CN115219602A - Method for determining volatile organic acid in tobacco and tobacco products - Google Patents
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Abstract
The invention discloses a method for measuring volatile organic acid in tobacco and tobacco products, which comprises the following steps: s1, mixing a sample to be detected with an extracting agent with an internal standard, placing the mixture in a closed medium tube, and performing mechanical collision extraction to obtain an extraction liquid; s2, performing membrane treatment on the extract liquor in the S1, placing the extract liquor in a closed medium pipe, and performing mechanical collision derivatization; and S3, detecting the content of the volatile organic acid in the derivatized extract liquor by adopting a gas chromatography-tandem mass spectrometry method. The precision RSD value of the method is less than 4.1 percent, the average standard addition recovery rate is 83.8 to 94.4 percent under the low, medium and high levels, the sensitivity and precision are better, the extraction time only needs 3min, the derivatization time only needs 6min, and the operation is simple and convenient.
Description
Technical Field
The invention relates to the field of tobacco detection, in particular to a method for measuring volatile organic acid in tobacco and tobacco products.
Background
Volatile organic acids, mainly lower fatty acids below C10, are an important class of flavor components in tobacco. The content of volatile organic acid in tobacco is 0.1% -0.2%, although the content is low, the volatile organic acid has important effect on the sensory quality of tobacco and cigarette, and has the effects of increasing the tobacco fragrance and improving the smoking taste. Therefore, the systematic research on the types and the content of the volatile organic acids in the tobacco has important significance for improving the internal quality of the tobacco and guiding the development of cigarette products.
At present, the industry mainly adopts a gas chromatography-mass spectrometry method for the determination of volatile organic acid, and the gas chromatography-mass spectrometry method is adopted for the determination after the tobacco leaves are pretreated. At present, two pretreatment modes mainly exist for analysis by adopting a gas chromatography-mass spectrometry method,
1) The method of the industry standard YC/T500-2014 is adopted, namely acetone is adopted to extract volatile organic acid in tobacco in an oscillating way, and then gas chromatography mass spectrometry is carried out by adopting a polar chromatographic column, so that the method has several problems, (1) the trailing of the chromatographic peak of part of volatile organic acid is indicated in the document 'analysis of free state and combined state organic acid content in tobacco leaves'; (2) volatile organic acids in the tobacco products are extracted by adopting an oscillation extraction mode, the oscillation time is generally 30min, and organic acids with strong volatility, such as formic acid, acetic acid and the like, are easy to escape and lose in the oscillation extraction process, so that the determination result is low;
2) The tobacco product is extracted with dichloromethane and then subjected to silane-based derivatization. The method adopts a gas chromatography-mass spectrometry method to measure the content of the volatile organic acid, and when the derivatization method is used for pretreatment, an extracting agent, an internal standard substance derivatization reagent and gas chromatography-mass spectrometry analysis conditions are generally the prior art, for example, in a patent with the publication number of CN 1092120066A, the method mainly comprises the following steps: adding an extracting agent and an internal standard substance into tobacco leaves or tobacco shreds for extraction, wherein the extracting agent is dichloromethane, n-hexane and acetonitrile, the internal standard substance is usually trans-2-hexenoic acid and cinnamic acid, and filtering to obtain an extraction liquid; adding a derivatization reagent N, O-bis (trimethylsilyl) trifluoroacetamide into the extract liquor for derivatization to obtain a solution to be detected; the gas chromatography-mass spectrometry analysis conditions of the liquid to be detected comprise the following temperature rise program: keeping the temperature at 40-60 ℃ for 1-3 min, then heating to 140-160 ℃ at the speed of 3-10 ℃/min, then heating to 190-210 ℃ at the speed of 5-10 ℃/min, finally heating to 270-290 ℃ at the speed of 20-30 ℃/min, and keeping the temperature for 15-25 min.
