / mL.

[0015] Furthermore, in the above step S3, the mass ratio of the powder, saturated NaCl solution, and internal standard solution is (0.4 - 1.2):6:(0.03 - 0.05).

[0016] Furthermore, in the above step S3, the temperature for heating extraction is 50 - 100 °C, and the extraction time is 10 - 60 min.

[0017] Furthermore, the GC / MS analysis conditions in the above step S4 are as follows: chromatographic column, TG - 5MS (30 m × 0.25 mm × 0.25 μm); carrier gas, He; flow rate, 0.8 mL / min; inlet temperature, 250 °C; transfer line temperature, 280 °C; ion source temperature, 230 °C; programmed temperature rise; ionization energy 70 eV; mass number range 135 - 350 amu.

[0018] Furthermore, the programmed temperature rise includes the following steps: initial temperature 60 °C, hold for 2 min, increase to 180 °C at a rate of 2 °C / min, hold for 2 min, increase to 260 °C at a rate of 10 °C / min, and hold for 5 min.

[0019] Furthermore, the following process is also included in the above step S4: Prepare a n - alkane mixture solution with a concentration of 10 ppm as the external standard, and perform GC - MS analysis together with the sample to be measured extracted in step S3.

[0020] The internal standard solution is a method used for accurate quantification in chromatographic analysis. A certain weight of pure substance is added as the internal standard to a certain amount of the analyzed sample mixture. According to the mass ratio of the test sample to the internal standard, the ratio of their corresponding chromatographic peak areas, and the relative correction factor, the content of the component to be measured is calculated.

[0021] Generally, the selection criteria for the internal standard solution are as follows:

[0022] a. The original sample does not contain the component

[0023] b. The retention time should be close to that of the analyte, but not overlap

[0024] c. It is a high-purity reference substance or a substance with a known content

[0025] d. It has a certain chemical stability under the given chromatographic conditions

[0026] Ethyl caprate is a colorless oily liquid at room temperature, with fruity and wine-like aromas, and a fragrance similar to that of pears and brandy. Therefore, the present invention selects ethyl caprate as the isotope internal standard, and its accuracy is very high, and it will not cause any impact on the detection of tobacco leaf aroma components.

[0027] Due to the different polarities of acids, alcohols, and phenols, different chromatographic columns are often required and cannot be analyzed simultaneously. The silanization reaction can solve this problem. Its principle is to make trimethylsilyl groups replace the active hydrogens of hydroxyl, carboxyl, mercapto, amino, and imino groups of the derivative object through silanization derivatization reaction, improve the volatility of the derivative object, thereby expanding the detection range of gas chromatography, and overcoming the disadvantage that gas chromatography cannot directly inject and analyze substances with strong polarity, low volatility, and poor thermal stability. In addition, the method of converting into derivatives also has the advantages of improving the separation selectivity of structurally similar compounds, overcoming the adsorption of high-polarity and low-volatility samples by the carrier and column wall, and improving the sample peak shape.

[0028] The present invention uses a silanization reagent to treat tobacco leaves. However, since the liner of the GC injection port itself is passivated, if there is a silanization reagent in the added sample, at the high temperature of the injection port, it is easy to form high active centers, which will cause part of the sample to react with the residual silanization reagent in the liner. Therefore, in the MS results, if searched with the NIST library, impurities will be seen, but the sample treatment cannot completely remove the silanization reagent. At the same time, silanization reagents are all strongly polar, while GC analysis generally analyzes weakly polar or medium-polar compounds. Therefore, over time, the silanization reagent will block the packing on the capillary column. During the high-temperature analysis process, its activity decreases, which will definitely lead to an increase in column bleeding and a decrease in column efficiency, thereby reducing the detection efficiency and accuracy.

[0029] Therefore, in the present invention, hexamethyldisilazane is used as a silylation reagent. The tobacco leaves are heated in a water bath with an excessive amount of hexamethyldisilazane until ammonia escapes. The active hydrogen atoms in the organic compound are replaced by silyl groups. The physical properties of its silyl derivatives change and the boiling point decreases, etc., and chromatography can be used for analysis. Since hexamethyldisilazane is soluble in water, in the present invention, hydrolysis is adopted to quickly remove the excessive hexamethyldisilazane, which can avoid interference with subsequent detections and further improve the accuracy of detections.

