CN111679008B

CN111679008B - GC-MS-MS method for simultaneously detecting volatile and semi-volatile acids, alcohols and phenols in tobacco leaves and cut tobacco

Info

Publication number: CN111679008B
Application number: CN202010569427.9A
Authority: CN
Inventors: 王冰; 秦亚琼; 崔华鹏; 王晓瑜; 赵俊伟; 蔡君兰; 余晶晶; 谢复炜; 刘绍锋; 刘惠民
Original assignee: Zhengzhou Tobacco Research Institute of CNTC
Current assignee: Zhengzhou Tobacco Research Institute of CNTC
Priority date: 2020-06-20
Filing date: 2020-06-20
Publication date: 2022-09-16
Anticipated expiration: 2040-06-20
Also published as: CN111679008A

Abstract

A GC-MS-MS method for simultaneously detecting volatile and semi-volatile acids, alcohols and phenols in tobacco leaves and cut tobacco is characterized in that: drying and crushing tobacco leaves, soaking and foaming the tobacco leaves by using an acid buffer solution, performing vortex extraction by using a solvent, diluting an extracting solution by using DMF, adding a mixed adsorbent to purify, centrifuging, taking supernate, and filtering by using an organic phase filter membrane. Taking the filtrate, adding a silanization reagent for derivatization, and finally analyzing by adopting GC-MS-MS. The outstanding advantage is: can simultaneously and accurately quantitatively analyze 118 volatile and semi-volatile acid, alcohol and phenol aroma components in the tobacco. The tobacco is soaked by the acidic buffer solution, so that on one hand, the combined acid, alcohol and phenol in the tobacco are converted into free states, the total amount of the free states and the combined states in the tobacco can be measured simultaneously, and the quantitative accuracy is improved; on the other hand, the dissolved amount of nicotine is reduced, and the interference of high content substance of nicotine to other trace components is reduced. The mixed adsorbent is adopted to purify the sample, the operation is simple, the repeatability is good, the accuracy is high, and the rapid analysis can be carried out.

Description

GC-MS-MS method for simultaneously detecting volatile and semi-volatile acids, alcohols and phenols in tobacco leaves and cut tobacco

Technical Field

The invention belongs to the technical field of tobacco flavor component detection, and particularly relates to an analysis method for simultaneously determining volatile semi-volatile acids, alcohols and phenols in tobacco by using acidic buffer solution soaking, organic solvent extraction, DMF (dimethyl formamide) dilution, mixed adsorbent purification and silanization derivatization-gas chromatography-tandem mass spectrometry (GC-MS/MS).

Background

Volatile and semi-volatile acids, alcohols and phenols in the tobacco leaves are important aroma substances in the smoke. Volatile acid is mostly C ₁₂ The following lower fatty acids and partially aromatic acids, which directly enter the smoke during cigarette smoking, have a significant impact on flavor. Alcohol compounds in tobacco comprise aliphatic alcohol, alicyclic alcohol, aromatic alcohol, terpene alcohol and the like, and some alcohols such as menthol, benzyl alcohol, phenethyl alcohol and the like have obvious effect on cigarette fragrance and are used for flavoring the tobacco. Tobacco leaves contain a small amount of simple phenol, and during smoking, the compounds enter smoke through ways such as evaporation and the like, directly affect the fragrance of the smoke, have specific fragrance for some, increase the strange taste of the smoke for some, and are not easily lightened by other aroma-causing agents (see: research and application of heat and mass transfer model in capillary porous medium drying process [ D ] [)]University of major graduates, 2003). The content level and mutual proportion of the components have a key influence on the style and quality of tobacco leaves and cigarettes, and are important chemical components influencing the aroma quality, the aroma quantity and the aroma type of the tobacco leaves, so that the components are also concerned more and more.

