Method for detecting tobacco-specific N-nitrosamine component in tobacco

Abstract

The invention discloses an extraction method of a tobacco-specific N-nitrosamine component and an acetonitrile-water extracting solution of the tobacco-specific N-nitrosamine component of a tobacco sample obtained by the extraction method. The extraction method is based on volatile component collection equipment, and comprises the following operations: heating a tobacco sample at 260-380 DEG C for 6.0-8.0 minutes, and conveying the volatilized components into an acetonitrile-water solution at 0 + / -5 DEG C by using carrier gas to obtain an acetonitrile-water extracting solution of the tobacco specific N-nitrosamine components. The extraction method disclosed by the invention is short in time consumption and simple and convenient to operate, the extracted N-nitrosamine component is high in purity and less in interference, and the N-nitrosamine component can be directly analyzed and determined without subsequent purification. The invention also discloses a method for detecting the tobacco-specific N-nitrosamine component in the tobacco based on the extraction method.

Description

Technical Field

[0001] The invention belongs to the field of testing technology, and specifically relates to a novel method for extracting tobacco-specific N-nitrosamine components and a method for detecting tobacco-specific N-nitrosamine components in tobacco based on the method. Background Art

[0002] Tobacco-specific N-nitrosamines (TSNAs) are a group of carcinogens found only in tobacco, tobacco products and smoke. They are the product of the reaction of tobacco alkaloids with nitrosating agents under acidic conditions. At present, eight TSNAs have been identified: N-nitrosononicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoanatabine (NAT), N-nitrosoanabasine (NAB), 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanal (NNA), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) and 4-(methylnitrosamino)-4-(3-pyridyl)-butyric acid (iso-NNAC). Among them, the first four compounds have been studied most deeply and extensively. TSNAs are not only important compounds in the Hoffman List and the U.S. Food and Drug Administration's "List of Harmful and Potentially Harmful Substances in Tobacco Products and Smoke", but also important components in the International Agency for Research on Cancer's "28 Harmful Substances in Smokeless Tobacco Products" list. It is generally believed that TSNAs will cause oxidative damage to DNA during metabolism, and NNN and NNK are confirmed as Class I carcinogens by the International Agency for Research on Cancer. At present, studies have shown that TSNAs in smoke mainly come from tobacco leaves. Therefore, it is of great significance to measure the content of TSNAs in tobacco.

[0003] Due to the complexity of tobacco matrix and the extremely low TSNAs content (usually 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. The current standard methods at home and abroad all use solvent extraction, purification, and concentration sample pretreatment methods. For example, the industry standard YC / T 184 "Determination of tobacco-specific N-nitrosamines in tobacco and tobacco products" stipulates that each gram of tobacco sample is ultrasonically extracted with 20mL of dichloromethane and 1mL of 10% sodium hydroxide solution for 20 minutes. ISO 22304:2008 "Alkaline dichloromethane extraction method for the determination of tobacco-specific nitrosamines in tobacco" stipulates that each 1.5g of tobacco sample is oscillated with 10mL of dichloromethane and 0.5mL of 10% sodium hydroxide solution for 44 minutes. The CORESTA (International Tobacco Research Cooperation Center) Recommended Method (CRM) No. 72 stipulates that each gram of tobacco sample should be shaken with 30 mL of 100 mM ammonium acetate solution for 40 minutes.

[0004] The above methods all have the following problems: due to the principle of solvent extraction (like dissolves like), when extracting TSNAs with solvents, impurities such as pigments in the smoke samples will inevitably be extracted together, and 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 of a single sample is relatively long, no less than 20 minutes, and with the subsequent purification treatment, it takes even longer to prepare a test sample for instrumental analysis.

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

[0006] In order to solve the above technical problems, the present invention provides a novel method for extracting tobacco-specific N-nitrosamine components from tobacco samples. The method is time-saving and easy to operate. The extracted N-nitrosamine components are of high purity and less interference, and can be directly analyzed and measured without subsequent purification. The present invention also provides a method for detecting tobacco-specific N-nitrosamine components in tobacco based on the extraction method.

[0007] In order to achieve the above technical effects, the present invention adopts the following technical solutions.

[0008] A method for extracting tobacco-specific N-nitrosamine components comprises the following operations:

[0009] The tobacco sample is heated at 260-380°C for 6.0-8.0 min, and the volatilized components are transported to an acetonitrile-water solution at 0±5°C by 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 collecting device; the volatile component collecting device includes a carrier gas pipeline, a gas flow meter, a heating device, a material storage cavity, a first gas circuit valve, a collecting device, and a cooling device; wherein the gas flow meter is connected and installed on the carrier gas pipeline; a storage cavity for placing the tobacco sample is provided in the material storage cavity, 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 surfaces; the heating device is used to heat the storage cavity; the first gas circuit valve is connected and arranged on the carrier gas pipeline after the material storage cavity; the collecting device includes a collecting bottle containing an acetonitrile-water solution, the collecting bottle is placed in the cooling device and placed at the end of the carrier gas pipeline, and the acetonitrile-water solution in the collecting bottle submerges the end opening of the carrier gas pipeline.

[0011] Preferably, the heating device comprises a heating layer, a temperature sensor and a heater for heating the heating layer, the heating layer is wrapped on the outer wall of the material storage cavity, and the temperature sensor is arranged in the storage cavity.

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

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

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

[0015] As a preferred embodiment, the volatile component collection equipment also includes a cleaning device, a second pneumatic valve and a three-way pipe joint, the carrier gas pipeline includes a conveying section, a cleaning section and a collecting section that are connected; the three-way pipe joint includes a first end, a second end and a third end, the first end is connected and installed in the conveying section, the second end is connected and installed in the collecting section, and the third end is connected and installed in the cleaning section; the gas flow meter, the material storage cavity and the first gas circuit valve are connected and arranged on the conveying section; the second gas circuit valve is connected and arranged on the cleaning section, and the collecting bottle is placed at the end of the collecting section; the cleaning device includes an infusion pump and a cleaning bottle with cleaning liquid, the suction port of the infusion pump is placed in the cleaning bottle, and the output port of the infusion pump is connected to the collecting section.

[0016] In the above-mentioned embodiment including a cleaning device, the volatile component collection device also includes a controller, and the controller is communicatively connected with the gas flow meter, the heating device, the first gas circuit valve, the cooling device, the second gas circuit valve and the infusion pump.