[0030] The beneficial effects of the present invention are as follows:

[0031] 1. A method for detecting volatile components in tobacco leaves during the fermentation process of cigars provided by the present invention specifically includes steps such as the preparation of cigar tobacco leaf samples, sample pretreatment, headspace solid-phase microextraction, GC-MS detection, etc. This method performs derivatization treatment on tobacco leaves with the silylation reagent hexamethyldisilazane, and then hexamethyldisilazane can be quickly removed by washing with water. While improving the detection accuracy of tobacco leaves, it can also avoid the interference of the silylation reagent on detections. This method has a wide detection range, is simple, convenient, fast and efficient in operation.

[0032] 2. A method for detecting volatile components in tobacco leaves during the fermentation process of cigars provided by the present invention uses hexamethyldisilazane as a silylation reagent to perform derivatization treatment on tobacco leaves, which can effectively improve the determination range of gas chromatography, and can also avoid the influence of excessive silylation reagent on detection results through hydrolysis. Description of the Drawings

[0033] Figure 1 is a flow chart of a method for detecting volatile components in tobacco leaves during the fermentation process of cigars in an embodiment of the present invention;

[0034] Figure 2 is the influence of different sample amounts on detection results in an embodiment of the present invention;

[0035] Figure 3 is the influence of different extraction temperatures on detection results in an embodiment of the present invention;

[0036] Figure 4 is the influence of different extraction times on detection results in an embodiment of the present invention;

[0037] Figure 5 is the detection result of cigar tobacco leaves in an embodiment of the present invention. Detailed Embodiments

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present invention and the prior art, the specific embodiments of the present invention will be described below with reference to the accompanying drawings.

[0039] Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings, and other implementation manners can also be obtained. The present invention is not limited to this embodiment.

[0040] In the embodiments of the present invention, a total of 15 samples were prepared and divided into 3 groups for detection. Each group of samples was subjected to single-factor variable detection for one factor respectively.

[0041] In the embodiments of the present invention, the GC / MS analysis conditions are as follows: chromatographic column, TG-5MS (30m×0.25 mm×0.25μm); carrier gas, He; flow rate, 0.8mL / min; inlet temperature, 250°C; transfer line temperature, 280°C; ion source temperature, 230°C; programmed temperature rise; ionization energy 70eV; mass number range 135~350amu. Among them, the programmed temperature rise includes the following steps: initial temperature 60°C, hold for 2min, rise to 180°C at a rate of 2°C / min, hold for 2min, and then rise to 260°C at a rate of 10°C / min, hold for 5min.

[0042] In the embodiments of the present invention, for the convenience of analysis, the following process is also included: preparing a n-alkane mixture with a concentration of 10ppm as an external standard and performing GC-MS analysis together with the tobacco leaf samples to be detected.

[0043] The specific embodiments of the present invention are as follows:

[0044] To measure the influence of different sample amounts on the test results, in Examples 1-5 of the present invention, different sample amounts were selected for experiments respectively.

[0045] Example 1

[0046] S1. Preparation of the sample: First, use a brush to remove the fine soil and sand on the leaves of the collected sample, remove the tobacco veins, and then cut the leaves into shreds; put the shredded tobacco into an oven and bake at 35°C for 4h until it can be crushed by hand.

[0047] S2. Sample pretreatment: Crush the tobacco leaf sample dried in step S1 above for 2min and pass through a 40-mesh sieve. The fine veins that did not pass through the sieve are re-ground and passed through the sieve. Put the sieved powder into a clean and dry reagent bottle, shake well, and then take 0.4g of the sieved powder and put it into a 1ml derivatization bottle, add 3g of the silylation reagent hexamethyldisilazane, perform derivatization at 70°C in a water bath for 60min, then wash with water, filter, and dry to obtain the derivatized powder.

[0048] S3. Headspace solid-phase microextraction: Take out the powder obtained after the derivation in step S2 above and put it into a 20 mL headspace vial. Add 6 mL of saturated NaCl solution and 0.03 mL of ethyl caprylate with a concentration of 50 μg / mL. Then use a manual SPME injector to insert a 70 μm CAR / PDM extraction head into the headspace vial, heat to 50 °C and extract for 10 min, and desorb to obtain the sample to be measured.

[0049] S4. GC-MS detection: Put the sample to be measured obtained in step S3 above into a GC-MS detection system for detection and analysis.

[0050] Example 2

[0051] S1. Preparation of the test sample: Use a brush to remove the fine soil and sand on the leaves of the collected sample, remove the tobacco veins, and then cut the leaves into shreds. Put the shredded tobacco into an oven and dry it at 36 °C for 3 h until it can be crushed by hand.