At present, the extraction methods of acids, alcohols, phenols and other aroma components in tobacco and smoke mainly comprise a simultaneous distillation extraction method (see isotope dilution-gas chromatography-mass spectrometry analysis of neutral aroma components [ J ] in smoke, journal of mass spectrometry 2019, 40 (6): 566 (573)), an ultrasonic extraction method (see ultrasonic extraction-gas chromatography/mass spectrometry analysis of volatile and semi-volatile organic acids [ J ] in smoke, journal of analytical science 2011, 27 (1): 35-38) and the like, and after extraction, the extraction method is further separated and prepared by liquid chromatography, and different fractions are collected for further analysis (CN 108627600A). The instrument used is typically GC-FID or GC-MS. The distillation extraction method is widely applied in practice, but has the problems of serious loss of low-boiling-point substances, more byproducts, low extraction efficiency, long time consumption and the like. Furthermore, due to the different polarities of the compounds, the acid components are generally analyzed by a polar analysis column, and part of the phenols and alcohol compounds are analyzed together with other neutral flavor components such as aldehyde, ketone and ester in the tobacco leaves by a non-polar column (see: analysis of flavor components in tobacco leaves with different ripeness [ J ] food science, 2007, 23 (2): 98-102 ]). Some acids, phenols and alcohols are difficult to volatilize and have strong polarity, so that few components can be analyzed, and only dozens of components can be analyzed at most. Moreover, most analysis methods adopt the peak area of the compound and the peak area of the internal standard for calculation, and only relative content can be obtained. Due to the limitations of pretreatment conditions and the performance of analytical instruments in the analytical method, the material basis of the style characteristics of tobacco leaves and cigarettes is difficult to reveal. There have been recent patents on the analysis of various organic acids in tobacco (see CN109406704A, CN109212066A), but alcohols and phenols have not been analyzed together. Therefore, there is an urgent need to establish a high-efficiency analysis method for analyzing acids, alcohols and phenols in tobacco leaves with high throughput, easy operation, high accuracy and good sensitivity.

Because of the different polarities of acids, alcohols and phenols, different chromatographic columns are often required to be applied, and the simultaneous analysis cannot be carried out, but the problem can be solved by the silanization reaction. The principle of the silanization gas chromatography is that active hydrogen of hydroxyl, carboxyl, sulfydryl, amino and imino of a derivative object is replaced by trimethylsilyl through silanization derivative reaction, and the volatility of the derivative object is improved, so that the measuring range of the gas chromatography is expanded, the defects that the gas chromatography cannot directly sample and analyze substances with strong polarity, low volatility and poor thermal stability are overcome, and in addition, the method for converting the trimethylsilyl into the derivative has the advantages of improving the separation selectivity of a structure approximate compound, overcoming the adsorption of a carrier and a column wall on a high-polarity low-volatility sample, improving the peak shape of the sample and the like (see the application of a tobacco shred silanization GC fingerprint in the cigarette quality judgment [ J ]. Chinese tobacco science and report, 2007, 13 (3): 18-20). The silanization technology is applied to the analysis of phenol in smoke and the analysis of volatile acid (see: silanization-GC/MS combined determination of 7 phenolic compounds J in cigarette mainstream smoke; tobacco science, 2017, 50 (6): 54-60). At present, no report exists for simultaneously determining acids, alcohols and phenols in tobacco through a silanization reaction.

Disclosure of Invention

The invention aims to establish a general and efficient pretreatment method which is simple to operate, high in universality, low in cost and environment-friendly and can simultaneously extract volatile semi-volatile acids, alcohols and phenols in tobacco leaves and cut tobacco, and to search an optimal chromatographic condition and tandem mass spectrometry MRM multi-reaction monitoring mode parameters by combining a gas chromatography-tandem mass spectrometry technology so as to realize simultaneous analysis of volatile semi-volatile acids, alcohols and phenols in tobacco. In the invention, the sample is silanized, and a GC-MS-MS instrument with higher sensitivity and better selectivity is combined to establish a high-throughput analysis method for simultaneously analyzing the acid, alcohol and phenol substances in the tobacco leaves and the cut tobacco, thereby meeting the requirement of simultaneously carrying out rapid and high-throughput analysis and research on the acid, alcohol and phenol fragrance components in the tobacco. The method is simple, accurate, easy to operate and high in flux, can be used for simultaneously measuring 118 flavor components, and provides technical support for the research of chemical components of tobacco.

