A method for detecting tobacco-specific n-nitrosamine components in tobacco

Abstract

The application discloses a tobacco-specific N-nitrosamine component extraction method and an acetonitrile-water extract of tobacco-specific N-nitrosamine components of a tobacco sample obtained by the extraction method. The extraction method is based on a volatile component collection device and comprises the following operations: heating a tobacco sample at 260-380 DEG C for 6.0-8.0 min, transporting the volatilized components to an acetonitrile-water solution at 0 DEG C + / - 5 DEG C by using a carrier gas, and obtaining an acetonitrile-water extract of tobacco-specific N-nitrosamine components. The extraction method disclosed by the application is short in time consumption, simple in operation, high in purity of extracted N-nitrosamine components, and low in interference, and can be directly analyzed and determined without subsequent purification. The application further discloses a detection method for tobacco-specific N-nitrosamine components in tobacco based on the above extraction method.

Description

Technical Field

[0001] This invention belongs to the field of testing technology, specifically relating to a novel method for extracting tobacco-specific N-nitrosamine components and a method for detecting tobacco-specific N-nitrosamine components based on this method. Background Technology

[0002] Tobacco-specific N-nitrosamines (TSNAs) are a group of carcinogens found only in tobacco, tobacco products, and tobacco smoke. They are products formed by the reaction of tobacco alkaloids with nitrosating agents under acidic conditions. Currently, eight TSNAs have been identified: N-nitrosonicotinic acid (NNN), 4-(methylnitroso)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosonechoic acid (NAT), N-nitrosopseudoestiline (NAB), 4-(methylnitroso)-4-(3-pyridyl)-1-butanal (NNA), 4-(methylnitroso)-1-(3-pyridyl)-1-butanol (NNAL), 4-(methylnitroso)-4-(3-pyridyl)-1-butanol (iso-NNAL), and 4-(methylnitroso)-4-(3-pyridyl)-butyric acid (iso-NNAC). Among them, the first four compounds have been studied most extensively and in-depth. TSNAs are not only important compounds on the Hoffman List and the U.S. Food and Drug Administration's list of "Hazardous and Potentially Hazardous Substances in Tobacco Products and Smoke," but also key components on the International Agency for Research on Cancer's list of "28 Hazardous Substances in Smokeless Tobacco Products." It is generally believed that TSNAs cause oxidative damage to DNA during metabolism, and NNN and NNK have been classified as Group 1 carcinogens by the International Agency for Research on Cancer. Current research indicates that TSNAs in tobacco smoke mainly originate from tobacco leaves. Therefore, determining the TSNA content in tobacco is of great significance.

[0003] Due to the complex matrix of tobacco and the extremely low content of TSNAs (typically at the μg / g or even ng / g level), all current analytical methods for TSNAs in tobacco require appropriate sample pretreatment before instrumental analysis to enrich TSNAs and remove interfering impurities. Current standard methods both domestically and internationally employ solvent extraction, purification, and concentration as sample pretreatment methods. For example, the industry standard YC / T 184, "Determination of Tobacco-Specific N-nitrosamines in Tobacco and Tobacco Products," specifies that each gram of tobacco sample should be ultrasonically extracted with 20 mL of dichloromethane and 1 mL of 10% sodium hydroxide solution for 20 minutes. ISO 22304:2008, "Determination of Tobacco-Specific Nitrosamines in Tobacco - Alkaline Dichloromethane Extraction Method," specifies that each 1.5 grams of tobacco sample should be shaken with 10 mL of dichloromethane and 0.5 mL of 10% sodium hydroxide solution for 44 minutes. CORESTA (Cooperation Centre for Tobacco Research) Recommended Method (CRM) No. 72 specifies that each gram of tobacco sample should be shaken for 40 minutes in 30 mL of 100 mM ammonium acetate solution.

[0004] The above methods all have the following problems: due to the principle of solvent extraction (like dissolves like), when extracting TSNAs with solvent, impurities such as pigments in the smoke sample are inevitably extracted along with them. Additional impurity removal steps are usually required before subsequent instrumental analysis; the amount of solvent consumed is large, especially organic reagents such as dichloromethane; the extraction time for a single sample is long, not less than 20 minutes, and with the subsequent purification process, the preparation of a test sample for instrumental analysis takes even longer.

[0005] Therefore, it is necessary to develop a rapid, solvent-free method for extracting TSNAs from smoke samples without subsequent purification steps. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a novel method for extracting tobacco-specific N-nitrosamines from tobacco samples. This method is time-efficient, simple to operate, and yields high-purity N-nitrosamines with minimal interference, requiring no subsequent purification and allowing for direct analysis. This invention also provides a method for detecting tobacco-specific N-nitrosamines in tobacco based on the aforementioned extraction method.

[0007] To achieve the above-mentioned technical effects, the present invention adopts the following technical solution.

[0008] A method for extracting N-nitrosamines specific to tobacco, comprising the following steps:

[0009] The tobacco sample was heated at 260~380℃ for 6.0~8.0 min, and the volatilized components were transported to an acetonitrile-water solution at 0℃±5℃ using a carrier gas to obtain an acetonitrile-water extract of tobacco-specific N-nitrosamine components.

[0010] Preferably, the extraction method is based on a volatile component collection device; the volatile component collection device includes a carrier gas pipeline, a gas flow meter, a heating device, a material storage chamber, a first gas valve, a collection device, and a cooling device; wherein, the gas flow meter is connected and installed on the carrier gas pipeline; the material storage chamber is provided with a storage cavity for placing the tobacco sample, the storage cavity is connected to the carrier gas pipeline, and the storage cavity is provided with a carrier gas inlet and a carrier gas outlet on two opposite sides; the heating device is used to heat the storage cavity; the first gas valve is connected and installed on the carrier gas pipeline after the material storage chamber; the collection device includes a collection bottle containing an acetonitrile-water solution, the collection bottle is placed in the cooling device and placed at the end of the carrier gas pipeline, and the acetonitrile-water solution in the collection bottle covers the opening at the end of the carrier gas pipeline.

[0011] Preferably, the heating device includes a heating layer, a temperature sensor, and a heater for heating the heating layer. The heating layer is wrapped around the outer wall of the material storage cavity, and the temperature sensor is located inside the storage cavity.

[0012] Preferably, the cooling device is selected from a cold trap or an ice bath, and more preferably a cold trap.

[0013] Preferably, the cold trap is a condensation trap.

[0014] Preferably, the volatile component collection device further includes a controller, which is communicatively connected to the gas flow meter, the heating device, the first gas path valve, and the cooling device.

[0015] In a preferred embodiment, the volatile component collection device further includes a cleaning device, a second gas valve, and a three-way connector. The carrier gas pipeline includes a connected conveying section, a cleaning section, and a collection section. The three-way connector includes a first end, a second end, and a third end. The first end is connected to the conveying section, the second end is connected to the collection section, and the third end is connected to the cleaning section. A gas flow meter, a material storage chamber, and the first gas valve are connected to the conveying section. The second gas valve is connected to the cleaning section, and the collection bottle is placed at the end of the collection section. The cleaning device includes an infusion pump and a cleaning bottle containing cleaning fluid. The inlet of the infusion pump is placed inside the cleaning bottle, and the outlet of the infusion pump is connected to the collection section.

[0016] In the above-described embodiments including a cleaning device, the volatile component collection device further includes a controller, which is communicatively connected to a gas flow meter, a heating device, a first gas path valve, a cooling device, a second gas path valve, and a delivery pump.

