CN110646536A - Method for measuring tobacco-specific nitrosamine on-line two-dimensional chromatography tandem mass spectrometry in cigarette mainstream smoke - Google Patents

Method for measuring tobacco-specific nitrosamine on-line two-dimensional chromatography tandem mass spectrometry in cigarette mainstream smoke Download PDF

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CN110646536A
CN110646536A CN201910919845.3A CN201910919845A CN110646536A CN 110646536 A CN110646536 A CN 110646536A CN 201910919845 A CN201910919845 A CN 201910919845A CN 110646536 A CN110646536 A CN 110646536A
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phase
interface
tobacco
chromatography
mass spectrometry
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周妍
戚大伟
罗辰
费婷
吴达
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Shanghai Tobacco Group Co Ltd
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Shanghai Tobacco Group Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/14—Preparation by elimination of some components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/16—Injection
    • G01N30/20—Injection using a sampling valve
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N2030/042—Standards
    • G01N2030/045—Standards internal
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/062—Preparation extracting sample from raw material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/16—Injection
    • G01N30/20—Injection using a sampling valve
    • G01N2030/201—Injection using a sampling valve multiport valves, i.e. having more than two ports

Abstract

The invention provides a method for measuring tobacco-specific nitrosamine on-line two-dimensional chromatography tandem mass spectrometry in cigarette mainstream smoke, which comprises the following steps: the method comprises the steps of collecting and extracting cigarette mainstream smoke to obtain a sample solution, and then carrying out qualitative and quantitative analysis on nitrosamine components in the sample solution by adopting an ultra performance convergence chromatography-liquid chromatography-mass spectrometry (UPCC-HPLC-MS/MS) method. The invention further provides an online two-dimensional chromatography tandem mass spectrometry system for tobacco-specific nitrosamine in cigarette mainstream smoke and a using method thereof. The method for measuring the on-line two-dimensional chromatography tandem mass spectrum of the tobacco-specific nitrosamine in the mainstream smoke of the cigarette has the advantages of simplicity and convenience in operation, rapidness in detection and high sensitivity, and is suitable for daily analysis and detection of the tobacco-specific nitrosamine in the cigarette smoke, particularly accurate quantification of trace nitrosamine in a medium and thin cigarette smoke sample.

Description

Method for measuring tobacco-specific nitrosamine on-line two-dimensional chromatography tandem mass spectrometry in cigarette mainstream smoke
Technical Field
The invention belongs to the technical field of chemical component analysis in tobacco, and relates to a method for determining tobacco-specific nitrosamine in cigarette mainstream smoke by using an online two-dimensional chromatography tandem mass spectrum, in particular to a method for determining tobacco-specific nitrosamine in cigarette mainstream smoke by using an ultra performance integrated chromatography-liquid chromatography-mass spectrometry (UPCC-HPLC-MS/MS) method.
Background
The Tobacco Specific Nitrosamines (TSNAs) comprise 4- (N-methyl nitrosamine) -1- (3-adjacent amine solution group) -1-butanone (NNK), N-nitrosonornicotine (NNN), N-Nitrosoanabasine (NAB) and Nitrosoanabasine (NAT), are tobacco specific harmful substances in tobacco products and smoke, are always hot spots concerned by tobacco researchers at home and abroad due to strong carcinogenicity, and have important significance for evaluating the tobacco products and guaranteeing the consumption safety of the cigarette products by accurately measuring the content of the tobacco specific nitrosamines in the cigarette smoke.
The content of the tobacco-specific nitrosamine in the cigarette smoke, particularly the flue-cured tobacco type cigarette, is extremely low, the smoke components are very complex, and the matrix interference is serious. Meanwhile, with the concern of people on smoking and health problems, medium and fine cigarettes are more and more popular in domestic markets, and the accurate quantification of the specific nitrosamine in trace tobacco in the mainstream smoke of the medium and fine cigarettes is quite difficult.