The purpose of the invention is realized by the following technical scheme:

a method for simultaneously detecting volatile semi-volatile acids, alcohols and phenols fragrance components in tobacco comprises soaking tobacco sample to be detected in acidic buffer solution, adding organic solvent for extraction, diluting the extract with DMF, adding mixed adsorbent for purifying the sample, centrifuging, collecting supernatant, and filtering with organic phase filter membrane. Taking the filtrate, adding a silanization reagent for derivatization, and finally analyzing by adopting GC-MS-MS. The method comprises the following specific steps:

(1) crushing tobacco leaves: drying flue-cured tobacco leaves, crushing and sieving to 40-60 meshes, and storing at room temperature;

(2) adjusting the pH value of the tobacco leaf sample: 60g of sodium dihydrogen phosphate containing two crystal waters is added to 90mL of deionized water, and then 9.5g of phosphoric acid is added dropwise and shaken well for later use. Weighing 2g of tobacco powder, adding 3mL of prepared acidic buffer solution, stirring uniformly by using a fine glass rod, and soaking for 30min.

(3) Sample extraction: adding 10mL of extraction solvent (dichloromethane) and 50 μ L of internal standard working solution (dichloromethane solution of diethylstilbestrol acid, the concentration is 100 μ g/mL), swirling at 2500r/min for 1-5 min, and centrifuging at 5000-8000 r/min for 3-5 min;

(4) sample purification: putting 500 mu L of organic phase solution into a 2mL centrifuge tube, adding 500 mu L of LDMF, shaking uniformly, adding 5-10 mg of adsorbent (C18 and GCB, the particle size is 40-60 mu m, the mass ratio is 1:1), immediately whirling at 2500r/min for 1-5 min, and centrifuging at 5000-8000 r/min for 3-5 min; the supernatant was filtered through a 0.22 μm organic phase filter.

(5) Derivation of a sample: mu.L of the filtrate was taken and put into a 1ml derivatization flask, and 20. mu.L of derivatization reagent BSTFA was added, and the mixture was subjected to derivatization in a water bath at 60 ℃ for 40 min.

(6) And (3) sample analysis: analyzing the liquid to be detected by gas chromatography-tandem mass spectrometry, preparing a standard curve by adopting a standard working solution, and quantifying by using the standard curve;

the GC-MS/MS analysis conditions were as follows:

a chromatographic column: an elastic quartz capillary chromatographic column, wherein the stationary phase is 5% phenyl-methyl polysiloxane, the specification is 60m multiplied by 0.25mm multiplied by 0.25 μm, and a pre-column (1m multiplied by 0.25mm multiplied by 0.25 μm) is connected in series at the sample inlet end;

sample inlet temperature: 280 ℃;

sample injection amount: 0.8-1.0 μ L;

and (3) sample introduction mode: split-flow sample introduction, split-flow ratio: 10: 1;

carrier gas: helium, constant flow mode, flow rate 1.0 mL/min;

temperature programming; the initial temperature is 40 ℃, then the temperature is increased to 280 ℃ at the temperature increasing speed of 4 ℃/min, and then the temperature is increased to 290 ℃ at the temperature increasing speed of 10 ℃/min, and the temperature is kept for 20 min.

Ionization mode: electron bombardment ionization, wherein the ionization energy is 70 eV;

filament current: 35 muA;

ion source temperature: 280 ℃; quadrupole temperature: 150 ℃; transmission line temperature: 280 ℃;

q2 collision gas: nitrogen (purity 99.999%), flow 1.5 mL/min; quenching gas: helium (purity 99.999%), flow 2.25 mL/min; the scanning mode is as follows: multiple Reaction Monitoring (MRM) mode. MRM parameters include determination of retention time, optimization of parent, daughter and collision energies. Firstly, carrying out Full scanning (Full Scan) (scanning range m/z 40-500) on each compound, determining retention time, and screening 2-3 ions with high mass-to-charge ratios and abundance as alternative parent ions; performing Product Ion Scan (Product Ion Scan) on the parent ions under different collision energies (5, 10, 15, 20, 25 and 30eV), screening 2 ions with high mass-to-charge ratio and abundance as daughter ions, and screening 2-3 pairs of Ion pairs and corresponding optimal collision energy for each compound; and finally, analyzing the standard solution, the matrix extracting solution and the matrix extracting solution added with the standard substance by using an MRM mode, and selecting two pairs of ion pairs without matrix interference and with high sensitivity as quantitative and qualitative ion pairs respectively. The MRM parameters of the target are shown in table 1.