[0017] When the first gas path valve is closed and the second gas path valve is opened, the pipelines of the cleaning section and the collection section can be cleaned, avoiding the pollution of the next collection of volatile components by the residual gas in the pipeline, and improving the collection purity of the volatile component collection equipment.

[0018] Preferably, the tobacco sample is 0.10g ± 0.05g.

[0019] Preferably, the acetonitrile-water solution is 1 mL.

[0020] Preferably, the temperature of the acetonitrile-water solution is 0℃±2℃; more preferably, it is -2℃.

[0021] Preferably, the tobacco sample is heated at 320°C.

[0022] Preferably, the tobacco sample is heated for 6.0 min.

[0023] Preferably, the flow rate of the carrier gas is 2 mL / min.

[0024] More preferably, the carrier gas is selected from compressed air, nitrogen, and helium; more preferably, nitrogen.

[0025] Preferably, after heating, the collection section is washed with 0.25~0.5mL, more preferably 0.25mL of acetonitrile-water solution. The washing solution is combined with the acetonitrile-water solution in the collection bottle to obtain an acetonitrile-water extract of tobacco-specific N-nitrosamine components.

[0026] Preferably, the acetonitrile-water solution has a volume ratio of acetonitrile to water of 1:1.

[0027] The method for extracting tobacco-specific N-nitrosamines provided by this invention is based on a novel volatile component collection device. For example... Figure 1 As shown, in the volatile component collection device of the present invention, when the sample in the storage chamber (e.g., the tobacco sample to be tested according to the present invention) is uniformly wrapped in the heating layer and heated to a specific temperature, the tobacco-specific N-nitrosamine components, which are thermally volatile, are carried by the carrier gas to the collection bottle and absorbed by the cooled absorption solvent (e.g., acetonitrile-water (1:1, v / v) of the present invention), thus achieving continuous collection of volatile components at this specific temperature. The first gas path valve and the second gas path valve allow for cleaning of the collection section pipeline, which not only reduces memory effect and interference with subsequent sample analysis but also allows for more thorough extraction of N-nitrosamine components. When the first gas path valve is opened and the second gas path valve is closed, the volatile components (e.g., the N-nitrosamine components of the present invention) are absorbed; when the first gas path valve is closed and the second gas path valve is opened, the collection section pipeline is cleaned.

[0028] Therefore, the present invention also provides an acetonitrile-water extract of tobacco-specific N-nitrosamine components from tobacco samples prepared by the above extraction method.

[0029] Another objective of this invention is to provide a method for detecting tobacco-specific N-nitrosamines in tobacco, based on liquid chromatography-tandem mass spectrometry; wherein, the tobacco sample to be tested is pretreated using the extraction method for tobacco-specific N-nitrosamines described in this invention to obtain a test solution; the standard is selected from at least one of NNN, NNK, NAT and NAB, and the deuterated product of the standard is used as an internal standard.

[0030] Preferably, the detection method includes the following steps:

[0031] (1) Preparation of the test solution

[0032] The tobacco sample was heated at 320°C for 6.0-8.0 min, and the volatilized components were transported to an acetonitrile-water solution at 0°C±5°C, preferably 0°C±2°C, more preferably -2°C, to obtain an acetonitrile-water extract of tobacco-specific N-nitrosamine components. The pipeline through which the carrier gas flowed was cleaned with a blank acetonitrile-water solution, and the cleaning solution was combined with the acetonitrile-water extract. An internal standard was added, and the volume was adjusted to the specified volume with acetonitrile-water solution to obtain the test solution.

[0033] (2) Preparation of a series of mixed standard solutions

[0034] A series of mixed standard solutions comprising the aforementioned standard and the corresponding internal standard were prepared using methanol and 0.1 mol / L ammonium acetate solution as solvents.

[0035] (3) Measurement

[0036] Liquid chromatography conditions include:

[0037] Column: ACQUITY UPLC ® BEH C18 column, 2.1 × 100 mm, 1.7 μm;

[0038] Column temperature: 40℃;

[0039] Injection volume: 5 μL;

[0040] Mobile phase: 0.01 mol / L ammonium acetate as phase A, 0.1% formic acid methanol solution as phase B, gradient elution, elution program as follows:

[0041] t=0 min, 90%A, 10%B;

[0042] t=1.0 min, 90%A, 10%B;

[0043] t=5.0min, 10%A, 90%B;

[0044] t=6.0min, 10%A, 90%B;

[0045] t=6.1 min, 90%A, 10%B;

[0046] t=8.0 min, 90%A, 10%B;

[0047] Flow rate: 0.25 mL / min;

[0048] Mass spectrometry conditions include:

[0049] Ionization mode: positive ion mode ionization;

[0050] Electrospray voltage: 3000 V;

[0051] Tapered hole voltage: 80 V;

[0052] Ion source temperature: 550℃;

[0053] Desolventizing gas flow: 800 L / hour;

[0054] Conical orifice airflow: 50 L / hour;

[0055] Dwell time: 24 ms;

[0056] Monitoring method: Multiple response monitoring;

[0057] Under the conditions of liquid chromatography and mass spectrometry, the test solution and the series of standard solutions were precisely pipetted into the liquid chromatography-tandem mass spectrometer for determination, and the chromatograms were recorded.

[0058] (4) Data processing and analysis

[0059] The assay includes qualitative and quantitative determinations. The qualitative determination involves determining the presence of the corresponding N-nitrosamine component in the test sample solution based on the retention time, quantitative and qualitative ion pairs, and corresponding collision energies of the standard. The quantitative determination employs the internal standard method, plotting a working curve with the ratio of the peak area of ​​the N-nitrosamine component in the series of mixed standard solutions to the peak area of ​​the internal standard as the ordinate and the concentration of the N-nitrosamine component in the series of mixed standard solutions as the abscissa. Based on the ratio of the peak area of ​​the N-nitrosamine component in the test sample solution to the peak area of ​​the internal standard, and according to the working curve, the content of the target N-nitrosamine component per gram of tobacco sample is calculated using Formula I.

[0060] I,

[0061] Where m: the content of the target N-nitrosamine component per gram of tobacco sample, ng / g.

[0062] x: The ratio of the peak area of ​​the target N-nitrosamine to that of the internal standard.

[0063] a: The slope of the working curve.

[0064] b: Intercept of the working curve

[0065] V: Volume of the test solution, mL

[0066] n: Mass of the tobacco sample, in grams.

[0067] Preferably, step (1) is performed in the volatile component collection device of the present invention; the tobacco sample is placed in the storage chamber, and the acetonitrile-water solution that absorbs the tobacco-specific N-nitrosamine components is placed in the collection bottle, the collection bottle is placed in the cooling device and positioned below the end opening of the collection section of the carrier gas pipeline, so that the end opening of the collection section is submerged in the acetonitrile-water solution; the temperature of the cooling device is set to 0℃±5℃, preferably 0℃±2℃, more preferably -2℃.

[0068] Preferably, in step (1), the mass of the tobacco sample is 0.10g ± 0.05g.

[0069] Preferably, in step (1), the volume of the acetonitrile-water solution that absorbs the tobacco-specific N-nitrosamine components is 1 mL.

[0070] Preferably, in step (1), the heating time is 6 minutes.

[0071] Preferably, in step (1), the flow rate of the carrier gas is 2 mL / min.

[0072] More preferably, the carrier gas is selected from compressed air, nitrogen, and helium; more preferably, nitrogen.