The derivatization treatment mainly comprises two modes, namely water bath heating derivatization treatment and microwave-assisted derivatization treatment, the derivatization treatment method can effectively solve the problem of chromatographic peak tailing, but still has several problems, (1) the water bath heating derivatization treatment has longer reaction time, and the report in the literature that dichloromethane is adopted to extract tobacco products in the process of GC-MS (gas chromatography-mass spectrometry) rapid detection of volatile organic acids and semi-volatile organic acids in tobaccos is subjected to water bath heating derivatization for 50min at the water bath heating temperature of 60 ℃; in addition, the patent application number 201811106974.2 discloses a method for measuring non-fatty organic acids in tobacco and tobacco products, wherein the non-fatty organic acids in the tobacco products are extracted by extract liquor and then subjected to water bath heating derivatization treatment, the reaction time is 10-60 min, and the water bath heating temperature is 15-40 ℃; the derivatization time is prolonged, side reactions are easy to occur, such as esterification reaction between alcohols and acids, and certain interference is caused to test results. (2) Microwave-assisted derivatization treatment is dangerous and temperature control is not easy. For example, in patent application No. 201911361110.X, the tobacco sample powder, the internal standard solution and the methanol solution of sulfuric acid are added into a microwave reaction tank for microwave-assisted derivatization treatment, although the reaction rate can be effectively increased and can be completed in only 10min, the microwave reaction conditions are as follows: the pressure was 200kPa, the temperature was 150 ℃ and the power was 600W. Since the side reaction is accelerated at a temperature of 150 ℃, the reaction temperature cannot be controlled although the time for the microwave-assisted derivatization treatment is shortened, which may aggravate the side reaction and affect the accuracy of the result.
Therefore, it is urgently needed to develop a novel volatile organic acid derivatization processing method, which can not only accurately measure and reduce the occurrence of side reactions caused by the rise of reaction temperature and the extension of reaction time, but also can be operated easily and conveniently and improve the working efficiency of scientific research personnel.
Disclosure of Invention
The invention aims to solve the technical problem that the method for determining the volatile organic acid in the tobacco and the tobacco products is provided aiming at the defects of the prior art, so that the content of the volatile organic acid in the tobacco and the tobacco products can be determined quickly and accurately.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a method for measuring volatile organic acid in tobacco and tobacco products comprises the following steps:
s1, mixing a tobacco sample to be detected with an extracting agent with an internal standard, placing the mixture into a closed medium pipe, and performing mechanical collision extraction to obtain an extraction liquid;
s2, mixing the extract liquor obtained in the step S1 with a silanization reagent after membrane treatment, placing the mixture into a sealed medium tube, performing mechanical collision derivatization, placing 1-2 microbeads in the sealed medium tube, performing mechanical collision derivatization reaction on a sealed centrifugal tube in one or more modes of a vortex mode, a vibration mode and/or a suction mode at a linear velocity range of 2.0-10.0m/S, and obtaining a liquid to be detected after the reaction;
and S3, detecting the content of the volatile organic acid in the liquid to be detected by adopting a gas chromatography-tandem mass spectrometry method.
Further, in the step S1, the tobacco sample to be detected and an extracting agent with an internal standard are mixed and then placed in a sealed medium pipe for extraction, the capacity of the sealed medium pipe is 2-5mL, 1-2 microbeads are arranged in the sealed medium pipe, the sealed medium pipe moves in one or more modes of a vortex mode, a vibration mode and/or a suction mode, the linear speed range is 2.0-10.0m/S, and the tobacco microparticles are larger than 400 meshes after extraction.
Further, the concentration of the internal standard substance in the extracting agent with the internal standard substance in S1 is 8-15 mug/mL, and the material-liquid ratio of the tobacco sample to be detected to the extracting agent is 0.1-0.5:2-5g/mL, and the extraction time is 2-3min.
Furthermore, in S2, 1-2 ceramic beads are arranged in a sealed medium pipe, the capacity of the sealed medium pipe is 2-8mL, and the linear velocity is 2-6m/S.
Further, the material-liquid ratio of the tobacco sample to be detected to the internal standard substance in S2 is 0.1-0.5, 40-60 g/mu L, and the derivatization time is 2-7min.
Furthermore, the closed and sealed medium pipe is made of tetrafluoroethylene.