[0017] When the first gas circuit valve is closed and the second gas circuit valve is opened, the pipelines of the cleaning section and the collection section can be cleaned, avoiding the residual gas in the pipe from contaminating the next collection of volatile components, thereby improving the collection purity of the volatile component collection equipment.

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

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

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

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

[0022] Also 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 one of compressed air, nitrogen and helium; more preferably nitrogen.

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

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

[0027] The method for extracting tobacco-specific N-nitrosamine components provided by the present invention is based on a novel volatile component collection device. Figure 1 As shown, when the volatile component collection device of the present invention is used, the sample in the storage chamber (for example, the tobacco sample to be tested of the present invention) is uniformly wrapped in the outer heating layer and heated to a specific temperature, and the tobacco-specific N-nitrosamine components with thermal volatility are carried to the collection bottle by the carrier gas and absorbed by the absorption solvent (for example, acetonitrile-water (1:1, v / v) of the present invention) that has been cooled therein, so that the continuous collection of volatile components at the specific temperature can be achieved. The first gas circuit valve and the second gas circuit valve can be set to clean the collection section pipeline, which can not only reduce the memory effect and the interference with the subsequent sample analysis, but also more thoroughly extract the N-nitrosamine components. When the first gas circuit valve is opened and the second gas circuit valve is closed, the volatile components (for example, the N-nitrosamine components of the present invention) are absorbed; when the first gas circuit valve is closed and the second gas circuit 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 of a tobacco sample prepared by the above extraction method.

[0029] Another object of the present invention is to provide a method for detecting tobacco-specific N-nitrosamine components in tobacco, based on liquid chromatography-tandem mass spectrometry; wherein the tobacco sample to be tested is pre-treated using the method for extracting tobacco-specific N-nitrosamine components of the present invention to obtain a test solution; the standard is selected from at least one of NNN, NNK, NAT and NAB, and the deuterated substance of the standard is used as an internal standard.

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

[0031] (1) Preparation of test solution

[0032] The tobacco sample is heated at 320°C for 6.0-8.0 min, and the volatilized components are transported to an acetonitrile-water solution at 0°C±5°C, preferably 0°C±2°C, and more preferably -2°C by a carrier gas to obtain an acetonitrile-water extract of tobacco-specific N-nitrosamine components; a blank acetonitrile-water solution is used to clean the pipeline through which the carrier gas flows, the cleaning solution is combined with the acetonitrile-water extract, an internal standard is added, and the acetonitrile-water solution is used to make up to a specified volume to obtain a test solution;

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

[0034] Using methanol and 0.1 mol / L ammonium acetate solution as solvents, preparing a series of mixed standard solutions including the standard and the corresponding internal standard;

[0035] (4) Determination

[0036] Liquid chromatography conditions included:

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

[0038] Column temperature: 40°C;

[0039] Injection volume: 5 μL;

[0040] Mobile phase: 0.01 mol / L ammonium acetate as phase A, 0.1% formic acid in methanol as phase B, titration elution, elution procedure:

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

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

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

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

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

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

[0047] Flow rate: 0.25 mL / min;

[0048] Mass spectrometry conditions included:

[0049] Ionization mode: positive ion mode ionization;

[0050] Electrospray voltage: 3000 V;

[0051] Cone voltage: 80V;

[0052] Ion source temperature: 550°C;

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

[0054] Cone airflow: 50L / hour;

[0055] Dwell time: 24ms;

[0056] Monitoring method: Multiple reaction monitoring;

[0057] Under the conditions of the liquid chromatography and mass spectrometry, the test solution and the series of standard solutions are respectively accurately aspirated and injected into the liquid chromatography-tandem mass spectrometer for measurement, and the chromatogram is recorded;

[0058] (5) Data processing and analysis

[0059] The method comprises qualitative and quantitative determination; wherein the qualitative determination comprises determining whether the corresponding N-nitrosamine components exist in the test solution according to the retention time, quantitative and qualitative ion pairs and corresponding collision energy of the standard; the quantitative determination adopts the internal standard method, and the working curve is drawn with the ratio of the peak area of ​​the N-nitrosamine components 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 components in the series of mixed standard solutions as the abscissa; the content of the target N-nitrosamine components in each cigarette is calculated according to the working curve by formula I according to the ratio of the peak area of ​​the N-nitrosamine components in the test solution to the peak area of ​​the internal standard.

[0060]

[0061] Where, m: the content of target N-nitrosamine components in each gram of tobacco sample, ng / g,

[0062] x: The peak area ratio of the target N-nitrosamine component to the internal standard,

[0063] a: slope of the working curve,

[0064] b: intercept of the working curve,

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

[0066] n: mass of tobacco sample, g.

[0067] Preferably, the step (1) is carried out in the volatile component collection device of the present invention; the tobacco sample is placed in the storage chamber, the acetonitrile-water solution for absorbing tobacco-specific N-nitrosamine components is placed in a collecting bottle, the collecting bottle is placed in the cooling device and placed below the end opening of the collecting section of the carrier gas pipeline, so that the end opening of the collecting section is immersed in the acetonitrile-water solution; the temperature of the cooling device is set to 0°C±5°C, preferably 0°C±2°C, and more preferably -2°C.

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

[0069] Preferably, in step (1), the volume of the acetonitrile-water solution for absorbing 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 one of compressed air, nitrogen and helium; more preferably nitrogen.

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

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

[0075] Preferably, the specific operations of step (1) are:

[0076] Place 0.10±0.05g of tobacco sample in the storage chamber of the volatile component collection device, and set the temperature of the cooling device to -2°C; accurately draw 1mL of acetonitrile-water solution with a volume ratio of 1:1, and transfer it to a collecting bottle as an absorption liquid, then put the collecting bottle into the cooling device and place it below the end opening of the collecting section of the carrier gas pipeline, so that the end opening of the collecting section is immersed in the acetonitrile-water solution; close the second gas circuit valve; open the first gas circuit valve, adjust the gas flowmeter to set the nitrogen flow rate to 2mL / min; set the temperature of the heating layer to 320°C, and keep warm for 6.0min; then stop heating the heating layer, close the first gas circuit valve, open the second gas circuit valve, clean the collecting section of the carrier gas pipeline with 0.25mL of acetonitrile-water solution with a volume ratio of 1:1, combine the cleaning liquid with the absorption liquid in the collecting bottle, add internal standard, and make the volume to 2mL with acetonitrile-water solution with a volume ratio of 1:1 to obtain the test solution.