[0073] Preferably, after heating, the collection section of the carrier gas pipeline is cleaned with 0.25~0.5mL, more preferably 0.25mL of acetonitrile-water solution, and the cleaning solution is combined with the acetonitrile-water solution in the collection bottle to obtain the test solution.

[0074] Preferably, the acetonitrile-water solution has a volume ratio of acetonitrile to water of 1:1.

[0075] Preferably, the specific operation of step (1) is as follows:

[0076] Place 0.10 g ± 0.05 g of tobacco sample in the storage chamber of the volatile component collection device, and set the cooling device to -2 °C. Accurately pipette 1 mL of acetonitrile-water solution (1:1 volume ratio) into a collection bottle as the absorbent. Then place the collection bottle in the cooling device and position it below the end opening of the collection section of the carrier gas pipeline, ensuring that the end opening of the collection section is submerged in the acetonitrile-water solution. Close the second gas path valve. Open the first gas path valve and adjust the gas flow meter to set the nitrogen flow rate to 2 mL / min. Set the heating layer temperature to 320 °C and maintain the temperature for 6.0 min. Then stop heating the heating layer, close the first gas path valve, open the second gas path valve, and clean the collection section of the carrier gas pipeline with 0.25 mL of acetonitrile-water solution (1:1 volume ratio). Combine the cleaning solution with the absorbent in the collection bottle, add an internal standard, and dilute to 2 mL with acetonitrile-water solution (1:1 volume ratio) to obtain the test solution.

[0077] Preferably, the concentration of the deuterated product of the standard in the test solution is 10 ng / mL.

[0078] Preferably, in step (2), the standard is all of NNN, NNK, NAT and NAB.

[0079] Preferably, in step (2), the concentration gradient of the standard in the series of mixed standard solutions is 0.5, 1.0, 2.0, 5.0, 10.0, 20.0 and 50 ng / mL.

[0080] Preferably, in step (2), the concentration of the deuterated product of the standard in the series of mixed standard solutions is 10 ng / mL.

[0081] Preferably, in step (4), the retention time of NNN is 3.44 min ± 0.10 min, the quantitative ion pair and collision energy are 178 > 148, 11 eV, and the qualitative ion pair and collision energy are 178 > 120, 17 eV, respectively; the retention time of NNN-d4 is 3.42 min ± 0.10 min, the quantitative ion pair and collision energy are 182 > 152, 11 eV, respectively; the retention time of NNK is 3.64 min ± 0.10 min, the quantitative ion pair and collision energy are 208 > 122, 11 eV, and the qualitative ion pair and collision energy are 208 > 106, 21 eV, respectively; the retention time of NNK-d4 is 3.63 min ± 0.10 min, the quantitative ion pair and collision energy are 212 > 126, 11 eV, respectively; and the retention time of NAT is 3.99 min ± 0.10 min. The retention times of NAT-d4 were 3.98 min ± 0.10 min, with quantitative ion pair and collision energies of 194 > 164 and 10 eV, respectively; the retention times of NAB were 4.10 min ± 0.10 min, with quantitative ion pair and collision energies of 192 > 162 and 12 eV, respectively; the retention times of NAB-d4 were 4.09 min ± 0.10 min, with quantitative ion pair and collision energies of 196 > 166 and 12 eV, respectively.

[0082] Unless otherwise specified and limited, "tobacco sample" in this document refers to tobacco leaves or shredded tobacco. All solvents (e.g., the solvents for the absorption liquid, standard solutions, and mobile phases used in the preparation of liquid chromatography) and reagents are of chromatographic purity.

[0083] This invention employs heating to directly volatilize tobacco-specific N-nitrosamines from tobacco samples. These compounds are then absorbed by a cold solution under a carrier gas and analyzed directly using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Internal standard method is used for qualitative and quantitative determination of the tobacco-specific N-nitrosamine content in the tobacco sample. The method of this invention requires no more than 2 mL of acetonitrile-water (1:1, v / v) to complete the determination of one sample, taking approximately 15-20 minutes. This method overcomes the shortcomings of existing techniques, such as the need for large amounts of organic solvents for sample extraction, the requirement for purification and concentration after extraction, long pretreatment time, and poor purification effect. It offers advantages such as simple and rapid operation, green and efficient operation, and strong matrix removal capability. Attached Figure Description

[0084] The present invention will be further described below with reference to the accompanying drawings.

[0085] Figure 1 This is a schematic diagram of a volatile component collection device. The reference numerals in the diagram are explained as follows:

[0086] 1: Carrier gas pipeline; 2: Gas flow meter; 3: Material storage cavity; 4: Storage cavity; 5: First gas path valve; 6: Collection bottle; 7: Heating layer; 8: Cooling device; 9: Cleaning device; 10: Second gas path valve; 13: Collection section; 14: Cleaning section.

[0087] Figure 2 The effects of different absorbents on the analytical results are shown. A uses pure acetonitrile as the absorbent, and B uses an acetonitrile / water mixture (1 / 1, v / v) as the absorbent.

[0088] Figure 3 The effect of absorbent temperature on the analytical results is shown.

[0089] Figure 4 The effect of sample heating time on the analytical results is shown in the figure. In the figure, 1st: heating for 2 min; 2nd: heating for 4 min; 3rd: heating for 6 min; 4th: heating for 8 min.

[0090] Figure 5 The effect of sample heating temperature on the analytical results is shown.

[0091] Figure 6 The effect of carrier gas type on the analysis results is shown.

[0092] Figure 7 The effect of carrier gas velocity on the analysis results is shown.

[0093] Figure 8 The effect of sample quality on the analytical results is shown.

[0094] Figure 9 Typical chromatograms of four tobacco-specific N-nitrosamines obtained by analyzing the same batch of tobacco samples using the method of this invention and current standard methods are shown. Figure A shows the chromatogram obtained by the method of this invention, and Figure B shows the chromatogram obtained by the CORESTA recommended method CRM No. 72.

[0095] Figure 10 Photographs of the extracts obtained by this method and the CORESTA recommended method CRM No. 72 are shown.

[0096] Figure 11 It shows Figure 10 The UV-Vis absorption spectrum of the extract after dilution by 50 times. Detailed Implementation

[0097] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0098] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all raw materials, reagents, and other materials used in the following examples are commercially available products. Details of some instruments and accessories are as follows:

[0099] Liquid Chromatography-Tandem Mass Spectrometry: Waters ACQUITY UPLC I-Class Xevo TQD;

[0100] Liquid chromatography column: ACQUITY UPLC ® BEH C18 column, 2.1 × 100 mm, 1.7 μm.

[0101] In the following examples and studies, the tobacco samples used were all shredded tobacco.

[0102] In the following examples or studies, the qualitative and quantitative determination of the test solution (containing tobacco-specific N-nitrosamine components and internal standard) was performed according to the following procedures:

[0103] Preparation of a series of standard solutions

[0104] Using methanol and 0.1 mol / L ammonium acetate solution as solvents, and standards of four tobacco-specific N-nitrosamines (N-nitrosonornicotinamide (NNN), 4-(methylnitroso)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoneonicotinamide (NAT), and N-nitrosopseudoestiline (NAB)) as solutes, and deuterated derivatives of the four tobacco-specific N-nitrosamines (NNN-d4, NNK-d4, NAT-d4, and NAB-d4) as internal standards, a series of mixed standard solutions with gradient concentrations containing internal standards were prepared. In the series of mixed standard solutions, the concentration gradients of each standard were 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, and 50 ng / mL, and the concentration of each internal standard was 10 ng / mL.