[0052] S2. Sample pretreatment: Crush the dried tobacco leaf sample in step S1 above for 2 min and pass through a 40-mesh sieve. Re-grind the fine veins that did not pass through the sieve and pass them through the sieve. Put the sieved powder into a clean and dry reagent bottle, shake well, and then take 0.6 g of the sieved powder and put it into a 1 mL derivation bottle. Add 3 g of the silylation reagent hexamethyldisilazane, water bath at 70 °C for 60 min, then wash with water, filter, and dry to obtain the derived powder.

[0053] S3. Headspace solid-phase microextraction: Take out the powder obtained after the derivation in step S2 above and put it into a 20 mL headspace vial. Add 6 mL of saturated NaCl solution and 0.04 mL of ethyl caprylate with a concentration of 50 μg / mL. Then use a manual SPME injector to insert a 70 μm CAR / PDM extraction head into the headspace vial, heat to 50 °C and extract for 10 min, and desorb to obtain the sample to be measured.

[0054] S4. GC-MS detection: Put the sample to be measured obtained in step S3 above into a GC-MS detection system for detection and analysis.

[0055] Example 3

[0056] S1. Preparation of the test sample: Use a brush to remove the fine soil and sand on the leaves of the collected sample, remove the tobacco veins, and then cut the leaves into shreds. Put the shredded tobacco into an oven and dry it at 37 °C for 3 h until it can be crushed by hand.

[0057] S2. Sample pretreatment: Pulverize the tobacco leaf sample dried in step S1 above for 1 minute and sieve it through a 40-mesh sieve. Re-grind and sieve the fine veins that did not pass through the sieve. Put the sieved powder into a clean and dry reagent bottle, shake it well to mix evenly. Then take 0.8 g of the sieved powder and put it into a 1-ml derivatization bottle, add 3 g of the silanization reagent hexamethyldisilazane, perform derivatization in a water bath at 70 °C for 60 minutes, then wash with water, filter, and dry to obtain the derivatized powder;

[0058] S3. Headspace solid-phase microextraction: Take out the powder derivatized in step S2 above and put it into a 20-mL headspace injection vial, add 6 mL of saturated NaCl solution and 0.04 mL of ethyl caprate with a concentration of 50 μg / mL. Then use a manual SPME injector to insert a 70-μm CAR / PDM extraction head into the headspace vial, heat to 50 °C and extract for 10 minutes, and desorb to obtain the sample to be tested;

[0059] S4. GC-MS detection: Put the sample to be tested obtained in step S3 above into a GC-MS detection system for detection and analysis.

[0060] Example 4

[0061] S1. Preparation of the test sample: Use a brush to remove the fine soil and sand particles on the leaves of the collected sample, remove the tobacco veins, and then cut the leaves into shreds; put the shredded tobacco into an oven and bake at 39 °C for 3 hours until it can be crushed by hand;

[0062] S2. Sample pretreatment: Pulverize the tobacco leaf sample dried in step S1 above for 1 minute and sieve it through a 40-mesh sieve. Re-grind and sieve the fine veins that did not pass through the sieve. Put the sieved powder into a clean and dry reagent bottle, shake it well to mix evenly. Then take 1.0 g of the sieved powder and put it into a 1-ml derivatization bottle, add 3 g of the silanization reagent hexamethyldisilazane, perform derivatization in a water bath at 70 °C for 60 minutes, then wash with water, filter, and dry to obtain the derivatized powder;

[0063] S3. Headspace solid-phase microextraction: Take out the powder derivatized in step S2 above and put it into a 20-mL headspace injection vial, add 6 mL of saturated NaCl solution and 0.05 mL of ethyl caprate with a concentration of 50 μg / mL. Then use a manual SPME injector to insert a 70-μm CAR / PDM extraction head into the headspace vial, heat to 50 °C and extract for 10 minutes, and desorb to obtain the sample to be tested;

[0064] S4. GC-MS detection: Put the sample to be tested obtained in step S3 above into a GC-MS detection system for detection and analysis.

[0065] Example 5

[0066] S1. Preparation of samples: First, use a brush to remove the fine soil and sand grains on the leaves of the collected samples, remove the tobacco veins, and then cut the leaves into shreds. Put the shredded tobacco into an oven and bake at 40 °C for 3 h until it can be crushed by hand.

[0067] S2. Pretreatment of samples: Crush the dried tobacco leaf samples in the above step S1 for 1 min and pass through a 40-mesh sieve. Re-grind the fine veins that did not pass through the sieve and pass them through the sieve. Put the sieved powder into a clean and dry reagent bottle, shake well, and then take 1.2 g of the sieved powder and put it into a 1-ml derivatization bottle. Add 3 g of the silylation reagent hexamethyldisilazane, perform derivatization in a water bath at 70 °C for 60 min, then wash with water, filter, and dry to obtain the derivatized powder.