At present, the method for analyzing tobacco-specific nitrosamines in cigarette mainstream smoke mainly comprises a gas chromatography-thermal energy analyzer method (GC-TEA), a gas chromatography-mass spectrometry combined method (GC-MS/MS) and a liquid chromatography-mass spectrometry combined method (LC-MS/MS). The GC-TEA method has the defects of complicated pretreatment steps, low detection efficiency and low sensitivity, and is difficult to meet the detection requirement of the flue-cured tobacco type cigarette with low TSNAs content. The GC-MS/MS method has higher sensitivity and selectivity, can simplify the pretreatment step and improve the accuracy of the analysis result, but the flue gas sample is easy to cause the pollution of the gas chromatographic column and increase the maintenance cost. The LC-MS/MS method has the problems of serious interference, low sensitivity, low selectivity and the like in the analysis of a flue gas sample with a complex matrix. Therefore, it is very important to find an analysis method capable of accurately measuring the trace TSNAs in the mainstream smoke of the cigarettes.
The two-dimensional chromatographic technology is a separation system formed by connecting two chromatographic columns which have different separation mechanisms and are mutually independent in series, the two chromatographic columns are used as interfaces through a switching valve or a pressure balancing device, a target substance separated from a one-dimensional column is introduced into the two-dimensional column for secondary analysis through flow path change, the separation capacity can be obviously improved for the analysis of a complex system sample, and the method is widely applied to the fields of proteomics, natural products, polymer analysis and the like.
The ultra-high performance integrated chromatography (UPCC) is developed based on a supercritical fluid chromatography technology, uses supercritical carbon dioxide and a small amount of organic solvent as a mobile phase, and has the advantages of high separation efficiency, wide application range, environmental protection and the like.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide an online two-dimensional chromatography tandem mass spectrometry method for measuring tobacco-specific nitrosamine in cigarette mainstream smoke, which can effectively solve the problems of low content of tobacco-specific nitrosamine, serious matrix interference and difficulty in accurate quantification in cigarette smoke, and is particularly suitable for accurately measuring trace nitrosamine in medium and thin cigarette smoke.
In order to achieve the above objects and other related objects, a first aspect of the present invention provides a method for determining an online two-dimensional chromatography-tandem mass spectrometry of tobacco-specific nitrosamines in mainstream smoke of a cigarette, comprising: the method comprises the steps of collecting and extracting cigarette mainstream smoke to obtain a sample solution, and then carrying out qualitative and quantitative analysis on nitrosamine components in the sample solution by adopting an ultra performance convergence chromatography-liquid chromatography-mass spectrometry (UPCC-HPLC-MS/MS) method.
Preferably, the tobacco specific nitrosamines in the mainstream smoke of the cigarette comprise N-nitrosonornicotine (NNN), 4- (methylnitrosoamino) -l- (3-bipyridyl) -1-butanone (NNK), N-Nitrosoanabasine (NAB) and N-Nitrosoanatabine (NAT).
Preferably, the method for obtaining the sample solution after the cigarette mainstream smoke is captured and extracted comprises the following steps:
1) selecting cigarette, igniting and then smoking, and trapping cigarette smoke by adopting a filter disc trapping mode;
2) and adding an ammonium acetate aqueous solution and an internal standard solution into the filter disc, oscillating, extracting and filtering to obtain a sample solution.
Preferably, in step 1), the selected number of the cigarette rods is 20/time.
Preferably, in step 1), the smoking is performed using a multi-channel carousel smoking machine. More preferably, the multi-hole carousel smoking machine is a 20-hole carousel smoking machine. The 20-channel rotary disc type smoking machine can smoke 20 cigarettes at a time.
Preferably, in step 1), the filter is a cambridge filter. The diameter of the cambridge filter disc is 92 mm.
Preferably, in step 1), said trapped cigarette smoke is trapped according to the method specified in standard ISO 3308: 2000.