Table 1118 conditions for mass spectrometry detection of target species

The invention has the outstanding advantages that:

1. in the invention, the sample is foamed by using the prepared phosphate buffer solution according to the proportion, on one hand, the pH value of a sample system is adjusted to about 3, and most of combined acid, alcohol and phenolic compounds are dissociated for detection. On one hand, the extraction amount of nicotine in the sample is reduced, and the influence of reducing the column effect of the chromatographic column by a large amount of nicotine is avoided. Finally, the density of the sample is increased after the sample is soaked and foamed by the buffer salt solution, and the sample can be well layered with dichloromethane after the subsequent extraction by the organic solvent and is deposited at the bottom, so that the sampling is convenient.

2. The method adopts dichloromethane extraction, C18 and GCB purification, and GC-MS-MS analysis is carried out after silanization derivatization, so that the operation is simple and rapid, the flux is high, the solvent dosage is less, and the simultaneous extraction and purification of volatile semi-volatile acids, alcohols and phenols in the tobacco can be met; compared with the prior simultaneous distillation extraction method for tobacco flavor components, the method has the advantages of no heating link, no problems of serious loss of low-boiling-point substances and generation of byproducts, short time consumption, low cost, small solvent dosage and high flux.

3. The purifying adsorbent adopted in the invention is a mixture of C18 and GCB with the mass ratio of 1:1, C18 has good adsorption on nonpolar compounds, and GCB mainly adsorbs pigments in samples. The two are matched for use, so that an ideal purifying effect is achieved, a cleaner on-machine solution is provided, and the maintenance of an instrument in the using process is reduced.

4. After the dichloromethane extraction liquid is diluted by DMF, the polarity of the system is enhanced, the adsorption capacity of macromolecular compounds and nonpolar compounds on a purification material (a mixture of C18 and GCB in a mass ratio of 1:1) is increased, the adsorption of polar target substances such as acids, alcohols, phenols and the like is reduced, the purification effect is increased, and the accuracy of the method is improved.

5. In the invention, the sample is silanized, active hydrogen on carboxyl and hydroxyl in acids, alcohols and phenols with different volatile and semi-volatile properties is replaced by trimethylsilyl, the volatility of a derivative object is improved, nonvolatile substances are converted into volatile derivatives, and the defects of strong polarity, low volatility and thermal stability of a gas chromatography are overcome

The defect that substances with poor properties cannot be directly subjected to sample injection analysis is overcome, the adsorption of a carrier and a column wall to a sample with high polarity and low volatility is overcome, and the sample peak shape is improved. The simultaneous determination of 117 compounds such as volatile semi-volatile acids, alcohols, phenols and the like in the tobacco by a set of chromatographic system is realized. Is a high-flux analysis method.

6. In the invention, the function of the pre-column is as follows: high molecular weight impurities in the sample are deposited in the pre-column, so that the pollution at the front end of the analysis column is reduced, and the service life of the column is prolonged; to obtain better peak shape and experimental stability.

7. The method simultaneously measures 119 volatile semi-volatile acids, alcohols and phenols fragrance components in the tobacco by using gas chromatography-tandem mass spectrometry (GC-MS/MS), and compared with the prior method, each compound selects and optimizes a corresponding quantitative ion pair and a corresponding qualitative ion pair, standard spectrum library retrieval is not needed, and the compounds are more accurate in qualitative sense; and the method has higher sensitivity and better precision and repeatability. The matrix effect of 119 compounds is examined, and the experimental result shows that the matrix effect is not obvious in the method, and the solvent preparation standard curve can be adopted for quantification. Establishing standard working curves for 119 compounds by an internal standard method, and calculating the content of corresponding components according to the area correspondence of each target object and the standard curve of each target object; most of the conventional methods for quantifying aroma components are semi-quantitative methods, which estimate the aroma components according to the peak area ratio of selected ions of compounds to the peak area of selected ions of internal standards and the amount of internal standard compounds. The accuracy of the invention is higher.