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

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

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

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

[0081] Preferably, in the step (4), the retention time of NNN is 3.44±0.10min, the quantitative ion pair and collision energy are 178>148, 11eV, respectively, and the qualitative ion pair and collision energy are 178>120, 17eV, respectively; the retention time of NNN-d4 is 3.42±0.10min, the quantitative ion pair and collision energy are 182>152, 11eV, respectively; the retention time of NNK is 3.64±0.10min, the quantitative ion pair and collision energy are 208>122, 11eV, respectively, and the qualitative ion pair and collision energy are 208>106, 21eV, respectively; the retention time of NNK-d4 is 3.63±0.10min, the quantitative ion pair and collision energy are 212>1 26, 11eV; the retention time of NAT was 3.99±0.10min, the quantitative ion pair and collision energy were 190>160, 10eV, and the qualitative ion pair and collision energy were 190>106, 16eV; the retention time of NAT-d4 was 3.98±0.10min, the quantitative ion pair and collision energy were 194>164, 10eV; the retention time of NAB was 4.10±0.10min, the quantitative ion pair and collision energy were 192>162, 12eV, and the qualitative ion pair and collision energy were 192>133, 22eV; the retention time of NAB-d4 was 4.09±0.10min, the quantitative ion pair and collision energy were 196>166, 12eV.

[0082] In this article, unless otherwise specified or limited, the "tobacco sample" refers to tobacco leaves or tobacco shreds. All solvents (eg, the absorption liquid, the solvent of the standard solution, the solvent used to prepare the liquid chromatography mobile phase) and reagents are chromatographically pure.

[0083] The present invention uses heating to make tobacco-specific N-nitrosamine components volatilize directly from tobacco samples, which are absorbed by cold solutions under carrier gas, and then directly analyzed by liquid chromatography-tandem mass spectrometry, and the content of tobacco-specific N-nitrosamine components in tobacco samples is qualitatively and quantitatively determined by internal standard method. The method of the present invention completes the determination of a sample, and only requires no more than 2mL of acetonitrile-water (1:1, v / v), which takes about 15 to 20 minutes. The method of the present invention overcomes the shortcomings of the existing determination technology that a large amount of organic solvents are needed to extract samples, purification and concentration treatment must be performed after extraction, the pre-treatment time is long, and the purification effect is poor, and has the advantages of simple and rapid operation, green and efficient, and strong matrix removal ability. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0086] 1: carrier gas pipeline; 2: gas flow meter; 3: material storage chamber; 4: storage chamber; 5: first gas circuit valve; 6: collecting bottle; 7: heating layer; 8: cooling device; 9: cleaning device; 10: second pneumatic valve; 13: collecting section; 14: cleaning section.

[0087] Figure 2 The effect of different absorption liquids on the analysis results is shown. A uses pure acetonitrile as the absorption liquid, and B uses a mixed solution of acetonitrile / water (1 / 1, v / v) as the absorption liquid.

[0088] Figure 3 The effect of the absorption liquid temperature on the analysis results is shown.

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

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

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

[0094] Fig. 9 The typical chromatograms of four tobacco-specific N-nitrosamine components obtained by analyzing the same batch of tobacco leaf samples using the method of the present invention and the current standard method are shown. Among them, Figure A is the chromatogram obtained by the method of the present invention, and Figure B is the chromatogram obtained by the CORESTA recommended method CRM No.72 method.

[0095] Fig.10 The photographs show the extracts obtained by this method and the CORESTA recommended method CRM No. 72.

[0096] Fig.11 Shows Fig.10 UV-visible absorption spectrum of the extract after dilution 50 times. DETAILED DESCRIPTION

[0097] The present invention is described below with reference to specific examples. It will be appreciated by those skilled in the art that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0098] The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified. Among them, some instruments and accessories are as follows:

[0099] Liquid chromatography-tandem mass spectrometer: Waters ACQUITY UPLC I-Class Xevo TQD;

[0100] HPLC columns: ACQUITY BEH C18 column, 2.1×100 mm, 1.7 μm.

[0101] In the following embodiments and research examples, the tobacco samples used are all shredded tobacco.

[0102] In the following examples or research examples, the qualitative and quantitative determination of the test solution (containing tobacco-specific N-nitrosamine components and internal standards) was carried out according to the following operating steps:

[0103] Preparation of series of standard solutions

[0104] Methanol and 0.1 mol / L ammonium acetate solution were used as solvents, 4 tobacco-specific N-nitrosamine components (N-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoneanabinine (NAT) and N-nitrosoanabasine (NAB)) were used as solutes, and 4 tobacco-specific N-nitrosamine components deuterated (NNN-d4, NNK-d4, NAT-d4 and NAB-d4) were used as internal standards to prepare a series of mixed standard solutions containing internal standards with gradient concentrations. 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] Determination

[0106] Liquid chromatography conditions included:

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

[0108] Column temperature: 40°C;

[0109] Injection volume: 5 μL;

[0110] Mobile phase: 0.01 mol / L ammonium acetate as phase A, 0.1% formic acid in methanol as phase B, titration elution, elution procedure:

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

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

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

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

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

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

[0117] Flow rate: 0.25 mL / min;

[0118] Mass spectrometry conditions included:

[0119] Ionization mode: positive ion mode ionization;

[0120] Electrospray voltage: 3000 V;

[0121] Cone voltage: 80V;

[0122] Ion source temperature: 550°C;

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

[0124] Cone airflow: 50L / hour;

[0125] Dwell time: 24ms;

[0126] Monitoring method: Multiple reaction monitoring;

[0127] Under the conditions of the liquid chromatography and mass spectrometry, the test solution and the series of standard solutions are respectively accurately aspirated and injected into the liquid chromatography-tandem mass spectrometer for measurement, and the chromatogram is recorded;

[0128] Data processing and analysis

[0129] Qualitative determination: Determine whether the corresponding tobacco-specific N-nitrosamine components are present in the test solution based on the retention time of the standard and its deuterated derivative, the quantitative / qualitative ion pairs, and the corresponding collision energy;

[0130] The retention time of NNN was 3.44±0.10min, the quantitative ion pair and collision energy were 178>148, 11eV, respectively, and the qualitative ion pair and collision energy were 178>120, 17eV, respectively; the retention time of NNN-d4 was 3.42±0.10min, the quantitative ion pair and collision energy were 182>152, 11eV, respectively; the retention time of NNK was 3.64±0.10min, the quantitative ion pair and collision energy were 208>122, 11eV, respectively, and the qualitative ion pair and collision energy were 208>106, 21eV, respectively; the retention time of NNK-d4 was 3.63±0.10min, the quantitative ion pair and collision energy were 212>126, 11 eV; the retention time of NAT was 3.99±0.10min, the quantitative ion pair and collision energy were 190>160, 10eV, and the qualitative ion pair and collision energy were 190>106, 16eV; the retention time of NAT-d4 was 3.98±0.10min, the quantitative ion pair and collision energy were 194>164, 10eV; the retention time of NAB was 4.10±0.10min, the quantitative ion pair and collision energy were 192>162, 12eV, and the qualitative ion pair and collision energy were 192>133, 22eV; the retention time of NAB-d4 was 4.09±0.10min, the quantitative ion pair and collision energy were 196>166, 12eV.