Preferably, in step 2), the ammonium acetate (NH)4Ac) was added to the aqueous solution at a concentration of 0.1 mol/L. The ammonium acetate aqueous solution is obtained by weighing ammonium acetate, completely dissolving the ammonium acetate with water, transferring the ammonium acetate into a volumetric flask, and adding water to a constant volume.
Preferably, in the step 2), the adding amount of the ammonium acetate aqueous solution is 10-30mL/20 cigarette smoke. More preferably, the addition amount of the ammonium acetate aqueous solution is 20mL/20 cigarette smoke.
Preferably, in the step 2), the addition amount of the internal standard solution is 50-150 μ L per 20 cigarettes. More preferably, the internal standard solution is added in an amount of 100 μ L per 20 cigarettes.
Preferably, in step 2), the internal standard solution is NNN-d4、NNK-d4、NAT-d4、NAB-d4The methanol solution of (1). The internal standard solution is prepared bySeparately weighing NNN-d4、NNK-d4、NAT-d4、NAB-d4Adding methanol to constant volume to obtain the product.
More preferably, NNN-d in the internal standard solution4、NNK-d4、NAT-d4、NAB-d4The concentration of (B) is 1-3. mu.g/mL. Most preferably, NNN-d in the internal standard solution4、NNK-d4、NAT-d4、NAB-d4The concentrations of (A) and (B) were all 2. mu.g/mL.
More preferably, the NNN-d4、NNK-d4、NAT-d4、NAB-d4Is a deuterated reagent. In particular, the NNN-d4、NNK-d4、NAT-d4、NAB-d4Respectively deuterated N-nitrosonornicotine, deuterated 4- (methylnitrosoamino) -l- (3-pyridyl) -1-butanone, deuterated N-nitrosoanatabine and deuterated N-nitrosoanabasine.
The NNN-d4、NNK-d4、NAT-d4、NAB-d4Is a commercial reagent, has stable chemical property and is suitable for being used as an internal standard.
Preferably, in step 2), the equipment adopted for the oscillatory extraction is a vortex mixing oscillator.
Preferably, in step 2), the time for shaking extraction is 20-40 min. More preferably, the time of the shaking extraction is 30 min.
Preferably, in step 2), the filtration is membrane filtration. More preferably, the filter is a 0.22 μm ptfe filter.
Preferably, the qualitative and quantitative analysis of the nitrosamine component in the sample solution by using ultra performance integrated chromatography-liquid chromatography-mass spectrometry (UPCC-HPLC-MS/MS) comprises the following steps:
A) preparing a standard solution: respectively taking standards of NAB, NAT, NNN and NNK, adding methanol to prepare stock solution, adding internal standard solution into the stock solution, and adopting ammonium acetate aqueous solution to fix volume to prepare standard solution;
B) and (3) qualitative and quantitative analysis: respectively carrying out ultra-high performance combined chromatography-liquid chromatography-mass spectrometry on the sample solution and the standard solution prepared in the step A), carrying out fraction through combined chromatography, then trapping through a Trap column, eluting Trap column trapping components, then separating through liquid chromatography, carrying out mass spectrometry, comparing retention time and mass spectrometry scanning to carry out MRM (MRM qualitative determination), determining 4 nitrosamine components in the sample solution, and carrying out MRM quantitative determination by adopting an internal standard curve method to obtain the content of the 4 nitrosamine components in the sample solution.
Preferably, in step A), the concentration of NAB, NAT, NNN, NNK in the stock solution is 1 μ g/ml.
Preferably, in the step A), the concentration range of NAB, NAT, NNN and NNK in the standard solution is 0.1-100 ng/mL.
Preferably, in the step A), the concentration of the internal standard solution added into the standard solution is 10 ng/mL. The internal standard solution is the same as the internal standard solution used in the sample solution.
Preferably, in step B), the determination conditions of the ultra-high performance phase-combination chromatography are as follows:
a chromatographic column: an amino chromatography column; column temperature: 30-50 ℃; back pressure control pressure: 1500-; sample introduction amount: 0.5-1.5 μ L; flow rate: 0.2-0.4 mL/min; mobile phase A: supercritical CO2(ii) a Mobile phase B: methanol containing 0.1-0.3% ammonia water; gradient elution; mobile phase of compensation pump: pure water; flow phase flow rate of compensation pump: 0.05-0.15 mL/min.