Further, the diameter of the microbeads is 0.5-1cm.
Further, the detection conditions of the gas chromatography-tandem mass spectrometry are that a chromatographic column: HP-5MS, with specification of 60m (length) × 250 μm (inner diameter) × 0.25 μm (film thickness), and temperature rising program of initial temperature, 40 deg.C, holding for 3min, rising to 75 deg.C at 4 deg.C/min, rising to 90 deg.C at 2 deg.C/min, and holding for 15min at 6 deg.C/min to 230 deg.C; the temperature of a sample inlet is 250 ℃; the sample injection amount is 1 mu L, and the split ratio is 15; the carrier gas is helium, and the flow rate is 1.8mL/min; the transmission line temperature is 280 ℃; the ion source temperature is 230 ℃; the temperature of a four-level bar is 150 ℃; ionization energy 70ev; the scanning mode is as follows: SIM mode; the solvent delay was 6min.
Further, the extractant is dichloromethane.
Further, the internal standard is trans-2-hexenoic acid and/or cinnamic acid.
Further, tobacco and tobacco products include, but are not limited to, flue-cured tobacco, burley tobacco, oriental tobacco, tobacco lamina. The volatile organic acid includes but is not limited to one or more of formic acid, acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, 3-methylvaleric acid, 4-methylvaleric acid, caproic acid, enanthic acid, caprylic acid and pelargonic acid.
In S1, tobacco and tobacco products are weighed to be 0.1-0.5 g in mass, an extraction solvent comprises but is not limited to dichloromethane containing an internal standard solution, and the concentration of the internal standard substance comprises but is not limited to 10 mug/mL;
in S1, after extraction, organic membrane filtration is carried out to obtain extract liquor.
Taking a liquid to be detected, and adopting a gas chromatography-tandem mass spectrometry method for detection, wherein the detection comprises the steps of preparing a volatile organic acid standard working solution, establishing a standard working curve and determining the content of the volatile organic acid in the liquid to be detected.
The standard working curve establishment method comprises the following steps: weighing an organic acid standard sample, and dissolving the organic acid standard sample into dichloromethane extraction liquid containing an internal standard substance, wherein the internal standard substance comprises but is not limited to trans-2-hexenoic acid and cinnamic acid, and the concentration comprises but is not limited to 10 mu g/mL; the standard working solution linear range includes, but is not limited to, the following: the linear range of the formic acid standard working solution is 32.3-516.2 mu g/mL, the linear range of the acetic acid standard working solution is 28.8-461.5 mu g/mL, the linear range of the propionic acid standard working solution is 5.03-80.5 mu g/mL, the linear range of the isobutyric acid standard working solution is 0.7-11.5 mu g/mL, the linear range of the butyric acid standard working solution is 0.5-7.5 mu g/mL, the linear range of the isovaleric acid standard working solution is 0.6-10.3 mu g/mL, the linear range of the valeric acid standard working solution is 0.9-14.2 mu g/mL, the linear range of the 3-methylvaleric acid standard working solution is 2.4-37.6 mu g/mL, the linear range of the 4-methylvaleric acid standard working solution is 0.9-13.7 mu g/mL, the linear range of the hexanoic acid standard working solution is 1.2-20.0 mu g/mL, the linear range of the heptanoic acid standard working solution is 3.2-12.7 mu g/mL, the linear range of the octanoic acid standard working solution is 2.1-32.0 mu g/mL, and the linear range of the nonanoic acid standard working solution is 0.0.0 mu g/mL.
Accurately transferring 1mL of the standard working solution with the highest concentration into a chromatographic bottle, transferring N, O-bis (trimethylsilyl) trifluoroacetamide into the standard working solution with the highest concentration by using a liquid transfer gun, wherein the volume of the N, O-bis (trimethylsilyl) trifluoroacetamide comprises but is not limited to 10-100 mu L, and performing derivatization reaction in a water bath heating environment. And determining optimal derivatization conditions of the standard working solution of the organic acid by optimizing the addition amount of a derivatization reagent, the derivatization time and the derivatization temperature, and performing derivatization on the standard working solution of the organic acid under the conditions to establish a standard working curve of the organic acid.