[0131] Quantitative determination: using the internal standard method, with the ratio of the peak area of ​​N-nitrosamine components in the series of standard solutions to the peak area of ​​the internal standard as the ordinate, and the concentration of N-nitrosamine components in the series of standard solutions as the abscissa, a working curve is drawn; according to the ratio of the peak area of ​​N-nitrosamine components in the test solution to the peak area of ​​the internal standard, according to the working curve, the content of the target N-nitrosamine components in each cigarette is calculated by formula I,

[0132]

[0133] Where, m: the content of target N-nitrosamine components in each gram of tobacco sample, ng / g,

[0134] x: The peak area ratio of the target N-nitrosamine component to the internal standard,

[0135] a: slope of the working curve,

[0136] b: intercept of the working curve,

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

[0138] n: mass of tobacco sample, g.

[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 cavity 3, a first gas circuit valve 5, a collecting device, a cooling device 8, a cleaning device 9, a second gas circuit valve 10, a three-way pipe joint and a controller. The carrier gas pipeline 1 is used for gas supply, and includes a conveying section, a cleaning section 14 and a collecting section 13 that are connected. The three-way pipe joint includes a first end, a second end and a third end, the first end is connected and installed in the conveying section of the carrier gas pipeline 1, the second end is connected and installed in the collecting section 13, and the third end is connected and installed in the cleaning section 14. The second gas circuit valves 10 are connected and installed on the cleaning section 14. The gas flow meter 2 is connected and installed on the conveying section of the carrier gas pipeline 1. A storage chamber 4 is arranged in the material storage cavity 3, and the storage chamber 4 is used to store solid or liquid samples. The storage chamber 4 is connected to the conveying section of the carrier gas pipeline 1, and a carrier gas inlet and a carrier gas outlet are arranged on two opposite surfaces of the storage chamber 4. The heating device includes a heating layer 7, a temperature sensor and a heater; the heater is used to heat the heating layer 7, the heating layer 7 is wrapped on the outer wall of the material storage cavity 3, and the temperature sensor is arranged in the storage cavity 4. The first gas circuit valve 5 is connected to the conveying section of the carrier gas pipeline 1 after the material storage cavity 3. The collecting device includes a collecting bottle 6 with an absorption liquid placed therein, the collecting bottle 6 is placed at the end of the collecting section 13 of the carrier gas pipeline 1, and the solvent used for absorption in the collecting bottle covers the end opening of the collecting section 13. The cleaning device 9 includes an infusion pump and a cleaning bottle with a cleaning liquid placed therein. The suction port of the infusion pump is placed in the cleaning bottle, and the output port of the infusion pump is connected to the collecting section 13. The controller is communicatively connected with the gas flowmeter 2, the heater, the temperature sensor, the first gas circuit valve 5, the cooling device 9 and the second gas circuit valve 10.

[0141] When using the volatile component collection device of this embodiment to collect volatile components, first, move an appropriate amount of absorption liquid (solvent) into the collection bottle 6, place the collection bottle 6 in the cooling device 8 and place it below the end opening of the collection section 13 of the carrier gas pipeline 1, the opening of the collection section 13 is immersed in the absorption liquid, and start the cooling device; then, place the solid or liquid sample in the storage chamber 4 of the material storage chamber 3; open the first gas circuit valve 5, and close the second gas circuit valve 10; finally, the controller turns on the heater, and the heater heats the solid or liquid sample in the storage chamber 4, and the gas in the carrier gas pipeline 1 at a specific flow rate will carry the volatile components in the storage chamber 4 to the collection bottle 6 through the collection section 13, and be absorbed by the absorption liquid. Thus, the volatile components can be collected at a specific temperature.

[0142] After the heating is completed, the first gas circuit valve 5 is closed, and the second gas circuit valve 10 and the infusion pump are opened, so that the pipelines of the cleaning section 14 and the collecting section 13 can be cleaned, which 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, thereby improving the collection purity and collection efficiency of the volatile component collection equipment.

[0143] Research Example 1: Preparation of Extracts of Tobacco-specific N-nitrosamines in Tobacco

[0144] This research example investigated the preparation of an extract of tobacco-specific N-nitrosamine components in tobacco using the volatile component collection device 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, 1mL of absorption liquid is precisely sucked and placed in the collection bottle 6, and then the collection bottle 6 is placed in the cooling device 8, and the end opening of the collection section 13 of the carrier gas pipeline 1 is immersed in the absorption liquid in the collection bottle 6; the first gas circuit valve 5 is opened and the second gas circuit valve 10 is closed, and the controller starts the heater to heat the heating layer 7. During the specific time period when the temperature of the heating layer 7 reaches the set temperature T and is maintained, the carrier gas with a specific flow rate in the carrier gas pipeline 1 carries the volatile components volatilized from the tobacco sample in the storage chamber 4 to the collection bottle 6 through the collection section 13, and is absorbed by the absorption liquid therein. In this way, the continuous collection of tobacco-specific N-nitrosamine components at the set temperature T (≤T) is achieved. After heating, close the first gas circuit valve 5 and open the second gas circuit valve 10, start the infusion pump, use the blank absorption liquid (0.25 mL) in the cleaning device to clean the collection section 13, combine the cleaning liquid and the absorption liquid to obtain the tobacco-specific N-nitrosamine component extract of the tobacco sample, add the internal standard, and make up to 2 mL with the blank absorption liquid, which can be directly used for liquid chromatography-tandem mass spectrometry analysis.

[0146] In the above collection process, factors such as the type and temperature of the absorption liquid, the tobacco heating temperature and time, the type and flow rate of the carrier gas will affect the extraction efficiency. This study investigated each of the above factors.