[0105] Measurement

[0106] Liquid chromatography conditions include:

[0107] Column: ACQUITY UPLC ® BEH C18 column, 2.1 × 100 mm, 1.7 μm;

[0108] Column temperature: 40℃;

[0109] Injection volume: 5 μL;

[0110] Mobile phase: 0.01 mol / L ammonium acetate as phase A, 0.1% formic acid methanol solution as phase B, gradient elution, elution program as follows:

[0111] t=0 min, 90%A, 10%B;

[0112] t=1.0 min, 90%A, 10%B;

[0113] t=5.0min, 10%A, 90%B;

[0114] t=6.0min, 10%A, 90%B;

[0115] t=6.1 min, 90%A, 10%B;

[0116] t=8.0 min, 90%A, 10%B;

[0117] Flow rate: 0.25 mL / min;

[0118] Mass spectrometry conditions include:

[0119] Ionization mode: positive ion mode ionization;

[0120] Electrospray voltage: 3000 V;

[0121] Tapered hole voltage: 80 V;

[0122] Ion source temperature: 550℃;

[0123] Desolventizing gas flow: 800 L / hour;

[0124] Conical orifice airflow: 50 L / hour;

[0125] Dwell time: 24 ms;

[0126] Monitoring method: Multiple response monitoring;

[0127] Under the conditions of liquid chromatography and mass spectrometry, the test solution and the series of standard solutions were precisely pipetted into the liquid chromatography-tandem mass spectrometer for determination, and the chromatograms were recorded.

[0128] Data processing and analysis

[0129] Qualitative determination: The presence of the corresponding tobacco-specific N-nitrosamine components in the test sample solution is determined based on the retention time of the standard and its deuterated derivative, quantitative / qualitative ion pairs, and corresponding collision energies.

[0130] The retention times of NNN were 3.44 min ± 0.10 min, with quantitative ion pair and collision energies of 178 > 148 and 11 eV, respectively, and qualitative ion pair and collision energies of 178 > 120 and 17 eV, respectively; the retention time of NNN-d4 was 3.42 min ± 0.10 min, with quantitative ion pair and collision energies of 182 > 152 and 11 eV, respectively; the retention time of NNK was 3.64 min ± 0.10 min, with quantitative ion pair and collision energies of 208 > 122 and 11 eV, respectively, and qualitative ion pair and collision energies of 208 > 106 and 21 eV, respectively; the retention time of NNK-d4 was 3.63 min ± 0.10 min, with quantitative ion pair and collision energies of 212 > 126 and 11 eV, respectively; and the retention time of NAT was 3.99 min ± 0.10 min. The retention times of NAT-d4 were 3.98 min ± 0.10 min, with quantitative ion pair and collision energies of 194 > 164 and 10 eV, respectively; the retention times of NAB were 4.10 min ± 0.10 min, with quantitative ion pair and collision energies of 192 > 162 and 12 eV, respectively; the retention times of NAB-d4 were 4.09 min ± 0.10 min, with quantitative ion pair and collision energies of 196 > 166 and 12 eV, respectively.

[0131] Quantitative determination: The internal standard method was used. A working curve was plotted with the ratio of the peak area of ​​N-nitrosamines in the series of standard solutions to the peak area of ​​the internal standard as the ordinate, and the concentration of N-nitrosamines in the series of standard solutions as the abscissa. Based on the ratio of the peak area of ​​N-nitrosamines in the test sample solution to the peak area of ​​the internal standard, the content of the target N-nitrosamine in each gram of tobacco sample was calculated according to Formula I based on the working curve.

[0132] I,

[0133] Where m: the content of the target N-nitrosamine component per gram of tobacco sample, ng / g.

[0134] x: The ratio of the peak area of ​​the target N-nitrosamine to that of the internal standard.

[0135] a: The slope of the working curve.

[0136] b: Intercept of the working curve

[0137] V: Volume of the test solution, mL

[0138] n: Mass of the tobacco sample, in grams.

[0139] Example 1: A volatile component collection device

[0140] like Figure 1 As shown, a volatile component collection device includes a carrier gas pipeline 1, a gas flow meter 2, a heating device, a material storage chamber 3, a first gas valve 5, a collection device, a cooling device 8, a cleaning device 9, a second gas valve 10, a tee connector, and a controller. The carrier gas pipeline 1 is used for gas supply and includes a connected conveying section, a cleaning section 14, and a collection section 13. The tee connector includes a first end, a second end, and a third end. The first end is connected to the conveying section of the carrier gas pipeline 1, the second end is connected to the collection section 13, and the third end is connected to the cleaning section 14. The second gas valve 10 is connected to the cleaning section 14. The gas flow meter 2 is connected to the conveying section of the carrier gas pipeline 1. The material storage chamber 3 has a storage cavity 4 for storing solid or liquid samples. The storage cavity 4 is connected to the conveying section of the carrier gas pipeline 1, and a carrier gas inlet and a carrier gas outlet are provided on two opposite surfaces of the storage cavity 4. The heating device includes a heating layer 7, a temperature sensor, and a heater. The heater heats the heating layer 7, which is wrapped around the outer wall of the material storage cavity 3. The temperature sensor is located inside the storage cavity 4. A first gas path valve 5 is connected to the conveying section of the carrier gas pipeline 1, which is located after the material storage cavity 3. The collection device includes a collection bottle 6 containing absorbent liquid, which is placed at the end of the collection section 13 of the carrier gas pipeline 1. The solvent used for absorption in the collection bottle covers the end opening of the collection section 13. The cleaning device 9 includes a pump and a cleaning bottle containing cleaning liquid. The suction port of the pump is placed inside the cleaning bottle, and the output port of the pump is connected to the collection section 13. The controller is communicatively connected to the gas flow meter 2, the heater, the temperature sensor, the first gas path valve 5, the cooling device 9, and the second gas path valve 10.

[0141] When collecting volatile components using the volatile component collection device of this embodiment, firstly, an appropriate amount of absorbent liquid (solvent) is transferred into the collection bottle 6. The collection bottle 6 is placed in the cooling device 8 and positioned below the end opening of the collection section 13 of the carrier gas pipeline 1, with the opening of the collection section 13 submerged in the absorbent liquid. The cooling device is then activated. Next, the solid or liquid sample is placed in the storage chamber 4 of the material storage chamber 3. The first gas path valve 5 is opened, and the second gas path valve 10 is closed. Finally, the controller activates the heater, which heats the solid or liquid sample in the storage chamber 4. The gas in the carrier gas pipeline 1 at a specific flow rate carries the volatile components in the storage chamber 4 through the collection section 13 to the collection bottle 6, where they are absorbed by the absorbent liquid. This achieves the collection of volatile components at a specific temperature.

[0142] After heating is completed, closing the first gas path valve 5 and opening the second gas path valve 10 and the infusion pump can clean the pipelines of the cleaning section 14 and the collection section 13. This not only avoids the residual gas in the pipe from contaminating the next collection of volatile components, but also makes the collection more complete and thorough, improving the collection purity and collection efficiency of the volatile component collection equipment.

[0143] Study Example 1: Preparation of extracts of tobacco-specific N-nitrosamines from tobacco

[0144] This study investigated the preparation of extracts of tobacco-specific N-nitrosamines from tobacco using the volatile component collection device described in Example 1.