[0068] S3. Headspace solid-phase microextraction: Take out the derivatized powder in the above step S2 and put it into a 20-mL headspace vial. Add 6 mL of saturated NaCl solution and 0.05 mL of ethyl decanoate with a concentration of 50 μg / mL. Then use a manual SPME injector to insert a 70-μm CAR / PDM extraction head into the headspace vial, heat to 50 °C and extract for 10 min, and desorb to obtain the sample to be measured.

[0069] S4. GC-MS detection: Put the sample to be measured obtained in the above step S3 into a GC-MS detection system for detection and analysis.

[0070] Data processing and analysis

[0071] In Examples 1-5 of the present invention, the main difference lies in the different amounts of each group of samples. The experimental data obtained from the detection of Examples 1-5 are retrieved through the NIST spectral library, combined with the analysis of the retention time of n-alkanes and manual parsing, and the identified compounds with a matching degree greater than 80% are reported. Using the internal standard method, the relative content of each volatile component is expressed as the ratio of the peak area of each volatile component to the peak area of ethyl decanoate for semi-quantitative analysis, and the total integrated area of the volatile components in the tobacco leaves is recorded. The results are as Figure 2 shown.

[0072] From Figure 2 it can be seen that the total integrated area of the volatile components in the tobacco leaves gradually increases with the increase of the sample amount and is basically stable after 0.8 g. Therefore, in the examples of the present invention, Example 3, that is, the optimal sample amount is 0.800 g.

[0073] To measure the influence of different headspace temperatures on the test results, Examples 6-10 of the present invention respectively select different headspace temperatures for experiments.

[0074] Example 6

[0075] S1. Preparation of the sample: First, use a brush to remove the fine soil and sand particles on the leaves of the collected sample, remove the tobacco veins, and then cut the leaves into shreds. Put the shredded tobacco into an oven and bake at 37 °C for 3 h until it can be crushed by hand.

[0076] S2. Pretreatment of the sample: Crush the tobacco sample dried in step S1 above for 2 min and pass through a 40-mesh sieve. Re-grind and sieve the fine veins that did not pass through the sieve. Put the sieved powder into a clean and dry reagent bottle, shake well to mix evenly, and then take 0.8 g of the sieved powder and put it into a 1-ml derivatization bottle. Add 3 g of the silylation reagent hexamethyldisilazane, perform derivatization in a water bath at 70 °C for 60 min, then wash with water, filter, and dry to obtain the derivatized powder.

[0077] S3. Headspace solid-phase microextraction: Take out the powder derivatized in step S2 above and put it into a 20-mL headspace vial. Add 6 mL of saturated NaCl solution and 0.03 mL of ethyl caprate with a concentration of 50 μg / mL. Then use a manual SPME injector to insert a 70-μm CAR / PDM extraction head into the headspace vial, heat to 60 °C and extract for 10 min, and desorb to obtain the sample to be tested.

[0078] S4. GC-MS detection: Put the sample to be tested obtained in step S3 above into a GC-MS detection system for detection and analysis.

[0079] Example 7

[0080] S1. Preparation of the sample: First, use a brush to remove the fine soil and sand particles on the leaves of the collected sample, remove the tobacco veins, and then cut the leaves into shreds. Put the shredded tobacco into an oven and bake at 38 °C for 3 h until it can be crushed by hand.

[0081] S2. Pretreatment of the sample: Crush the tobacco sample dried in step S1 above for 1 min and pass through a 40-mesh sieve. Re-grind and sieve the fine veins that did not pass through the sieve. Put the sieved powder into a clean and dry reagent bottle, shake well to mix evenly, and then take 0.8 g of the sieved powder and put it into a 1-ml derivatization bottle. Add 3 g of the silylation reagent hexamethyldisilazane, perform derivatization in a water bath at 70 °C for 60 min, then wash with water, filter, and dry to obtain the derivatized powder.

[0082] S3. Headspace solid-phase microextraction: Take out the powder derivatized in step S2 above and put it into a 20-mL headspace vial. Add 6 mL of saturated NaCl solution and 0.04 mL of ethyl caprate with a concentration of 50 μg / mL. Then use a manual SPME injector to insert a 70-μm CAR / PDM extraction head into the headspace vial, heat to 70 °C and extract for 10 min, and desorb to obtain the sample to be tested.

[0083] S4. GC-MS detection: Put the sample to be tested obtained in step S3 above into a GC-MS detection system for detection and analysis.