[0147] 1. Investigation of absorption liquid types

[0148] Acetonitrile and acetonitrile-water solution (1 / 1, v / v) at room temperature were used as absorption liquids, respectively, the heating temperature was 320°C, nitrogen was used as carrier gas, and the flow rate was 15 mL / min to prepare the extraction solutions. NNN-d4, NNK-d4, NAT-d4 and NAB-d4 were added to each extraction solution as internal standards to make the concentration of each deuterated product 10 ng / mL, and the blank absorption liquid was used to make the volume to 2 mL, and the qualitative and quantitative determination of tobacco-specific N-nitrosamine components was carried out. The results are shown in Figure 2Figure A and Figure B of the diagram.

[0149] Figure 2 Figure A shows that when pure acetonitrile is used as the absorption liquid, the chromatographic peaks of the four target analytes all show solvent effects, and the solvent effect increases as the retention time decreases. Figure 2 Figure B shows that when acetonitrile-water solution (1 / 1, v / v) is used as the absorption liquid, the chromatographic peaks of the four target analytes are symmetrical and sharp, and there is no solvent effect. Therefore, acetonitrile-water mixed solution (1 / 1, v / v) is preferably used as the absorption liquid.

[0150] 2. Investigation of absorption liquid temperature

[0151] Acetonitrile-water solution (1 / 1, v / v) was used as the absorption liquid, the tobacco sample was heated to 350°C, nitrogen was used as the carrier gas, and the flow rate was 5 mL / min. The extraction solutions were prepared at room temperature (25°C) and -2°C, respectively. NNN-d4, NNK-d4, NAT-d4 and NAB-d4 were added to each extraction solution as internal standards to make the concentration of each deuterated product 10 ng / mL, and the blank absorption solution was used to make the volume to 2 mL, and the qualitative and quantitative determination of tobacco-specific N-nitrosamine components was carried out. The results are shown in Figure 3 .

[0152] Figure 3 It is shown that the absorption efficiency of the absorption liquid at -2°C is higher. The reason may be that the high-temperature gas continuously flowing out of the material storage chamber will cause the temperature of the absorption liquid at room temperature to rise, which is not conducive to the condensation and absorption of the target analyte; while the temperature-controlled cold pool with condensation function can always maintain the temperature of the absorption liquid at a low temperature (-2°C), which can not only cool the high-temperature carrier gas, but also facilitate the absorption of the target analyte. Therefore, the preferred temperature of the absorption liquid is -2°C.

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

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

[0155] Figure 4 The results show that 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°C for 6 minutes, the four target compounds were basically all volatilized from the tobacco sample. Therefore, the heating time of the tobacco sample is preferably 6.0 minutes.

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

[0157] The extraction solutions were prepared using -2℃ acetonitrile-water solution (1 / 1, v / v) as the absorption solution, the tobacco sample heating time was 6 min, nitrogen was used as the carrier gas, the flow rate was 2 mL / min, and the heating temperatures were 260℃, 290℃, 320℃, 350℃ and 380℃, respectively. NNN-d4, NNK-d4, NAT-d4 and NAB-d4 were added to each extraction solution as internal standards to make the concentration of each deuterated product 10 ng / mL, and the blank absorption solution was used to make the volume to 2 mL, and the qualitative and quantitative determination of tobacco-specific N-nitrosamine components was carried out. The results are shown in Figure 5 .

[0158] Figure 5 It is shown that the contents of the four tobacco-specific N-nitrosamine components all reach the highest at 320°C. The reason may be that lower temperatures are not conducive to the volatilization of the target from the tobacco sample; too high a heating temperature will cause the degradation of the target to a certain extent, and at the same time, the high-temperature gas at a higher heating temperature will cause the temperature of the absorption liquid to rise, which is not conducive to the adsorption of the target at a lower temperature. Therefore, the heating temperature of the tobacco sample is preferably 320°C.

[0159] 5. Investigation of carrier gas type

[0160] The extraction solution was prepared by using -2℃ acetonitrile-water solution (1 / 1, v / v) as the absorption liquid, the tobacco sample heating temperature was 320℃, the time was 6min, the carrier gas flow rate was 15mL / min, and nitrogen and helium were used as carrier gases respectively. NNN-d4, NNK-d4, NAT-d4 and NAB-d4 were added to each extraction solution as internal standards, so that the concentration of each deuterated product was 10ng / mL, and the blank absorption solution was used to make the volume to 2mL, and the qualitative and quantitative determination of tobacco-specific N-nitrosamine components was carried out. The results are shown in Figure 6 .

[0161] Figure 6 The results show that there is no difference in the analysis results of the two carrier gases, which indicates that any inert gas can be used as a carrier gas, such as nitrogen, helium, compressed air, etc., and nitrogen, which has a high safety factor, is cheap, and is commonly used in laboratories, is preferably used as a carrier gas.

[0162] 6. Investigation of carrier gas flow rate

[0163] The extraction solutions were prepared using -2℃ acetonitrile-water solution (1 / 1, v / v) as the absorption solution, the tobacco sample was heated at 320℃ for 6 min, and nitrogen was used 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 to each extraction solution as internal standards to make the concentration of each deuterated product 10 ng / mL. The blank absorption solution was used to make the volume 2 mL, and the qualitative and quantitative determination of tobacco-specific N-nitrosamine components was performed. The results are shown in Figure 7 .

[0164] Figure 7 It is shown that when there is no carrier gas (carrier gas flow rate is 0mL / min), the device can still achieve the purpose of collecting volatile target substances. As the carrier gas flow rate increases, the absorption effect of the target substance shows a trend of first increasing and then decreasing, and reaches the maximum at 2mL / min. The above results not only prove that the absorption efficiency of the target substance can be enhanced under the auxiliary effect of carrier gas, but also illustrate that the carrier gas flow rate should not be too large. Therefore, the preferred carrier gas flow rate is 2mL / min.

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

[0166] 7. Inspection of tobacco sample quality

[0167] Using -2℃ acetonitrile-water solution (1 / 1, v / v) as the absorption liquid, the tobacco sample was heated at 320℃ for 6 minutes, nitrogen was used as the carrier gas, and the flow rate was 2mL / min. Accurately weighed 0.0506g, 0.1004g and 0.1801g of tobacco sample extraction solutions were prepared respectively. NNN-d4, NNK-d4, NAT-d4 and NAB-d4 were added to each extraction solution as internal standards to make the concentration of each deuterated substance 10ng / mL. The blank absorption liquid was used to make the volume to 2mL, and the qualitative and quantitative determination of tobacco-specific N-nitrosamine components was carried out. The results are shown in Figure 8 .