Drawings

FIG. 1 is a total ion flow diagram of a standard solution in the present invention on a GC-MS/MS.

Fig. 2 shows the results of discrimination analysis of flavor components having significant differences.

Detailed Description

The invention is further described below with reference to examples (figures) without restricting the invention thereto.

Example of content measurement:

(1) preparation of tobacco leaf samples

The tobacco leaves are dried and crushed, and then are screened by a 40-mesh sieve and packed in a sealing bag for later use at room temperature.

(2) Sample pretreatment

Weighing 2g of tobacco powder, adding 3mL of prepared acidic buffer solution, stirring uniformly by using a fine glass rod, and soaking for 30min. Adding 10mL of dichloromethane and 50 μ L of internal standard working solution, vortexing at 2500r/min for 5min, and centrifuging at 8000r/min for 3 min; putting 500 mu L of organic phase solution into a 2mL centrifuge tube, adding 500 mu L of LDMF, shaking uniformly, then adding 10mg of adsorbent (C18 and GCB, the particle size is 40-60 mu m, the mass ratio is 1:1), swirling at 2500r/min for 3min, and centrifuging at 8000r/min for 3 min; the supernatant was filtered through a 0.22 μm organic phase filter. mu.L of the filtrate was taken and put into a 1ml derivatization flask, and 20. mu.L of derivatization reagent BSTFA was added, and the mixture was subjected to derivatization in a water bath at 60 ℃ for 40 min.

(3) Gas chromatograph conditions and mass spectrometry conditions

Chromatographic conditions are as follows: a chromatographic column: an elastic quartz capillary chromatographic column, wherein the stationary phase is 5% phenyl-methyl polysiloxane, the specification is 60m multiplied by 0.25mm multiplied by 0.25 μm, and a pre-column (1m multiplied by 0.25mm multiplied by 0.25 μm) is connected in series at the sample inlet end; sample inlet temperature: 280 ℃; sample introduction amount: 1.0 μ L; and (3) sample introduction mode: split-flow sample introduction, split-flow ratio: 10: 1; carrier gas: helium, constant flow mode, flow rate 1.0 mL/min; temperature programming; the initial temperature is 40 ℃, then the temperature is increased to 280 ℃ at the temperature increasing speed of 4 ℃/min, and then the temperature is increased to 290 ℃ at the temperature increasing speed of 10 ℃/min, and the temperature is kept for 20 min.

Mass spectrum conditions: ionization mode: electron bombardment ionization, wherein the ionization energy is 70 eV; filament current: 35 muA;

ion source temperature: 280 ℃; quadrupole temperature: 150 ℃; transmission line temperature: 280 ℃; q2 collision gas: nitrogen (purity 99.999%), flow 1.5 mL/min; quenching gas: helium (purity 99.999%), flow 2.25 mL/min; the scanning mode is as follows: multiple Reaction Monitoring (MRM) mode. The MRM parameters are shown in Table 1.

(4) Determination of working curve and detection limit

The matrix effect of 119 flavour ingredients was investigated by the following formula: and ME is B/A, wherein A is the slope of the standard curve of the solvent standard working solution, and B is the slope of the standard curve of the matrix matching the standard working solution.