[0145] Using the novel volatile component collection device of Example 1, a precisely weighed 0.1g tobacco sample is placed in the storage chamber 4, and 1mL of absorbent liquid is precisely pipetted into the collection bottle 6. The collection bottle 6 is then placed in the cooling device 8, with the end opening of the collection section 13 of the carrier gas pipeline 1 submerged in the absorbent liquid in the collection bottle 6. The first gas valve 5 is opened and the second gas valve 10 is closed. The controller activates the heater to heat the heating layer 7. During a specific time period when the temperature of the heating layer 7 reaches and is maintained at a set temperature T, the carrier gas at a specific flow rate in the carrier gas pipeline 1 carries the volatile components volatilized from the tobacco sample in the storage chamber 4 through the collection section 13 to the collection bottle 6, where they are absorbed by the absorbent liquid. This achieves continuous collection of tobacco-specific N-nitrosamine components at the set temperature T (≤T). After heating is complete, close the first gas path valve 5 and open the second gas path valve 10. Start the infusion pump and clean the collection section 13 with the blank absorption liquid (0.25 mL) in the cleaning device. Combine the cleaning liquid with the absorption liquid to obtain the tobacco-specific N-nitrosamine component extract of the tobacco sample. Add the internal standard and dilute to 2 mL with the blank absorption liquid. It can then be directly used for liquid chromatography-tandem mass spectrometry analysis.

[0146] During the collection process described above, factors such as the type and temperature of the absorbent, the heating temperature and time of the tobacco, and the type and flow rate of the carrier gas all affect the extraction efficiency. This study investigated each of these factors individually.

[0147] 1. Investigation of the types of absorbent liquid

[0148] Extraction solutions were prepared using acetonitrile at room temperature and acetonitrile-water solution (1 / 1, v / v) as absorbents, heated to 320℃, with nitrogen as carrier gas and a flow rate of 15 mL / min. NNN-d4, NNK-d4, NAT-d4, and NAB-d4 were added as internal standards to each extract solution to achieve a concentration of 10 ng / mL for each deuterated compound. The solutions were then diluted to 2 mL with blank absorbent for qualitative and quantitative determination of tobacco-specific N-nitrosamines. The results are shown in the table below. Figure 2Figures A and B.

[0149] Figure 2 Figure A shows that when pure acetonitrile is used as the absorbent, the chromatographic peaks of the four target analytes all exhibit a solvent effect, and the solvent effect increases as the retention time decreases. Figure 2 Figure B shows that when an acetonitrile-water mixture (1 / 1, v / v) is used as the absorbent, the chromatographic peaks of the four target analytes are symmetrical and sharp, with no solvent effect. Therefore, an acetonitrile-water mixture (1 / 1, v / v) is preferred as the absorbent.

[0150] 2. Investigation of the temperature of the absorbent liquid

[0151] Extraction solutions were prepared at room temperature (25℃) and -2℃ using acetonitrile-water solution (1 / 1, v / v) as the absorbent, with tobacco samples heated to 350℃, nitrogen as the carrier gas, and a flow rate of 5 mL / min. NNN-d4, NNK-d4, NAT-d4, and NAB-d4 were added as internal standards to each extract solution to achieve a concentration of 10 ng / mL for each deuterated compound. The solutions were then diluted to 2 mL with blank absorbent for qualitative and quantitative determination of tobacco-specific N-nitrosamines. The results are shown in [Figure number missing]. Figure 3 .

[0152] Figure 3 The results show that the absorption efficiency of the absorbent is higher at -2℃. This may be because the high-temperature gas continuously flowing out of the material storage chamber causes the temperature of the absorbent at room temperature to rise, which is detrimental to the condensation and absorption of the target analyte. A temperature-controlled refrigeration tank with condensation function can maintain the temperature of the absorbent at a low temperature (-2℃), which not only cools the high-temperature carrier gas but also facilitates the absorption of the target analyte. Therefore, the preferred temperature for the absorbent is -2℃.

[0153] 3. Investigation of heating time for tobacco samples

[0154] Using acetonitrile-water solution (1 / 1, v / v) at -2℃ as the absorbent, the tobacco sample was heated to 350℃, nitrogen was used as the carrier gas, and the flow rate was 15 mL / min. The volatile components collected at 2, 4, 6, and 8 min of heating were qualitatively and quantitatively determined. The results are shown in the figure. Figure 4 .

[0155] Figure 4 As shown, the total proportions of the four target compounds in the first three collection stages were 98.3%, 98.0%, 98.6%, and 99.4%, respectively, indicating that after heating at 320℃ for 6 minutes, almost all four target compounds volatilized from the tobacco sample. Therefore, the preferred heating time for the tobacco sample is 6.0 minutes.

[0156] 4. Investigation of heating temperature for tobacco samples

[0157] Extraction solutions were prepared using acetonitrile-water solution (1 / 1, v / v) at -2℃ as the absorbent, with tobacco samples heated for 6 min, nitrogen as the carrier gas, and a flow rate of 2 mL / min, at heating temperatures of 260℃, 290℃, 320℃, 350℃, and 380℃, respectively. NNN-d4, NNK-d4, NAT-d4, and NAB-d4 were added as internal standards to each extract solution to achieve a concentration of 10 ng / mL for each deuterated compound. The solutions were then diluted to 2 mL with blank absorbent for qualitative and quantitative determination of tobacco-specific N-nitrosamines. The results are shown in [Figure number missing]. Figure 5 .

[0158] Figure 5 The results show that the content of the four tobacco-specific N-nitrosamine components reached its highest level at 320℃. The reason for this is likely that lower temperatures are not conducive to the volatilization of the target substances from the tobacco sample; excessively high heating temperatures can cause degradation of the target substances to some extent, and the high-temperature gases at higher heating temperatures will cause the temperature of the absorbent liquid to rise, which is not conducive to the adsorption of the target substances at lower temperatures. Therefore, the preferred heating temperature for tobacco samples is 320℃.

[0159] 5. Examination of carrier gas type

[0160] Using acetonitrile-water solution (1 / 1, v / v) at -2℃ as the absorbent, the tobacco sample was heated to 320℃ for 6 min, with a carrier gas flow rate of 15 mL / min. Extraction solutions were prepared using nitrogen and helium as carrier gases, respectively. NNN-d4, NNK-d4, NAT-d4, and NAB-d4 were added as internal standards to each extract solution to achieve a concentration of 10 ng / mL for each deuterated compound. The solution was then diluted to 2 mL with blank absorbent for qualitative and quantitative determination of tobacco-specific N-nitrosamines. The results are shown in [Figure number missing]. Figure 6 .

[0161] Figure 6 The results show no difference between the two carrier gases. This suggests that any inert gas can be used as a carrier gas, such as nitrogen, helium, or compressed air. Nitrogen is preferred due to its high safety factor, low price, and availability in laboratories.

[0162] 6. Examination of carrier gas velocity

[0163] Extraction solutions were prepared using acetonitrile-water solution (1 / 1, v / v) at -2℃ as the absorbent, with tobacco samples heated to 320℃ for 6 min, and nitrogen as the carrier gas at nitrogen flow rates of 0, 1, 2, 5, and 10 mL / min. NNN-d4, NNK-d4, NAT-d4, and NAB-d4 were added as internal standards to each extract solution to achieve a concentration of 10 ng / mL for each deuterated compound. The solutions were then diluted to 2 mL with blank absorbent for qualitative and quantitative determination of tobacco-specific N-nitrosamines. The results are shown in the figure. Figure 7 .