[0168] Figure 8 The results show that there is no difference in the test results of the four targets under different sample masses. Considering the sample uniformity and usage, the sample mass is finally determined to be 0.10±0.05g.

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

[0170] The absorption liquid is an acetonitrile-water mixed solution (1 / 1, v / v) at a temperature of -2°C, the sample heating temperature is 320°C, the heating time is 6.0 min, the carrier gas is nitrogen, the carrier gas flow rate is 2 mL / min, and the sample mass is 0.10±0.05 g.

[0171] Study Example 2: Methodological Review

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

[0173] (1) Preparation of test solution

[0174] 0.10±0.05g of tobacco sample was placed in the storage chamber of the volatile component collection device as described in Example 1, and the temperature of the cooling device was set to -2°C; 1mL of acetonitrile-water solution with a volume ratio of 1:1 was accurately drawn and transferred into the collection bottle as the absorption liquid, and then the collection bottle was placed in the cooling device and placed under the end opening of the collection section so that the end opening was immersed in the absorption liquid; the second gas circuit valve was closed; the first gas circuit valve was opened to adjust the gas flow meter to set the nitrogen flow rate to 2mL / min; the heating layer temperature was set to 320℃, keep warm for 6.0min; then stop heating the heating layer, close the first gas circuit valve, open the second gas circuit valve, start the infusion pump, wash the collecting section with 0.25mL of acetonitrile-water solution with a volume ratio of 1:1, combine the washing liquid with the absorption liquid in the collecting bottle, add NNN-d4, NNK-d4, NAT-d4 and NAB-d4 as internal standards, make the volume to 2mL with acetonitrile-water solution with a volume ratio of 1:1, the concentration of each internal standard is 10ng / mL, and obtain the test solution.

[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 the series of mixed standard solutions, the concentration gradients of NNN, NNK, NAT and NAB were all 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.

[0177] (3) Determination

[0178] Liquid chromatography conditions included:

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

[0180] Column temperature: 40°C;

[0181] Injection volume: 5 μL;

[0182] Mobile phase: 0.01 mol / L ammonium acetate as phase A, 0.1% formic acid in methanol as phase B, titration elution, elution procedure:

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

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

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

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

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

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

[0189] Flow rate: 0.25 mL / min;

[0190] Mass spectrometry conditions included:

[0191] Ionization mode: positive ion mode ionization;

[0192] Electrospray voltage: 3000 V;

[0193] Cone voltage: 80V;

[0194] Ion source temperature: 550°C;

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

[0196] Cone airflow: 50L / hour;

[0197] Dwell time: 24ms;

[0198] Monitoring method: Multiple reaction monitoring;

[0199] Under the conditions of the liquid chromatography and mass spectrometry, the test solution and the series of standard solutions are respectively accurately aspirated and injected into the liquid chromatography-tandem mass spectrometer for measurement, and the chromatogram is recorded;

[0200] (4) Data processing and analysis

[0201] Includes qualitative and quantitative determination;

[0202] Among them, qualitative determination: determine whether the corresponding tobacco-specific N-nitrosamine components are present in the test solution based on the retention time, quantitative and qualitative ion pairs and corresponding collision energy of the standard and the deuterated product of the standard;

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

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

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

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

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

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

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

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

[0211] Quantitative determination: using the internal standard method, with the ratio of the peak area of ​​N-nitrosamine components in the series of standard solutions to the peak area of ​​the internal standard as the ordinate, and the concentration of N-nitrosamine components in the series of standard solutions as the abscissa, a working curve is drawn; according to the ratio of the peak area of ​​N-nitrosamine components in the test solution to the peak area of ​​the internal standard, according to the working curve, the content of the target N-nitrosamine components in each cigarette is calculated by formula I,

[0212]

[0213] Where, m: the content of target N-nitrosamine components in each gram of tobacco sample, ng / g,

[0214] x: The peak area ratio of the target N-nitrosamine component to the internal standard,

[0215] a: slope of the working curve,

[0216] b: intercept of the working curve,

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

[0218] n: mass of tobacco sample, g.

[0219] 1. Comparison with existing methods

[0220] According to the above detection method, 0.10g of a tobacco leaf sample was measured, and the typical chromatogram of the target analyte was obtained. Fig. 9 Figure A.

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

[0222] Compare Fig. 9 From the A and B graphs, it can be seen that the baseline of graph A is flatter and has fewer interfering components. Therefore, compared with the current CRM No. 72 standard method, the test solution prepared by the method of the present invention has less matrix interference, which is beneficial to the qualitative and quantitative determination of the target compound.

[0223] The photos of the test solution prepared by the method of the present invention and the test solution prepared by the CRM No.72 standard method are shown in Fig.10 The color of the extract obtained by the method of the present invention is obviously lighter than that of the extract obtained by the CRM No. 72 method. This indicates that the method of the present invention carries out less tobacco impurities.

[0224] The extract was diluted 50 times and the UV-visible absorption spectrum in the wavelength range of 200-400 nm was measured. Fig.11 Compared with the ultraviolet spectrum of the extract obtained by the standard method CRM No.72, the absorption intensity of the absorption spectrum of the extract obtained by the method of the present invention in the wavelength range of 200 to 400 nm is significantly weakened, indicating that the extract obtained by the method of the present invention contains less interfering substances except pigments.

[0225] 2. Linear range, detection limit and quantification limit

[0226] The standard working curve (weighted as 1 / x) was made by comparing the peak area ratio of the four tobacco-specific N-nitrosamine standards and the corresponding internal standards to the concentration of the N-nitrosamine standards. The detection limit and quantification limit were calculated at 3 and 10 times the signal-to-noise ratio, respectively. The results are shown in Table 1.

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

[0228] Table 1 Linear range, working curve, detection limit and quantification limit of the detection method of the present invention

[0229]

[0230] 3. Accuracy and repeatability inspection

[0231] Take the same tobacco sample and accurately weigh 3 portions, each portion is 0.1 g; prepare the test solution according to the method established by the present invention on day 1, and then measure it, calculate the content of tobacco-specific N-nitrosamine components per gram of tobacco sample by standard working curve and formula I, and calculate the intra-day relative standard deviation.

[0232] In order to compare the accuracy of the determination results of the method of the present invention with that of the current standard method (such as CRM No.72), the same batch of tobacco samples were subjected to qualitative and quantitative determination of tobacco-specific N-nitrosamine components using CRM No.72 in parallel. The results are shown in Table 2.