Wherein the concentration of the internal standard substance trans-2-hexenoic acid is 500 ng/mL. According to the content of the target object in the sample, the content of the compounds is lower, and the concentration of the 6-grade marked line is 10, 20, 50, 100, 200 and 500ng/mL respectively; the total 7 compounds with higher content are respectively formic acid, acetic acid, palmitic acid, linoleic acid, oleic acid, linolenic acid and stearic acid, and the concentration of the 6-grade marked line is respectively 2, 4, 10, 20, 40 and 100 mu g/mL. Carrying out linear regression analysis by using the ratio of the peak area of each target object to the peak area of the internal standard and the ratio of the concentration of each target object to the concentration of the internal standard to obtain a standard working curve, wherein the experimental result shows that: the matrix effect of 118 targets was not significant (0.9< ME <1.1), so this experiment can be used to directly prepare standard curves using solvents. Within the linear concentration range, the linear relation of each standard curve is good, and the method is suitable for quantitative analysis. Detection Limit (LOD) and quantification Limit (LOQ) are calculated by using a 3-time signal-to-noise ratio and 10-time signal-to-noise ratio, the detection limit of all target objects is between 1 ng/g and 34ng/g, and the quantification limit is between 3ng/g and 103ng/g, so that the quantitative analysis requirement of the volatile semi-volatile acid components in the tobacco leaves is completely met.

TABLE 2 Standard working curves for the Compounds

(4) Recovery and precision

Weighing 2g of tobacco powder, adding 3mL of prepared acidic buffer solution, adding the standard solution to ensure that the adding level of the low-content target is 0.5 mu g/g and the adding level of the high-content target is 5 mu g/g, uniformly stirring by using a fine glass rod, and soaking for 30min. The other operations are as described in (2), 6 times in a day and 6 times in the day, the precision of the method is considered, and the sample standard addition recovery rate is measured at the high, middle and low content levels (the addition amount is 0.5 time, 1 time and 2 times of the addition amount by taking the content in the sample as reference). The results show that the RSD percent in the day of the 118 compounds is 1.03-8.22 percent, and the RSD percent in the daytime is 1.27-10.64 percent; results for the three-level process recovery ranged between 77.95% and 119.27%. The result shows that the method has good recovery rate, good precision, sensitivity and stability and can meet the requirements of analysis and detection.

TABLE 3119 recovery and precision of Compounds

(5) Sample assay

By adopting the established method, the tobacco leaves with four style characteristics of fresh sweet aroma, burnt sweet aroma and costustoot honey sweet aroma are analyzed, 59 target objects are detected out altogether, and 19 components with significant differences are screened out by adopting F test with the P value less than 0.05 as the standard (Table 4). And (3) carrying out Weierk (Lambda) discriminant analysis on the difference components, wherein the cigarettes of different styles present significant differences on discriminant graphs (see figure 2), which shows that the screened indexes can better distinguish the tobacco leaves of different styles and characteristics.

P values of Table 419 Difference Compounds

	Significance P value		Significance P value
				Formic acid	.015	Benzyl alcohol	.022
Acetic acid	.003	@3 methylfuroic acid	.011
				Butyric acid	.016	Phenylethanols	.006
Valeric acid	.043	Syringol	.021
				N-hexanol	.000	Salicylic acid methyl ether	.002
(Phyllol)	.005	Capric acid	.009
				Trans 2 methyl 2 butenoic acid	.015	@4 Oxononoic acid	.003
@13 butanediol	.035	Lauryl alcohol	.018
				@2 Ethyl hexanol	.044	Eicosanol	.022
N-octyl alcohol	.026

Claims

1. A GC-MS/MS method for simultaneously detecting volatile and semi-volatile acids, alcohols and phenols in tobacco leaves and cut tobacco is characterized in that: the method comprises the following steps of (1) drying and crushing tobacco leaves, soaking the tobacco leaves in an acidic buffer solution, performing vortex extraction by using an extraction solvent, diluting an extracting solution by using DMF, adding a mixed adsorbent for purification, centrifuging, taking supernate, filtering by using an organic phase filter membrane, taking filtrate, adding a silanization reagent for derivatization, and finally analyzing by using GC-MS/MS, wherein the method comprises the following specific steps:

(2) adjusting the pH value of the tobacco leaf sample: adding 60g of sodium dihydrogen phosphate containing two crystal waters into 90mL of deionized water, then dropwise adding 9.5g of phosphoric acid, and shaking up for later use; weighing 2g of tobacco powder, adding 3mL of prepared acidic buffer solution, uniformly stirring by using a fine glass rod, and soaking for 30 min;