The invention is described below:
(1) The tobacco and the tobacco products are put into a sealed centrifuge tube made of tetrafluoroethylene material, ceramic beads are arranged in the centrifuge tube, organic acid in the tobacco and the tobacco products is quickly extracted in a sealed mode preferably by adopting an American MP homogenizer FastPrep-24 5G, the tobacco and the tobacco products are crushed by the ceramic beads in a high-speed oscillation process, tobacco particles after crushing are larger than 400 meshes, and the particle size of the crushed tobacco is smaller, so that the extraction efficiency can be greatly improved, and the extraction time can be greatly shortened.
(2) Preferably, an American MP homogenizer FastPrep-24 5G is adopted to carry out silane-based derivatization treatment on volatile organic acids in tobacco and tobacco products, the reaction rate can be accelerated by continuous vibration in the high-speed continuous mechanical collision process, less heat is generated in the collision process, the temperature range is not more than 35 ℃, and the temperature is close to the room temperature. The derivatization reaction of the organic acid at normal temperature can be realized by using a little heat generated in the continuous beating process of the ceramic beads and the continuous stirring process, so that the time required by derivatization is greatly shortened, the occurrence of side reaction is avoided, and the measurement result is more appropriate and real.
Compared with the prior art, the invention has the following beneficial effects: the invention adopts an internal standard curve method for quantification, and the precision RSD values of the method are all less than 4.1%; the average standard addition recovery rate is 83.8% -94.4% under the low, medium and high 3 levels; has better sensitivity and precision. Meanwhile, compared with the conventional water bath derivatization method reported in the literature, the method disclosed by the invention has the advantages that the result of measuring the volatile organic acid in the tobacco and the tobacco products is more accurate, the extraction time only takes 3min, the derivatization time only takes 6min, and the operation is simple and convenient.
Drawings
FIG. 1 is a graph showing the effect of different water bath temperatures on the derivatization efficiency of an organic acid standard working curve.
FIG. 2 is a graph showing the effect of different heating times on the derivatization efficiency of an organic acid standard working curve.
FIG. 3 is a graph showing the effect of different addition amounts of derivatizing agents on the derivatization efficiency of an organic acid standard working curve.
Detailed Description
The invention is further described with reference to specific examples. The following examples are intended to illustrate the invention without further limiting it.
Examples
1 materials, reagents and instruments
1.1 materials, reagents
1.1.1 materials
Formic acid, acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, 3-methylvaleric acid, 4-methylvaleric acid, caproic acid, heptanoic acid, caprylic acid, nonanoic acid, carbofuran; trans-2-hexenoic acid, cinnamic acid, carbofuran; derivatization reagent: n, O-bis (trimethylsilyl) trifluoroacetamide, sigma, usa; one part of upper tobacco leaf, one part of middle tobacco leaf and one part of lower tobacco leaf.
1.1.2 reagents
Dichloromethane (chromatogram pure)
1.2 instruments
HP 6890/5973 gas chromatography-mass spectrometer, electronic balance, and constant temperature water bath kettle
2 organic acid standard working solution derivation conditions
2.1 temperature investigation of derivatization
Performing derivatization treatment on the organic acid standard working solution with the highest concentration by adopting a water bath heating derivatization method, accurately transferring 1mL of the organic acid standard working solution with the highest concentration, adding 60 muL of silanization derivatization reagent, performing derivatization for 50min at 40 ℃, 50 ℃,60 ℃, 70 ℃ and 80 ℃ respectively, and inspecting the derivatization efficiency of the organic acid standard working solution at different temperatures, wherein the result is shown in figure 1. The results show that the derivatization efficiency of the organic acid is higher when the water bath temperature reaches 60 ℃.
2.2 derivatization time study
Performing derivatization treatment on the organic acid standard working solution with the highest concentration by adopting a water bath heating derivatization method, accurately transferring 1mL of the organic acid standard working solution with the highest concentration, adding 60 muL of silanization derivatization reagent, performing derivatization for 20min, 30min, 40min, 50min and 60min at the temperature of 60 ℃, and inspecting the derivatization efficiency of the organic acid standard working solution under different heating time, wherein the result is shown in figure 2. The results show that the derivatization efficiency of the organic acid is higher when the heating time is 50 min.