[0233] Table 2 Comparison of the results of the method of the present invention and CRM No.72

[0234]

[0235]

[0236] The data in Table 2 show that the relative deviation of the results obtained by analyzing the same sample using the method of the present invention and the CORESTA recommended method is between 2.76% and 4.36%, both less than 10%, indicating that the method of the present invention has good consistency with the CORESTA recommended method, proving that the method is accurate and reliable. The relative standard deviation of the same sample tested by the method of the present invention is between 0.65% and 3.62%. The above shows that the accuracy and repeatability of the method of the present invention can meet the daily detection needs of tobacco-specific N-nitrosamine components in tobacco.

[0237] Embodiment 1:

[0238] The other two tobacco samples were subjected to qualitative and quantitative determination of tobacco-specific N-nitrosamine components according to the method described in Study Example 2. At the same time, the same batch of tobacco samples were also determined in parallel using the CORESTA recommended method CRM No. 72. The results of the two determinations are shown in Table 3.

[0239] Table 3 Detection results of TSNAs in two tobacco samples (ng / g)

[0240]

[0241] The data in Table 3 show that the results obtained by the method of the present invention and the CORESTA recommended method (CRM No.72) are consistent, which once again shows that the method is accurate and reliable. The detection method of the present invention takes 6-8 minutes to detect a tobacco sample and consumes 2 mL of extraction solvent (acetonitrile-water). The CRM No.72 method takes 40-60 minutes to detect a sample and consumes 30 mL of extraction solvent. In terms of ease of operation, efficiency, cost, environmental protection, etc., the method of the present invention has more advantages.

Claims

1. A method for extracting tobacco-specific N-nitrosamine components, comprising the following operations: The tobacco sample is heated at 260-380°C for 6.0-8.0 min, and the volatilized components are transported to an acetonitrile-water solution at 0±5°C by a carrier gas to obtain an acetonitrile-water extract of tobacco-specific N-nitrosamine components.

2. The extraction method according to claim 1, characterized in that 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 cavity, a first gas circuit valve, a collecting device, and a cooling device; wherein the gas flow meter is connected and installed on the carrier gas pipeline; a storage cavity for storing the tobacco sample is provided in the material storage cavity, 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 surfaces; the heating device is used to heat the storage cavity; the first gas circuit valve is connected and arranged on the carrier gas pipeline after the material storage cavity; the collecting device includes a collecting bottle with an acetonitrile-water solution, the collecting bottle is placed in the cooling device and placed at the end of the carrier gas pipeline, and the acetonitrile-water solution in the collecting bottle submerges the end opening of the carrier gas pipeline.

3. The extraction method according to claim 2, characterized in that The heating device comprises a heating layer, a temperature sensor and a heater for heating the heating layer, the heating layer is wrapped on the outer wall of the material storage cavity, and the temperature sensor is arranged in the storage cavity; Preferably, the cooling device is selected from a cold trap or an ice bath, more preferably a cold trap; More preferably, the cold trap is a condensation trap.

4. The extraction method according to claim 2 or 3, characterized in that The volatile component collection device also includes a controller, which is communicatively connected with the gas flow meter, the heating device, the first gas path valve and the cooling device.

5. The extraction method according to claim 2 or 3, characterized in that The volatile component collection device also includes a cleaning device, a second pneumatic valve and a three-way pipe joint, the carrier gas pipeline includes a conveying section, a cleaning section and a collecting section that are connected; the three-way pipe joint includes a first end, a second end and a third end, the first end is connected and installed in the conveying section, the second end is connected and installed in the collecting section, and the third end is connected and installed in the cleaning section; the gas flow meter, the material storage cavity and the first gas circuit valve are connected and arranged on the conveying section; the second gas circuit valve is connected and arranged on the cleaning section, and the collecting bottle is placed at the end of the collecting section; the cleaning device includes an infusion pump and a cleaning bottle with a cleaning liquid placed therein, the suction port of the infusion pump is placed in the cleaning bottle, and the output port of the infusion pump is connected to the collecting section; Preferably, the volatile component collection device further comprises a controller, and the controller is communicatively connected with the gas flow meter, the heating device, the first gas circuit valve, the cooling device, the second gas circuit valve and the infusion pump.

6. The extraction method according to claim 1, characterized in that The tobacco sample is 0.10±0.05g; Preferably, the acetonitrile-water solution is 1 mL; Preferably, the temperature of the acetonitrile-water solution is 0°C ± 2°C; more preferably -2°C; Preferably, the tobacco sample is heated at 320°C; Also preferably, the tobacco sample heating time is 6.0 min; Preferably, the flow rate of the carrier gas is 2 mL / min; More preferably, the carrier gas is selected from one of compressed air, nitrogen and helium; more preferably nitrogen; Preferably, after the heating is completed, the collection section is washed with 0.25-0.5 mL, more preferably 0.25 mL, of acetonitrile-water solution, and 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; Preferably, in the acetonitrile-water solution, the volume ratio of acetonitrile to water is 1:

1.

7. An acetonitrile-water extract of tobacco-specific N-nitrosamine components of a tobacco sample, prepared by the extraction method according to any one of claims 1 to 6.

8. A method for detecting tobacco-specific N-nitrosamine components in tobacco, based on liquid chromatography-tandem mass spectrometry; wherein: The tobacco sample to be tested is pre-treated by the extraction method according to any one of claims 1 to 6 to obtain a test solution; the standard is selected from at least one of NNN, NNK, NAT and NAB, and the deuterated substance of the standard is used as the internal standard.