[0164] Figure 7 The results show that the device can still collect volatile target substances even without carrier gas (carrier gas flow rate of 0 mL / min). As the carrier gas flow rate increases, the absorption efficiency of the target substance first increases and then decreases, reaching its maximum at 2 mL / min. These results not only demonstrate that carrier gas assistance can enhance the absorption efficiency of the target substance, but also indicate that the carrier gas flow rate should not be too high. Therefore, a carrier gas flow rate of 2 mL / min is preferred.

[0165] In summary, the optimized preparation conditions for the extract are as follows: the absorption solvent is an acetonitrile-water solution (1 / 1, v / v) at -2℃, the sample heating temperature and time are 320℃ and 6.0 min, and the carrier gas type and flow rate are nitrogen and 2 mL / min.

[0166] 7. Examination of tobacco sample quality

[0167] Using acetonitrile-water solution (1 / 1, v / v) at -2℃ as the absorbent, the tobacco samples were heated to 320℃ for 6 min, with nitrogen as the carrier gas at a flow rate of 2 mL / min. Accurately weighed extracts of 0.0506 g, 0.1004 g, and 0.1801 g of tobacco samples were prepared. NNN-d4, NNK-d4, NAT-d4, and NAB-d4 were added as internal standards to each extract to achieve a concentration of 10 ng / mL for each deuterated compound. The extracts were then diluted to 2 mL with blank absorbent for qualitative and quantitative determination of tobacco-specific N-nitrosamines. The results are shown in [Figure number missing]. Figure 8 .

[0168] Figure 8 The results show no difference in the test results of the four target substances under different sample masses. Considering the sample homogeneity and usage, the final sample mass was determined to be 0.10g ± 0.05g.

[0169] In summary, the optimal preparation conditions for the extract of tobacco-specific N-nitrosamine components are as follows:

[0170] The absorption solution was an acetonitrile-water mixture (1 / 1, v / v) at -2℃. The sample heating temperature was 320℃, the heating time was 6.0 min, the carrier gas was nitrogen, the carrier gas flow rate was 2 mL / min, and the sample mass was 0.10 g ± 0.05 g.

[0171] Study Example 2: Methodological Investigation

[0172] Based on Study Example 1, the present invention establishes a method for detecting tobacco-specific N-nitrosamine components in tobacco samples, comprising the following steps:

[0173] (1) Preparation of the test solution

[0174] 0.10 g ± 0.05 g of tobacco sample was placed in the storage chamber of the volatile component collection device as described in Example 1, with the cooling device set to -2°C. 1 mL of a 1:1 volume ratio acetonitrile-water solution was precisely pipetted into a collection bottle as the absorbent. The collection bottle was then placed in the cooling device and positioned below the end opening of the collection section, ensuring the end opening was submerged in the absorbent. The second gas path valve was closed. The first gas path valve was opened, and the gas flow meter was adjusted to a nitrogen flow rate of 2 mL / min. The heating layer was set to 320°C and maintained for 6.0 min. Heating of the heating layer was then stopped, the first gas path valve was closed, the second gas path valve was opened, and the infusion pump was started. 0.25 mL of a 1:1 volume ratio acetonitrile-water solution was used to clean the collection section. The cleaning solution was combined with the absorbent in the collection bottle. NNN-d4, NNK-d4, NAT-d4, and NAB-d4 were added as internal standards, and the volume was adjusted to 2 mL with a 1:1 volume ratio acetonitrile-water solution. The concentration of each internal standard was 10. The test solution was obtained by measuring ng / mL.

[0175] (2) Preparation of a series of mixed standard solutions

[0176] A series of mixed standard solutions containing internal standards of different concentrations were prepared using methanol and 0.1 mol / L ammonium acetate solution as solvents, NNN, NNK, NAT, and NAB as solutes, and NNN-d4, NNK-d4, NAT-d4, and NAB-d4 as internal standards. In these mixed standard solutions, the concentration gradients of NNN, NNK, NAT, and NAB were 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, and 50 ng / mL, respectively, and the concentration of each internal standard was 10 ng / mL.

[0177] (3) Measurement

[0178] Liquid chromatography conditions include:

[0179] Column: ACQUITY UPLC ®BEH C18 column, 2.1 × 100 mm, 1.7 μm;

[0180] Column temperature: 40℃;

[0181] Injection volume: 5 μL;

[0182] Mobile phase: 0.01 mol / L ammonium acetate as phase A, 0.1% formic acid methanol solution as phase B, gradient elution, elution program as follows:

[0183] t=0 min, 90%A, 10%B;

[0184] t=1.0 min, 90%A, 10%B;

[0185] t=5.0min, 10%A, 90%B;

[0186] t=6.0min, 10%A, 90%B;

[0187] t=6.1 min, 90%A, 10%B;

[0188] t=8.0 min, 90%A, 10%B;

[0189] Flow rate: 0.25 mL / min;

[0190] Mass spectrometry conditions include:

[0191] Ionization mode: positive ion mode ionization;

[0192] Electrospray voltage: 3000 V;

[0193] Tapered hole voltage: 80 V;

[0194] Ion source temperature: 550℃;

[0195] Desolventizing gas flow: 800 L / hour;

[0196] Conical orifice airflow: 50 L / hour;

[0197] Dwell time: 24 ms;

[0198] Monitoring method: Multiple response monitoring;

[0199] Under the conditions of liquid chromatography and mass spectrometry, the test solution and the series of standard solutions were precisely pipetted into the liquid chromatography-tandem mass spectrometer for determination, and the chromatograms were recorded.

[0200] (4) Data processing and analysis

[0201] Including qualitative and quantitative determination;

[0202] Qualitative determination: Based on the retention time, quantitative and qualitative ion pairs and corresponding collision energies of the standard and its deuterated derivative, the presence of the corresponding tobacco-specific N-nitrosamine components in the test sample solution is determined.

[0203] The retention time of NNN was 3.44 min ± 0.10 min, the quantitative ion pair and collision energy were 178 > 148 and 11 eV, respectively, and the qualitative ion pair and collision energy were 178 > 120 and 17 eV, respectively.

[0204] The retention time of NNN-d4 was 3.42 min ± 0.10 min, and the quantitative ion pair and collision energy were 182 > 152 and 11 eV, respectively.

[0205] The retention time of NNK was 3.64 min ± 0.10 min, the quantitative ion pair and collision energy were 208>122 and 11 eV, respectively, and the qualitative ion pair and collision energy were 208>106 and 21 eV, respectively.

[0206] The retention time of NNK-d4 was 3.63 min ± 0.10 min, and the quantitative ion pair and collision energy were 212 > 126 and 11 eV, respectively.

[0207] The retention time of NAT was 3.99 min ± 0.10 min, the quantitative ion pair and collision energy were 190 > 160 and 10 eV, respectively, and the qualitative ion pair and collision energy were 190 > 106 and 16 eV, respectively.

[0208] The retention time of NAT-d4 was 3.98 min ± 0.10 min, and the quantitative ion pair and collision energy were 194 > 164 and 10 eV, respectively.

[0209] The retention time of NAB was 4.10 min ± 0.10 min, the quantitative ion pair and collision energy were 192 > 162 and 12 eV, respectively, and the qualitative ion pair and collision energy were 192 > 133 and 22 eV, respectively.

[0210] The retention time of NAB-d4 was 4.09 min ± 0.10 min, and the quantitative ion pair and collision energy were 196 > 166, 12 eV.