[0084] Example 8

[0085] S1. Preparation of the sample: Use a brush to remove the fine soil and sand on the leaves of the collected sample, remove the tobacco veins, and then cut the leaves into shreds; put the shredded tobacco into an oven and dry it at 39 °C for 3 h until it can be crushed by hand;

[0086] S2. Pretreatment of the sample: Crush the dried tobacco leaf sample from step S1 for 1 min and sieve it through a 40-mesh sieve. Re-grind and sieve the un-sieved fine veins. Put the sieved powder into a clean and dry reagent bottle, shake it well to mix evenly, and then take 0.8 g of the sieved powder and put it into a 1-ml derivatization bottle. Add 3 g of the silylation reagent hexamethyldisilazane, perform derivatization in a water bath at 70 °C for 60 min, then wash with water, filter, and dry to obtain the derivatized powder;

[0087] S3. Headspace solid-phase microextraction: Take out the derivatized powder from step S2 and put it into a 20-mL headspace injection vial. Add 6 mL of saturated NaCl solution and 0.04 mL of ethyl caprate with a concentration of 50 μg / mL. Then use a manual SPME injector to insert a 70-μm CAR / PDM extraction head into the headspace vial, heat it to 80 °C and extract for 10 min, and desorb to obtain the sample to be tested;

[0088] S4. GC-MS detection: Put the sample to be tested obtained in step S3 into a GC-MS detection system for detection and analysis.

[0089] Example 9

[0090] S1. Preparation of the sample: Use a brush to remove the fine soil and sand on the leaves of the collected sample, remove the tobacco veins, and then cut the leaves into shreds; put the shredded tobacco into an oven and dry it at 40 °C for 3 h until it can be crushed by hand;

[0091] S2. Pretreatment of the sample: Crush the dried tobacco leaf sample from step S1 for 1 min and sieve it through a 40-mesh sieve. Re-grind and sieve the un-sieved fine veins. Put the sieved powder into a clean and dry reagent bottle, shake it well to mix evenly, and then take 0.8 g of the sieved powder and put it into a 1-ml derivatization bottle. Add 3 g of the silylation reagent hexamethyldisilazane, perform derivatization in a water bath at 70 °C for 60 min, then wash with water, filter, and dry to obtain the derivatized powder;

[0092] S3. Headspace solid-phase microextraction: Take out the derivatized powder from step S2 and put it into a 20-mL headspace injection vial. Add 6 mL of saturated NaCl solution and 0.04 mL of ethyl caprate with a concentration of 50 μg / mL. Then use a manual SPME injector to insert a 70-μm CAR / PDM extraction head into the headspace vial, heat it to 90 °C and extract for 10 min, and desorb to obtain the sample to be tested;

[0093] S4, GC-MS detection: Put the sample to be tested obtained in the above step S3 into the GC-MS detection system for detection and analysis.

[0094] Example 10

[0095] S1, Preparation of the test sample: First, use a brush to remove the fine soil and sand on the leaves of the collected sample, remove the tobacco veins, and then cut the leaves into shreds; put the shredded tobacco into an oven and dry it at 40°C for 3 hours until it can be crushed by hand.

[0096] S2, Sample pretreatment: Crush the dried tobacco leaf sample in the above step S1 for 1 minute and pass it through a 40-mesh sieve. Re-grind the fine veins that did not pass through the sieve and pass them through the sieve. Put the sieved powder into a clean and dry reagent bottle, shake it well to mix evenly, and then take 0.8 g of the sieved powder and put it into a 1-ml derivatization bottle. Add 3 g of the silylation reagent hexamethyldisilazane, perform derivatization at 70°C in a water bath for 60 minutes, then wash with water, filter, and dry to obtain the derivatized powder.

[0097] S3, Headspace solid-phase microextraction: Take out the derivatized powder in the above step S2 and put it into a 20-mL headspace injection vial. Add 6 mL of saturated NaCl solution and 0.04 mL of ethyl caprate with a concentration of 50 μg / mL. Then use a manual SPME injector to insert a 70-μm CAR / PDM extraction head into the headspace vial, heat to 100°C and extract for 10 minutes, and desorb to obtain the sample to be tested.

[0098] S4, GC-MS detection: Put the sample to be tested obtained in the above step S3 into the GC-MS detection system for detection and analysis.

[0099] Data processing and analysis

[0100] In Examples 6-10 of the present invention, the main difference lies in the different headspace extraction temperatures. The experimental data obtained from the detection of Example 3 and Examples 6-10 are retrieved through the NIST spectral library, combined with the analysis of the retention time of n-alkanes and manual parsing. Compounds with a matching degree greater than 80% are reported. Using the internal standard method, the relative content of each volatile component is expressed as the ratio of the peak area of each volatile component to the peak area of ethyl caprate for semi-quantitative analysis, and the total integrated area of the volatile components in the tobacco leaves is recorded. The results are as Figure 3 shown.