9. The detection method according to claim 8, characterized in that: The detection method comprises the following steps: (1) Preparation of test solution The tobacco sample is heated at 320°C for 6.0-8.0 min, and the volatilized components are transported to an acetonitrile-water solution at 0°C±5°C, preferably 0°C±2°C, and more preferably -2°C by a carrier gas to obtain an acetonitrile-water extract of tobacco-specific N-nitrosamine components; a blank acetonitrile-water solution is used to clean the pipeline through which the carrier gas flows, the cleaning solution is combined with the acetonitrile-water extract, an internal standard is added, and the acetonitrile-water solution is used to make up to a specified volume to obtain a test solution; (2) Preparation of a series of mixed standard solutions Using methanol and 0.1 mol / L ammonium acetate solution as solvents, preparing a series of mixed standard solutions including the standard and the corresponding internal standard; (4) Determination Liquid chromatography conditions included: Column: ACQUITY BEH C18 column, 2.1×100 mm, 1.7 μm; Column temperature: 40°C; Injection volume: 5 μL; Mobile phase: 0.01 mol / L ammonium acetate as phase A, 0.1% formic acid in methanol as phase B, titration elution, elution procedure: t = 0 min, 90% A, 10% B; t=1.0min, 90%A, 10%B; t=5.0min, 10%A, 90%B; t=6.0min, 10%A, 90%B; t=6.1min, 90%A, 10%B; t=8.0min, 90%A, 10%B; Flow rate: 0.25 mL / min; Mass spectrometry conditions included: Ionization mode: positive ion mode ionization; Electrospray voltage: 3000 V; Cone voltage: 80V; Ion source temperature: 550°C; Desolventizing gas flow: 800L / hour; Cone airflow: 50L / hour; Dwell time: 24ms; Monitoring method: Multiple reaction monitoring; Under the conditions of the liquid chromatography and mass spectrometry, the test solution and the series of standard solutions are respectively accurately aspirated and injected into the liquid chromatography-tandem mass spectrometer for measurement, and the chromatogram is recorded; (5) Data processing and analysis The method comprises qualitative and quantitative determination; wherein the qualitative determination comprises determining whether the corresponding N-nitrosamine components exist in the test solution according to the retention time, quantitative and qualitative ion pairs and corresponding collision energy of the standard; the quantitative determination adopts the internal standard method, and the working curve is drawn with the ratio of the peak area of ​​the N-nitrosamine components 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 components in the series of mixed standard solutions as the abscissa; the content of the target N-nitrosamine components in each cigarette is calculated according to the working curve by formula I according to the ratio of the peak area of ​​the N-nitrosamine components in the test solution to the peak area of ​​the internal standard. Where, m: the content of target N-nitrosamine components in each gram of tobacco sample, ng / g, x: The peak area ratio of the target N-nitrosamine component to the internal standard, a: slope of the working curve, b: intercept of the working curve, V: volume of the test solution, mL, n: mass of tobacco sample, g.

10. The detection method according to claim 9, characterized in that: The step (1) is performed in the volatile component collection device described in any one of claims 2 to 5; the tobacco sample is placed in the storage chamber, the acetonitrile-water solution for absorbing tobacco-specific N-nitrosamine components is placed in a collection bottle, the collection bottle is placed in the cooling device and placed below the end opening of the collection section of the carrier gas pipeline, so that the end opening of the collection section is immersed in the acetonitrile-water solution; the temperature of the cooling device is set to 0°C ± 5°C, preferably 0°C ± 2°C, and more preferably -2°C; Preferably, in step (1), the mass of the tobacco sample is 0.10±0.05 g; Preferably, in step (1), the volume of the acetonitrile-water solution for absorbing tobacco-specific N-nitrosamine components is 1 mL; Preferably, in step (1), the heating time is 6 min; Preferably, in step (1), the flow rate of the carrier gas is 2 mL / min; More preferably, the carrier gas is selected from one of compressed air, nitrogen and helium; more preferably nitrogen; Preferably, after heating, the collection section of the carrier gas pipeline is cleaned with 0.25-0.5 mL, more preferably 0.25 mL, of acetonitrile-water solution, and the cleaning solution is combined with the acetonitrile-water solution in the collection bottle to obtain a test solution; Preferably, in the acetonitrile-water solution, the volume ratio of acetonitrile to water is 1:

1.

11. The detection method according to claim 10, characterized in that: The specific operations of step (1) are: 0.10±0.05g tobacco sample is placed in the storage chamber of the volatile component collection device, and the temperature of the cooling device is set to -2°C; 1mL of acetonitrile-water solution with a volume ratio of 1:1 is accurately drawn and transferred into a collection bottle as an absorption liquid, and then the collection bottle is placed in the cooling device and placed below the end opening of the collection section of the carrier gas pipeline, so that the end opening of the collection section is immersed in the acetonitrile-water solution; the second gas circuit valve is closed; the first gas circuit valve is opened, and the gas flow meter is adjusted to set the nitrogen flow rate to 2mL / min; the heating layer is set to 320°C and kept warm for 6.0min; then the heating of the heating layer is stopped, the first gas circuit valve is closed, the second gas circuit valve is opened, and the collection section of the carrier gas pipeline is cleaned with 0.25mL of acetonitrile-water solution with a volume ratio of 1:1, the cleaning liquid is combined with the absorption liquid in the collection bottle, an internal standard is added, and the volume is adjusted to 2mL with acetonitrile-water solution with a volume ratio of 1:1 to obtain a test solution; Preferably, the concentration of the deuterated substance of the standard in the test solution is 10 ng / mL.

12. The detection method according to claim 9, characterized in that: In the step (2), the standard substances are all of NNN, NNK, NAT and NAB; Preferably, in the step (2), in the series of mixed standard solutions, the concentration gradients of the standards are 0.5, 1.0, 2.0, 5.0, 10.0, 20.0 and 50 ng / mL; Also preferably, in the step (2), in the series of mixed standard solutions, the concentration of the deuterated substance of the standard is 10 ng / mL.

13. The detection method according to claim 9, characterized in that: In the step (4), the retention time of NNN is 3.44±0.10min, the quantitative ion pair and collision energy are 178>148, 11eV, respectively, and the qualitative ion pair and collision energy are 178>120, 17eV, respectively; the retention time of NNN-d4 is 3.42±0.10min, the quantitative ion pair and collision energy are 182>152, 11eV, respectively; the retention time of NNK is 3.64±0.10min, the quantitative ion pair and collision energy are 208>122, 11eV, respectively, and the qualitative ion pair and collision energy are 208>106, 21eV, respectively; the retention time of NNK-d4 is 3.63±0.10min, the quantitative ion pair and collision energy are 212>126, respectively. , 11eV; the retention time of NAT was 3.99±0.10min, the quantitative ion pair and collision energy were 190>160, 10eV, and the qualitative ion pair and collision energy were 190>106, 16eV; the retention time of NAT-d4 was 3.98±0.10min, the quantitative ion pair and collision energy were 194>164, 10eV; the retention time of NAB was 4.10±0.10min, the quantitative ion pair and collision energy were 192>162, 12eV, and the qualitative ion pair and collision energy were 192>133, 22eV; the retention time of NAB-d4 was 4.09±0.10min, the quantitative ion pair and collision energy were 196>166, 12eV.

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