[0211] Quantitative determination: The internal standard method was used. A working curve was plotted with the ratio of the peak area of ​​N-nitrosamines in the series of standard solutions to the peak area of ​​the internal standard as the ordinate, and the concentration of N-nitrosamines in the series of standard solutions as the abscissa. Based on the ratio of the peak area of ​​N-nitrosamines in the test sample solution to the peak area of ​​the internal standard, the content of the target N-nitrosamine in each gram of tobacco sample was calculated according to Formula I based on the working curve.

[0212] I,

[0213] Where m: the content of the target N-nitrosamine component per gram of tobacco sample, ng / g.

[0214] x: The ratio of the peak area of ​​the target N-nitrosamine to that of the internal standard.

[0215] a: The slope of the working curve.

[0216] b: Intercept of the working curve

[0217] V: Volume of the test solution, mL

[0218] n: Mass of the tobacco sample, in grams.

[0219] 1. Comparison with existing methods

[0220] Following the above detection method, a 0.10 g sample of tobacco leaves was analyzed, and the typical chromatogram of the target analyte obtained is shown below. Figure 9 Figure A.

[0221] In parallel, 0.63 g of the same batch of tobacco sample was analyzed using the CORESTA recommended method CRM No. 72. The chromatogram of the target analyte is shown in [Figure 1]. Figure 9 Figure B. The preparation method of the test solution is as follows: each gram of smoke sample is shaken for 40 minutes with 30 mL of 100 mM ammonium acetate solution, filtered, and internal standard is added to obtain the test solution; the preparation, determination and data processing analysis of the series of mixed standard solutions are the same as those in (2) to (4) above.

[0222] Compare Figure 9 As shown in Figures A and B, Figure A has a flatter baseline and fewer interfering components. Therefore, compared with the current CRM No. 72 standard method, the test solution prepared by the method of this invention has less matrix interference, which is beneficial for the qualitative and quantitative determination of the target compound.

[0223] Photographs of the test solution prepared by the method of this invention and the test solution prepared by the CRM No. 72 standard method are shown below. Figure 10 The extract obtained by the method of this invention is significantly lighter in color than the extract obtained by the CRM No. 72 method. This indicates that the method of this invention carries away fewer tobacco impurities.

[0224] The extract was diluted 50 times, and the UV-Vis absorption spectrum in the wavelength range of 200–400 nm was measured. (See attached image.) Figure 11Compared with the UV spectrum of the extract obtained by the standard method CRM No.72, the absorption intensity of the extract obtained by the method of the present invention is significantly reduced in the wavelength range of 200~400 nm, indicating that there are fewer interfering substances other than pigments in the extract obtained by the method of the present invention.

[0225] 2. Linear range, limit of detection, and limit of quantitation

[0226] Standard curves (weighted at 1 / x) were plotted against the concentrations of four tobacco-specific N-nitrosamine standards and their corresponding internal standards, using the peak area ratios. The limits of detection (LOD) and quantitation (LOQ) were calculated using signal-to-noise ratios of 3 and 10, respectively. The results are shown in Table 1.

[0227] As shown in Table 1, the four target analytes have good linear relationships within their respective linear ranges, and the square of the linear correlation coefficient of their working curves is not less than 0.9994. The limits of detection (LOD) and limits of quantitation (LOQ) of the target analytes are between 0.004 and 0.010 ng / mL and 0.014 and 0.031 ng / mL, respectively.

[0228]

[0229] 3. Accuracy and repeatability assessment

[0230] Take the same tobacco sample, accurately weigh 3 portions, each 0.1g; prepare the test solution according to the method established in this invention for 1 day, and then perform the determination. Calculate the content of tobacco-specific N-nitrosamine components per gram of tobacco sample using the standard working curve and Formula I, and calculate the intra-day relative standard deviation.

[0231] To compare the accuracy of the results obtained by the method of this invention with those obtained by existing standard methods (such as CRM No. 72), the qualitative and quantitative determination of tobacco-specific N-nitrosamine components was performed in parallel using CRM No. 72 on the same batch of tobacco samples. The results are shown in Table 2.

[0232]

[0233] Table 2 shows that the relative deviations of the results obtained by analyzing the same sample using the method of this invention and the CORESTA recommended method are between 2.76% and 4.36%, both less than 10%, indicating that the method of this invention and the CORESTA recommended method have good consistency, proving that this method is accurate and reliable. The relative standard deviation of the method of this invention for testing the same sample is between 0.65% and 3.62%. These findings demonstrate that the accuracy and repeatability of the method of this invention can meet the routine detection needs of tobacco-specific N-nitrosamine components in tobacco.

[0234] Example 2:

[0235] The qualitative and quantitative determination of tobacco-specific N-nitrosamines in two other tobacco samples was performed using the method described in Example 2. Simultaneously, the same batch of tobacco samples was measured using the CORESTA recommended method CRM No. 72. The results of the two determinations are shown in Table 3.

[0236]

[0237] Table 3 shows that the results obtained by the method of this invention are in good agreement with those obtained by the CORESTA recommended method (CRM No. 72), further demonstrating the accuracy and reliability of this method. The detection method of this invention takes 6-8 minutes to detect one tobacco sample and consumes 2 mL of extraction solvent (acetonitrile-water). In contrast, the CRM No. 72 method takes 40-60 minutes to detect one sample and consumes 30 mL of extraction solvent. The method of this invention has advantages in terms of ease of operation, efficiency, cost, and environmental friendliness.

Claims

1. A method for extracting N-nitrosamines specific to tobacco, based on a volatile component collection device; the volatile component collection device includes a carrier gas pipeline, a gas flow meter, a heating device, a material storage chamber, a first gas path valve, a collection device, a cooling device, a cleaning device, a second gas path valve, a tee connector, and a controller; the carrier gas pipeline includes a connected conveying section, a cleaning section, and a collection section; the tee connector includes a first end, a second end, and a third end, the first end being connected to the conveying section of the carrier gas pipeline, the second end being connected to the collection section, and the third end being connected to the cleaning section; the second gas path valve is connected to the cleaning section; the gas flow meter is connected to the conveying section of the carrier gas pipeline; the material storage chamber is provided with a storage cavity for placing the tobacco sample, the storage cavity being connected to the carrier gas pipeline, and a carrier gas inlet and a carrier gas outlet are provided on two opposite sides of the storage cavity; the heating device... The system includes a heating layer, a temperature sensor, and a heater for heating the heating layer. The heating layer is wrapped around the outer wall of the material storage cavity, and the temperature sensor is located inside the storage cavity. A first gas path valve is connected to the delivery section of the carrier gas pipeline after the material storage cavity. The collection device includes a collection bottle containing an acetonitrile-water solution, which is placed in the cooling device and at the end of the collection section of the carrier gas pipeline. The acetonitrile-water solution in the collection bottle covers the end opening of the collection section. The cleaning device includes an infusion pump and a cleaning bottle containing cleaning fluid. The inlet of the infusion pump is placed inside the cleaning bottle, and the outlet of the infusion pump is connected to the collection section. The controller is communicatively connected to the gas flow meter, the heating device, the first gas path valve, the cooling device, the second gas path valve, and the infusion pump. The extraction method includes the following operations: 0.10 g ± 0.05 g of tobacco sample was heated at 260 ~ 380 °C for 6.0 ~ 8.0 min. The volatilized components were transported to 1 mL of acetonitrile-water solution at 0 °C ± 5 °C using a carrier gas. After heating, the collection section was washed with 0.25 ~ 0.5 mL of acetonitrile-water solution. The washing solution was combined with the acetonitrile-water solution in the collection bottle to obtain an acetonitrile-water extract of tobacco-specific N-nitrosamine components. The acetonitrile-water solution has an acetonitrile to water volume ratio of 1:

1.