[0101] It can be Figure 3 seen that the total integrated area of the volatile components in the tobacco leaves gradually increases with the increase of the extraction temperature, reaches the peak value at 90°C, and then decreases slightly and remains basically stable. Therefore, in the examples of the present invention, Example 9, that is, the optimal headspace extraction temperature is 90°C.

[0102] To measure the influence of different headspace extraction times on the test results, in Examples 11 - 15 of the present invention, different headspace extraction times were respectively selected for experiments.

[0103] Example 11

[0104] S1. Preparation of the test sample: First, use a brush to remove the fine soil and sand particles on the leaves of the collected sample, remove the tobacco veins, and then cut the leaves into shreds; put the shredded tobacco into an oven and dry it at 35 °C for 4 h until it can be crushed by hand;

[0105] S2. Pretreatment of the sample: Crush the tobacco leaf sample dried in step S1 above for 2 min and pass it through a 40 - mesh sieve. Re - grind the fine veins that did not pass through the sieve and pass them through the sieve. Put the sieved powder into a clean and dry reagent bottle, shake it well, and then take 0.8 g of the sieved powder and put it into a 1 - ml derivatization bottle. Add 3 g of the silylation reagent hexamethyldisilazane, perform derivatization in a water bath at 70 °C for 60 min, then wash with water, filter, and dry to obtain the derivatized powder;

[0106] S3. Headspace solid - phase microextraction: Take out the powder derivatized in step S2 above and put it into a 20 - mL headspace injection vial. Add 6 mL of saturated NaCl solution and 0.04 mL of ethyl caprylate with a concentration of 50 μg / mL. Then use a manual SPME injector to insert a 70 - μm CAR / PDM extraction head into the headspace vial, heat to 90 °C and extract for 20 min, and desorb to obtain the sample to be tested;

[0107] S4. GC - MS detection: Put the sample to be tested obtained in step S3 above into a GC - MS detection system for detection and analysis.

[0108] Example 12

[0109] S1. Preparation of the test sample: First, use a brush to remove the fine soil and sand particles on the leaves of the collected sample, remove the tobacco veins, and then cut the leaves into shreds; put the shredded tobacco into an oven and dry it at 36 °C for 3 h until it can be crushed by hand;

[0110] S2. Pretreatment of the sample: Crush the tobacco leaf sample dried in step S1 above for 2 min and pass it through a 40 - mesh sieve. Re - grind the fine veins that did not pass through the sieve and pass them through the sieve. Put the sieved powder into a clean and dry reagent bottle, shake it well, and then take 0.8 g of the sieved powder and put it into a 1 - ml derivatization bottle. Add 3 g of the silylation reagent hexamethyldisilazane, perform derivatization in a water bath at 70 °C for 60 min, then wash with water, filter, and dry to obtain the derivatized powder;

[0111] S3. Headspace solid-phase microextraction: Take out the powder derived in step S2 above and put it into a 20 mL headspace vial. Add 6 mL of saturated NaCl solution and 0.04 mL of ethyl caprylate with a concentration of 50 μg / mL. Then use a manual SPME injector to insert a 70 μm CAR / PDM extraction head into the headspace vial, heat to 90 °C and extract for 30 min, and desorb to obtain the sample to be tested;

[0112] S4. GC-MS detection: Put the sample to be tested obtained in step S3 above into a GC-MS detection system for detection and analysis.

[0113] Example 13

[0114] S1. Preparation of the test sample: First, use a brush to remove the fine soil and sand on the leaves of the collected sample, remove the tobacco veins, and then cut the leaves into shreds; put the shredded tobacco into an oven and bake at 37 °C for 3 h until it can be crushed by hand;

[0115] S2. Sample pretreatment: Crush the dried tobacco leaf sample in step S1 above for 2 min and pass through a 40-mesh sieve. Re-grind the fine veins that did not pass through the sieve and pass through the sieve. Put the sieved powder into a clean and dry reagent bottle, shake well, and then take 0.8 g of the sieved powder and put it into a 1 ml derivatization bottle. Add 3 g of the silylation reagent hexamethyldisilazane, perform derivatization in a water bath at 70 °C for 60 min, then wash with water, filter, and dry to obtain the derivatized powder;

[0116] S3. Headspace solid-phase microextraction: Take out the powder derived in step S2 above and put it into a 20 mL headspace vial. Add 6 mL of saturated NaCl solution and 0.04 mL of ethyl caprylate with a concentration of 50 μg / mL. Then use a manual SPME injector to insert a 70 μm CAR / PDM extraction head into the headspace vial, heat to 90 °C and extract for 40 min, and desorb to obtain the sample to be tested;

[0117] S4. GC-MS detection: Put the sample to be tested obtained in step S3 above into a GC-MS detection system for detection and analysis.