2.3 derivatization reagent addition amount investigation
Performing derivatization treatment on the organic acid standard working solution with the highest concentration by adopting a water bath heating derivatization method, accurately transferring 1mL of the organic acid standard working solution with the highest concentration, respectively adding 40 muL, 50 muL, 60 muL, 70 muL and 80 muL of silanization derivatization reagents, derivatizing for 50min at the temperature of 60 ℃, and examining the influence of the addition amount of the derivatization reagents on the derivatization efficiency, wherein the result is shown in FIG. 3. The results show that the derivatization efficiency of the organic acid is high when the addition amount of the derivatization reagent is 60. Mu.L.
2.4 establishment of organic acid Standard working Curve
Taking 1mL of 5 organic acid mixed standard solutions with different concentration gradients containing internal standards (trans-2-hexenoic acid and cinnamic acid), respectively adding 60 muL of BSTFA derivatization reagent, reacting in a water bath at 60 ℃ for 50min, cooling to room temperature, and then carrying out GC-MS analysis and determination. And the corresponding concentration is plotted according to the ratio of the peak area of the organic acid derivatization product to the peak area of the internal standard, so as to obtain the standard working curve equation of the organic acid, and the result is shown in table 1. Taking 6 parts of organic acid standard solution with the lowest concentration, performing derivatization, then introducing a sample to obtain a standard deviation, and calculating a detection line and a quantification limit by using 3 times and 10 times of standard deviation respectively, wherein the results are shown in table 1.
TABLE 1 organic acid Standard Curve, determinant coefficient, detection Limit and quantification Limit
3. Organic acid sample derivation conditions
3.1 Investigation of liquid-to-feed ratio
Compared with the lower tobacco leaves and the middle tobacco leaves, the upper tobacco leaves have more organic acid content, so the upper tobacco leaves are considered as objects. Accurately weighing 0.1g, 0.15g, 0.2g, 0.25g and 0.3g of upper tobacco leaves, adding 3mL of dichloromethane extractant containing internal standard, and extracting organic acid components in the tobacco leaves by adopting a mechanical collision technology for 3min. And adding 60 mu L of BSTFA derivatization reagent into 1mL of extract liquor after membrane filtration, and performing derivatization by adopting a mechanical collision technology, wherein the derivatization time is 5min. The solution after derivatization was subjected to GC-MS analysis, and the results are shown in Table 2. As can be seen from Table 2, the organic acid content measured for the tobacco samples of 0.1g and 0.15g was better, but the content of 9 of the 13 volatile organic acids was higher than that measured for the tobacco sample of 0.15g, so the tobacco sample of 0.15g was selected.
TABLE 2 Effect of different quality samples on the results of organic acid determination
3.2 examination of derivatization time
Accurately weighing 0.15g of upper tobacco leaves, adding 3mL of dichloromethane extractant containing internal standard, and extracting organic acid components in the tobacco leaves by adopting a homogenate instrument technology for 3min. And adding 60 mu L of BSTFA derivatization reagent into 1mL of extract liquor after the membrane is filtered, and performing derivatization by adopting a homogenizer technology, wherein the derivatization time is respectively 2min, 3min, 4min, 5min, 6min and 7min. The solution after derivatization was subjected to GC-MS analysis, and the results are shown in Table 3. As can be seen from Table 3, the effect of measuring the content of organic acid in the derivatization time of 6min was better.
TABLE 3 Effect of different derivatization times on the results of the organic acid assay
3.3 examination of the amount of derivatizing agent
Accurately weighing 0.15g of upper tobacco leaves, adding 3mL of dichloromethane extracting agent containing internal standard, and extracting organic acid components in the tobacco leaves by adopting a homogenizer technology for 3min. And (3) respectively adding 10 mu L, 20 mu L, 30 mu L, 40 mu L, 50 mu L and 60 mu L of BSTFA derivatization reagent into 1mL of extract liquor after membrane passing, and performing derivatization by adopting a homogenizer technology, wherein the derivatization time is 6min. The solution after derivatization was subjected to GC-MS analysis, and the results are shown in Table 4. As is clear from Table 4, the effect of measuring the content of organic acid was better when the amount of the derivatizing reagent was 50. Mu.L.