2. The extraction method according to claim 1, characterized in that, The cooling device is selected from a cold trap or an ice bath.

3. The extraction method according to claim 2, characterized in that, The cooling device is a cold trap.

4. The extraction method according to claim 3, characterized in that, The cold trap is a condensation trap.

5. The extraction method according to claim 1, characterized in that, The temperature of the acetonitrile-water solution is 0℃±2℃.

6. The extraction method according to claim 1, characterized in that, The temperature of the acetonitrile-water solution is -2℃.

7. The extraction method according to claim 1, characterized in that, The tobacco sample was heated at 320°C.

8. The extraction method according to claim 1, characterized in that, The tobacco sample was heated for 6.0 min.

9. The extraction method according to claim 1, characterized in that, The flow rate of the carrier gas is 2 mL / min.

10. The extraction method according to claim 1 or 9, characterized in that, The carrier gas is selected from compressed air, nitrogen, and helium.

11. The extraction method according to claim 10, characterized in that, The carrier gas is nitrogen.

12. An acetonitrile-water extract of tobacco-specific N-nitrosamine components from a tobacco sample, prepared by the extraction method according to any one of claims 1 to 11.

13. A method for detecting tobacco-specific N-nitrosamines in tobacco, based on liquid chromatography-tandem mass spectrometry; the detection method includes the following steps: (1) Preparation of the test solution The sample of tobacco to be tested is pretreated by the extraction method according to any one of claims 1 to 11 to obtain a test solution; (2) Preparation of a series of mixed standard solutions The standard is selected from at least one of NNN, NNK, NAT and NAB, and the deuterated product of the standard is used as an internal standard; a series of mixed standard solutions including the standard and the corresponding internal standard are prepared using methanol and 0.1 mol / L ammonium acetate solution as solvents. (3) Measurement Liquid chromatography conditions include: Column: ACQUITY UPLC ® BEH C18 column, 2.1 × 100 mm, 1.7 μm; Column temperature: 40℃; Injection volume: 5 μL; Mobile phase: 0.01 mol / L ammonium acetate as phase A, 0.1% formic acid methanol solution as phase B, gradient elution, elution program as follows: t=0 min, 90%A, 10%B; t=1.0 min, 90%A, 10%B; t=5.0min, 10%A, 90%B; t=6.0min, 10%A, 90%B; t=6.1 min, 90%A, 10%B; t=8.0 min, 90%A, 10%B; Flow rate: 0.25 mL / min; Mass spectrometry conditions include: Ionization mode: positive ion mode ionization; Electrospray voltage: 3000 V; Tapered hole voltage: 80 V; Ion source temperature: 550℃; Desolventizing gas flow: 800 L / hour; Conical orifice airflow: 50 L / hour; Dwell time: 24 ms; Monitoring method: Multiple response monitoring; Under the conditions of liquid chromatography and mass spectrometry, the test solution and the series of standard solutions were precisely pipetted into the liquid chromatography-tandem mass spectrometer for determination, and the chromatograms were recorded. (4) Data processing and analysis The assay includes qualitative and quantitative determinations. The qualitative determination involves determining the presence of the corresponding N-nitrosamine component in the test sample solution based on the retention time, quantitative and qualitative ion pairs, and corresponding collision energies of the standard. The quantitative determination employs the internal standard method, plotting a working curve with the ratio of the peak area of ​​the N-nitrosamine component in the series of mixed standard solutions to the peak area of ​​the internal standard as the ordinate and the concentration of the N-nitrosamine component in the series of mixed standard solutions as the abscissa. Based on the ratio of the peak area of ​​the N-nitrosamine component in the test sample solution to the peak area of ​​the internal standard, and according to the working curve, the content of the target N-nitrosamine component per gram of tobacco sample is calculated using Formula I. I， Where m: the content of the target N-nitrosamine component per gram of tobacco sample, ng / g. x: The ratio of the peak area of ​​the target N-nitrosamine to that of the internal standard. a: The slope of the working curve. b: Intercept of the working curve V: Volume of the test solution, mL n: Mass of the tobacco sample, in grams.

14. The detection method according to claim 13, characterized in that, The specific operation of step (1) is as follows: Place 0.10 g ± 0.05 g of tobacco sample in the storage chamber of the volatile component collection device, and set the cooling device to -2 °C. Accurately pipette 1 mL of acetonitrile-water solution (1:1 volume ratio) into a collection bottle as the absorbent. Then place the collection bottle in the cooling device and position it below the end opening of the collection section of the carrier gas pipeline, ensuring that the end opening of the collection section is submerged in the acetonitrile-water solution. Close the second gas path valve. Open the first gas path valve and adjust the gas flow meter to set the nitrogen flow rate to 2 mL / min. Set the heating layer temperature to 320 °C and maintain the temperature for 6.0 min. Then stop heating the heating layer, close the first gas path valve, open the second gas path valve, and clean the collection section of the carrier gas pipeline with 0.25 mL of acetonitrile-water solution (1:1 volume ratio). Combine the cleaning solution with the absorbent in the collection bottle, add an internal standard, and dilute to 2 mL with acetonitrile-water solution (1:1 volume ratio) to obtain the test solution.

15. The detection method according to claim 14, characterized in that, In the test solution, the concentration of the deuterated product of the standard was 10 ng / mL.

16. The detection method according to claim 13, characterized in that, In step (2), the standard products are all of NNN, NNK, NAT and NAB.

17. The detection method according to claim 13 or 16, characterized in that, In step (2), the concentration gradient of the standard in the series of mixed standard solutions is 0.5, 1.0, 2.0, 5.0, 10.0, 20.0 and 50 ng / mL.

18. The detection method according to claim 13 or 16, characterized in that, In step (2), the concentration of the deuterated product of the standard in the series of mixed standard solutions is 10 ng / mL.

19. The detection method according to claim 13, characterized in that, In step (4), the retention time of NNN is 3.44 min ± 0.10 min, the quantitative ion pair and collision energy are 178 > 148, 11 eV, and the qualitative ion pair and collision energy are 178 > 120, 17 eV, respectively; the retention time of NNN-d4 is 3.42 min ± 0.10 min, the quantitative ion pair and collision energy are 182 > 152, 11 eV, respectively; the retention time of NNK is 3.64 min ± 0.10 min, the quantitative ion pair and collision energy are 208 > 122, 11 eV, and the qualitative ion pair and collision energy are 208 > 106, 21 eV, respectively; the retention time of NNK-d4 is 3.63 min ± 0.10 min, the quantitative ion pair and collision energy are 212 > 126, 11 eV, respectively; and the retention time of NAT is 3.99 min ± 0.10 min. The retention times were as follows: NAT-d4: 3.98 min ± 0.10 min, quantitative ion pair and collision energy: 190 > 160, 10 eV; NAB: 4.10 min ± 0.10 min, quantitative ion pair and collision energy: 192 > 162, 12 eV; qualitative ion pair and collision energy: 192 > 133, 22 eV; NAB-d4: 4.09 min ± 0.10 min, quantitative ion pair and collision energy: 196 > 166, 12 eV.

Images