[0118] Example 14

[0119] S1. Preparation of the test sample: First, use a brush to remove the fine soil and sand on the leaves of the collected sample, remove the tobacco veins, and then cut the leaves into shreds; put the shredded tobacco into an oven and bake at 38 °C for 3 h until it can be crushed by hand;

[0120] S2. Sample pretreatment: Crush the tobacco leaf samples dried in step S1 above for 1 minute and sieve through a 40-mesh sieve. Re-grind and sieve the fine veins that did not pass through the sieve. Put the sieved powder into a clean and dry reagent bottle, shake well to mix evenly. Then take 0.8 g of the sieved powder and put it into a 1-ml derivatization bottle, add 3 g of the silylation reagent hexamethyldisilazane, perform derivatization in a water bath at 70 °C for 60 minutes, then wash with water, filter, and dry to obtain the derivatized powder;

[0121] S3. Headspace solid-phase microextraction: Take out the powder derivatized in step S2 above and put it into a 20-mL headspace vial, add 6 mL of saturated NaCl solution and 0.04 mL of ethyl caprate with a concentration of 50 μg / mL. Then use a manual SPME injector to insert a 70-μm CAR / PDM extraction head into the headspace vial, heat to 90 °C and extract for 50 minutes, and desorb to obtain the sample to be tested;

[0122] S4. GC-MS detection: Put the sample to be tested obtained in step S3 above into a GC-MS detection system for detection and analysis.

[0123] Example 15

[0124] S1. Preparation of the test sample: First, use a brush to remove the fine soil and sand grains on the leaves of the collected samples, remove the tobacco stems, and then cut the leaves into filaments; put the cut tobacco filaments into an oven and dry at 39 °C for 3 hours until they can be crushed by hand;

[0125] S2. Sample pretreatment: Crush the tobacco leaf samples dried in step S1 above for 1 minute and sieve through a 40-mesh sieve. Re-grind and sieve the fine veins that did not pass through the sieve. Put the sieved powder into a clean and dry reagent bottle, shake well to mix evenly. Then take 0.8 g of the sieved powder and put it into a 1-ml derivatization bottle, add 3 g of the silylation reagent hexamethyldisilazane, perform derivatization in a water bath at 70 °C for 60 minutes, then wash with water, filter, and dry to obtain the derivatized powder;

[0126] S3. Headspace solid-phase microextraction: Take out the powder derivatized in step S2 above and put it into a 20-mL headspace vial, add 6 mL of saturated NaCl solution and 0.04 mL of ethyl caprate with a concentration of 50 μg / mL. Then use a manual SPME injector to insert a 70-μm CAR / PDM extraction head into the headspace vial, heat to 90 °C and extract for 60 minutes, and desorb to obtain the sample to be tested;

[0127] S4. GC-MS detection: Put the sample to be tested obtained in step S3 above into a GC-MS detection system for detection and analysis.

[0128] Data processing and analysis

[0129] In Examples 11-15 of the present invention, the main difference lies in the different headspace extraction times. The experimental data obtained from the detection of Example 9 and Examples 11-15 are retrieved through the NIST spectral library, combined with the analysis of the retention time of n-alkanes and manual analysis, and the identified compounds with a matching degree greater than 80% are reported. Using the internal standard method, the relative content of each component is expressed as the ratio of the peak area of each volatile component to the peak area of ethyl caprate, and semi-quantitative analysis is carried out, and the total integrated area of the volatile components in the tobacco leaves is recorded. The results are as Figure 4 shown.

[0130] It can be Figure 4 seen that the total integrated area of the volatile components in the tobacco leaves gradually increases with the increase of the headspace extraction time and is basically stable after 40 min. Therefore, in Example 13 of the present invention, the optimal headspace extraction time is 40 min.

[0131] The volatile components of the tobacco leaves during the fermentation process of cigars are detected under the detection conditions of Example 13 of the present invention. The experimental data are retrieved through the NIST spectral library, combined with the analysis of the retention time of n-alkanes and manual analysis, and the identified compounds with a matching degree greater than 80% are reported. Open the mass spectrometry file of the sample to be tested using the MassHunter quantitative software, and perform the integration and calibration of the chromatographic peaks. The peak area (Area) of each chromatographic peak represents the relative content of the corresponding substance, and finally export all the chromatographic peak area integration data for storage.