TABLE 4 Effect of different amounts of derivatizing reagents on organic acid assay results
4. Methodology investigation
4.1 Stability of the derivatized product
Accurately weighing 0.15g of upper tobacco leaves, adding 3mL of dichloromethane extracting agent containing internal standard, and extracting organic acid components in the tobacco leaves by adopting a homogenizer technology for 3min. Taking 1mL of extract after membrane passing, adding 50 mu L of BSTFA derivatization reagent, and adopting a homogenizer technology to perform derivatization, wherein the derivatization time is 6min.
The extraction method adopts a homogenizer to perform ball milling extraction, the homogenizer preferably selects an American MP homogenizer FastPrep-24 5G, the linear velocity is preferably 4m/s, a sealed centrifuge tube made of tetrafluoroethylene material is selected, 2 ceramic beads are arranged in the centrifuge tube, and the capacity of the centrifuge tube is 5mL. After the extract was passed through an organic membrane, 1mL of the extract was transferred to a 2mL sealed centrifuge tube made of tetrafluoroethylene, and 1 ceramic bead was placed in the tube. The linear velocity of ball milling derivatization ball milling is 4m/s.
Carrying out a derivatization test on an organic acid sample under an optimized pretreatment condition, cooling to room temperature, and carrying out GC-MS analysis; after the injection, the sample is placed in a refrigerator, and the sample is injected for 8 times at intervals of 12 hours. The results show that the RSD of the peak area of each organic acid derivative and the peak area of the internal standard is not more than 4.1 percent, which indicates that the organic acid silanization derivative product is stable and is suitable for quantitative analysis.
The method adopts an American MP homogenizer FastPrep-24 5G for derivatization, has small solvent usage amount, can process 24 samples at one time, is suitable for measuring the content of volatile organic acid in tobacco and tobacco products on a large scale, and improves the efficiency of workers.
4.2 reproducibility and recovery
And (3) quantitatively analyzing the volatile organic acid in the tobacco and the tobacco products by adopting an internal standard curve method. Gas chromatography sample introduction conditions include, but are not limited to, chromatography column: HP-5MS, its specification is 60m (length) × 250 μm (inner diameter) × 0.25 μm (film thickness), the temperature program is initial temperature, 40 deg.C, keep 3min, rise to 75 deg.C with 4 deg.C/min, rise to 90 deg.C with 2 deg.C/min, keep 15min with 6 deg.C/min to 230 deg.C; the temperature of a sample inlet is 250 ℃; the sample injection amount is 1 mu L, and the split ratio is 15; the carrier gas is helium, and the flow rate is 1.8mL/min; the transmission line temperature is 280 ℃; the ion source temperature is 230 ℃; the temperature of a four-level bar is 150 ℃; ionization energy 70ev; the scanning mode is as follows: SIM mode; the solvent delay was 6min.
The same sample was subjected to 5 replicates using the above parameters and the results are shown in table 5. The standard sample is added into the sample according to three different levels of low, medium and high respectively, each level is subjected to 3 times of repeated tests, the standard adding recovery rate is calculated according to the measurement result, and the standard adding recovery rate is compared with the standard adding recovery rate of volatile organic acid in the literature 'GC-MS method rapid detection of volatile organic acid and semi-volatile organic acid in tobacco', and the result is shown in Table 5. As can be seen from Table 5, the recovery rate of spiking in the present invention is 89.3% to 97.7%, and the recovery rate of spiking in the literature is 81.4% to 93.8%. The average recovery rate of the method is higher than that of the literature for the same organic acid, and the method is more accurate in measurement result.