(3) sample extraction: adding 10mL of extraction solvent and 50 muL of internal standard working solution, swirling at 2500r/min for 1-5 min, and centrifuging at 5000-8000 r/min for 3-5 min, wherein the internal standard working solution is dichloromethane solution of diethylstilbestrol acid, and the extraction solvent is dichloromethane;

(4) sample purification: adding 500 mu L of organic phase solution into a 2mL centrifuge tube, adding 500 mu LDMF, shaking up, then adding 5-10 mg of adsorbent, immediately performing vortex centrifugation for 1-5 min at 2500r/min, and centrifuging for 3-5 min at 5000-8000 r/min; filtering the supernatant with 0.22 μm organic phase filter membrane; the adsorbent comprises C18 and GCB, the granularity is 40-60 mu m, and the mass ratio is 1: 1;

(5) derivation of a sample: taking 500 mu L of filtrate, adding derivatization test BSTFA20 mu L into a 1ml derivatization bottle, carrying out water bath at 60 ℃, and derivatizing for 40 min;

(6) and (3) sample analysis: analyzing the liquid to be detected by GC-MS/MS, preparing a standard curve by adopting a standard working solution, and quantifying by using the standard curve;

the GC-MS/MS analysis conditions were as follows:

a chromatographic column: an elastic quartz capillary chromatographic column, wherein the stationary phase is 5% phenyl-methyl polysiloxane, the specification is 60m multiplied by 0.25mm multiplied by 0.25 mu m, the injection port end is connected with a pre-column in series, and the specification is 1m multiplied by 0.25mm multiplied by 0.25 mu m;

sample inlet temperature: 280 ℃;

sample introduction amount: 0.8-1.0 μ L;

carrier gas: helium, constant flow mode, flow rate 1.0 mL/min;

temperature programming: the initial temperature is 40 ℃, then the temperature is increased to 280 ℃ at the temperature increasing speed of 4 ℃/min, then the temperature is increased to 290 ℃ at the temperature increasing speed of 10 ℃/min, and the temperature is kept for 20 min;

filament current: 35 muA;

q2 collision gas: nitrogen with the flow rate of 1.5 mL/min; quenching gas: helium with a flow rate of 2.25 mL/min; the scanning mode is as follows: multiple Reaction Monitoring (MRM) mode.

2. The GC-MS/MS method for simultaneously detecting volatile and semi-volatile acids, alcohols and phenols in tobacco leaves and cut tobacco according to claim 1, which is characterized in that: the pH value in the step (2) is adjusted to 3.

3. The GC-MS/MS method for simultaneously detecting volatile and semi-volatile acids, alcohols and phenols in tobacco leaves and cut tobacco according to claim 1, which is characterized in that: the concentration of the dichloromethane solution of the internal standard working solution trans-dihexenic acid is 100 mug/mL.

4. The GC-MS/MS method for simultaneously detecting volatile and semi-volatile acids, alcohols and phenols in tobacco leaves and cut tobacco according to claim 1, which is characterized in that: the MRM parameters comprise determination of retention time and optimization of parent ions, daughter ions and collision energy, and specifically comprise the following steps: firstly, carrying out Full scanning (Full Scan) on each compound, determining retention time, and screening 2-3 ions with large mass-to-charge ratios and abundance as alternative parent ions; performing Product Ion Scan (Product Ion Scan) on the parent ions at different collision energies including 5 eV, 10 eV, 15 eV, 20 eV, 25 eV and 30eV, screening 2 ions with high mass-to-charge ratio and abundance as daughter ions, and screening 2-3 pairs of Ion pairs and corresponding optimal collision energy for each compound; and finally, analyzing the standard solution, the matrix extracting solution and the matrix extracting solution added with the standard substance by using an MRM mode, and selecting two pairs of ion pairs without matrix interference and with high sensitivity as quantitative and qualitative ion pairs respectively.

5. The GC-MS/MS method for simultaneously detecting volatile and semi-volatile acids, alcohols and phenols in tobacco leaves and cut tobacco according to claim 1, which is characterized in that: the number of volatile and semi-volatile acids, alcohol and phenol in the tobacco leaves and the cut tobacco is detected at the same time, and the number is 118.