[0132] Taking the total integrated area and the response value as the main indicators, observe the number and shape of the peaks in the chromatogram, etc., and comprehensively judge the detection results of the GC-MS analysis conditions. The detection results are as Figure 5 shown. The total integrated area is 1.54×10^10, the number of peaks is relatively large, the peak shape is good, the spacing between peaks is clear, there is no obvious overlap, and the overall evaluation of the detection results is excellent. It shows that the detection method provided by the embodiment of the present invention can accurately detect the content of the volatile components of cigars.

[0133] The content described above can be implemented alone or in various combinations, and these variant methods are all within the protection scope of the present invention.

[0134] It should be noted that, in this text, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising an..." does not exclude the presence of additional identical elements in the process, method, article or device comprising the element.

[0135] The above content is a further detailed description of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific embodiments of the present invention are only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention pertains, without departing from the concept of the present invention, several simple deductions or substitutions can still be made, and all should be regarded as belonging to the protection scope of the present invention.

Claims

1. A method for detecting volatile components in tobacco leaves during the fermentation process of cigars, characterized in that, It includes the following steps: S1. Preparation of the sample: Remove the tobacco veins from the collected cigar tobacco leaf samples, then cut the leaves into pieces or shreds, and put the tobacco leaves or shredded tobacco into an oven for drying; S2. Pretreatment of the sample: Crush and sieve the tobacco leaf sample dried in the above step S1, put the sieved powder into a derivatization bottle, add the silylation reagent hexamethyldisilazane, perform derivatization at 70 °C in a water bath for 60 min, then wash with water, filter, and dry to obtain the derivatized powder; S3. Headspace solid-phase microextraction: Take out the powder derivatized in the above step S2 and put it into a 20 mL headspace vial, add saturated NaCl solution and internal standard solution, then use a manual SPME injector to insert a 70 μm CAR / PDM extraction head into the headspace vial, heat for extraction, and desorb to obtain the sample to be tested; S4. GC-MS detection: Put the sample to be tested obtained in the above step S3 into a GC-MS detection system for detection and analysis.

2. The detection method of tobacco leaf volatile components during the cigar fermentation process according to claim 1, characterized in that, In the above step S1, the tobacco leaves or shredded tobacco are put into an oven and dried at 35 - 40 °C for 3 - 4 h.

3. The detection method of tobacco leaf volatile components during the cigar fermentation process according to claim 1, characterized in that, In the above step S2, the crushing time of the tobacco leaf sample is 1 - 2 min, and after crushing, it is sieved through a 40-mesh sieve.

4. The detection method of tobacco leaf volatile components during the cigar fermentation process according to claim 1, wherein In the above step S2, the mass ratio of the sieved powder to hexamethyldisilazane is (0.4 - 1.2):

3.

5. The detection method of tobacco leaf volatile components during the cigar fermentation process according to claim 1, characterized in that, In the above step S3, the internal standard solution is ethyl caprate with a concentration of 50 μg / mL.

6. The detection method of tobacco leaf volatile components during the cigar fermentation process according to claim 1, characterized in that In the above step S3, the mass ratio of the powder, saturated NaCl solution, and internal standard solution is (0.4 - 1.2):6:(0.03 - 0.05).

7. The detection method of tobacco leaf volatile components during the cigar fermentation process according to claim 1, characterized in that, In the above step S3, the temperature for heating extraction is 50 - 100 °C, and the extraction time is 10 - 60 min.

8. The detection method of tobacco leaf volatile components during the cigar fermentation process according to claim 1, characterized in that In the above step S4, the GC / MS analysis conditions are as follows: chromatographic column, TG-5MS 30 m × 0.25 mm × 0.25 μm; carrier gas, He; flow rate, 0.8 mL / min; inlet temperature, 250 °C; transfer line temperature, 280 °C; ion source temperature, 230 °C; programmed temperature rise; ionization energy 70 eV; mass number range 135 - 350 amu.

9. The detection method of tobacco leaf volatile components during the cigar fermentation process according to claim 8, wherein The programmed temperature rise includes the following steps: initial temperature 60 °C, hold for 2 min, increase to 180 °C at a rate of 2 °C / min, hold for 2 min, increase to 260 °C at a rate of 10 °C / min, and hold for 5 min.

10. The detection method of tobacco leaf volatile components during the cigar fermentation process according to claim 1, characterized in that, In the above step S4, the following process is also included: Prepare a n-alkane mixture solution with a concentration of 10 ppm as an external standard, and perform GC-MS analysis together with the sample to be tested extracted in step S3.

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