TABLE 5 relative standard deviation and spiked recovery for organic acid determination
5 comparison of the methods
The results of 3 types of tobacco shreds of flue-cured cigarettes are used as detection objects, the optimized conditions of the invention are compared with the method reported in the literature "GC-MS method rapid detection of volatile organic acids and semi-volatile organic acids in tobacco", and the results of the two methods are compared, as shown in Table 6. As can be seen from Table 6, the method for determining the content of volatile organic acid in the patent of the invention is shorter than the method reported in the literature except that the pretreatment time is shorter, and the method is mostly higher than the method reported in the literature due to the escape of part of volatile organic acid and the occurrence of side reactions in the processes of vibration extraction and water bath derivatization, which also indicates that the method of the invention is better in accuracy than the method used in the literature.
TABLE 6 results of determination of volatile organic acids in tobacco samples by the methods
Claims (9)
1. A method for measuring volatile organic acid in tobacco and tobacco products is characterized by comprising the following steps:
s1, mixing a tobacco sample to be detected with an extracting agent with an internal standard, placing the mixture into a closed medium pipe, and performing mechanical collision extraction to obtain an extraction liquid;
s2, mixing the extract liquor obtained in the step S1 with a silanization reagent after membrane treatment, placing the mixture into a sealed medium tube, and performing mechanical collision derivatization, wherein 1-2 micro-beads are arranged in the sealed medium tube, the sealed centrifugal tube performs mechanical collision derivatization reaction in one or more modes of a vortex mode, a vibration mode and/or a suction mode, the linear velocity range is 2.0-10.0m/S, and the liquid to be detected is obtained after the reaction;
and S3, detecting the content of the volatile organic acid in the liquid to be detected by adopting a gas chromatography-tandem mass spectrometry method.
2. The determination method according to claim 1, wherein in S1, the tobacco sample to be determined and the extracting agent with the internal standard are mixed and then placed in a sealed medium tube for extraction, the capacity of the sealed medium tube is 2-5mL, 1-2 microbeads are arranged in the sealed medium tube, the sealed medium tube moves in one or more modes of a vortex mode, a vibration mode and/or a suction mode, the linear velocity range is 2.0-10.0m/S, and the tobacco microparticles are larger than 400 meshes after extraction.
3. The method for measuring according to claim 2, wherein: the concentration of the internal standard in the extractant with the internal standard in S1 is 8 to 15μg/mL, wherein the material-liquid ratio of the tobacco sample to be detected to the extracting agent is 0.1-0.5:2-5g/mL, and the extraction time is 2-3min.
4. The method according to claim 1, wherein in S2, 1 to 2 ceramic beads are placed in a closed medium tube having a capacity of 2 to 8mL and a linear velocity of 2 to 6m/S.
5. The method for measuring according to claim 1, wherein: the material-liquid ratio of the tobacco sample to be detected to the internal standard substance in S2 is 0.1-0.5μL, and the derivatization time is 2-7min.
6. The method for measuring according to claim 5, wherein: the closed medium pipe is made of tetrafluoroethylene.
7. The assay method according to any one of claims 1 to 5, wherein: the detection conditions of the gas chromatography-tandem mass spectrometry are that a chromatographic column: HP-5MS, with specification of 60m (length) × 250 μm (inner diameter) × 0.25 μm (film thickness), and temperature rising program of initial temperature, 40 deg.C, holding for 3min, rising to 75 deg.C at 4 deg.C/min, rising to 90 deg.C at 2 deg.C/min, and holding for 15min at 6 deg.C/min to 230 deg.C; the temperature of a sample inlet is 250 ℃; the sample injection amount is 1 mu L, and the split ratio is 15; the carrier gas is helium, and the flow rate is 1.8mL/min; the transmission line temperature is 280 ℃; the ion source temperature is 230 ℃; the temperature of a four-level bar is 150 ℃; ionization energy 70ev; the scanning mode comprises the following steps: a SIM mode; the solvent delay was 6min.
8. The assay method according to any one of claims 1 to 5, wherein: the extractant is dichloromethane.
9. The assay method according to any one of claims 1 to 5, wherein: the internal standard is trans-2-hexenoic acid and/or cinnamic acid.
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