More preferably, the determination conditions of the ultra-high performance phase-compatible chromatography are as follows:
a chromatographic column: 1-amino anthracene chromatographic column (1-AA) (column length 100mm X inner diameter 2.1mm, particle size 1.7 μm); column temperature: 40 ℃; back pressure control pressure: 2000 psi; sample introduction amount: 1 mu L of the solution; flow rate: 0.3 mL/min; mobile phase A: supercritical CO2(ii) a Mobile phase B: methanol containing 0.2% ammonia; gradient elution; mobile phase of compensation pump: pure water; flow phase flow rate of compensation pump: 0.1 mL/min.
More preferably, the specific procedure of the gradient elution is:
0-1min, phase A: the volume ratio of the phase B is 95: 5-90: 10;
1-4min, phase A: the volume ratio of the phase B is 90: 10-70: 30, of a nitrogen-containing gas;
4-6min, phase A: the volume ratio of the phase B is 70: 30-70: 30, of a nitrogen-containing gas;
6-6.1min, phase A: the volume ratio of the phase B is 70: 30-95: 5.
preferably, in step B), the Trap column is a C8 chromatography column.
More preferably, the Trap column is an Agilent ZORBAX Eclipse Plus C8 column (column length 50 mm. times. inner diameter 4.6mm, particle size 5 μm).
Preferably, in step B), the determination conditions of the liquid chromatography are:
a chromatographic column: a C18 chromatography column; column temperature: 30-50 ℃; flow rate: 0.3-0.5 mL/min; sample introduction amount: 0.5-10 μ l; mobile phase A: 5-15mmol/L ammonium formate aqueous solution; mobile phase B: is acetonitrile containing 5-15mmol/L ammonium formate; gradient elution.
More preferably, the determination conditions of the liquid chromatography are:
a chromatographic column: poroshell 120EC-C18 column (column length 100 mm. times.inner diameter 3mm, particle size 2.7 μm); column temperature: 40 ℃; flow rate: 0.4 mL/min; sample introduction amount: 1 mul; mobile phase A: 10mmol/L ammonium formate aqueous solution; mobile phase B: is acetonitrile containing 10mmol/L ammonium formate; gradient elution.
More preferably, the specific procedure of the gradient elution is:
0-10min, phase A: the volume ratio of the phase B is 90: 10-90: 10;
10-12min, phase A: the volume ratio of the phase B is 90: 10-50: 50;
12-14min, phase A: the volume ratio of the phase B is 50: 50-30: 70;
14-16min, phase A: the volume ratio of the phase B is 30: 70-0: 100, respectively;
16-18min, phase A: the volume ratio of the phase B is 0: 100-0: 100, respectively;
18-18.1min, phase A: the volume ratio of the phase B is 0: 100-90: 10.
preferably, in step B), the mass spectrum is determined under the following conditions:
an ionization mode: electrospray ion source (H-ESI), positive ion mode; temperature of the drying gas: 190 ℃ to 210 ℃; flow rate of drying gas: 13-15L/min; temperature of sheath gas: 390 ℃ and 410 ℃; flow rate of sheath gas: 11-13L/min; sheath gas pressure: 45-55 psi; spraying voltage: 3000-4000V; taper hole voltage: 0V; the scanning mode is as follows: multiple Reaction Monitoring (MRM); MRM conditions: see table 1.
More preferably, the measurement conditions of the mass spectrum are:
an ionization mode: electrospray ion source (H-ESI), positive ion mode; temperature of the drying gas: 200 ℃; flow rate of drying gas: 14L/min; temperature of sheath gas: 400 ℃; flow rate of sheath gas: 12L/min; sheath gas pressure: 50 psi; spraying voltage: 3500V; taper hole voltage: 0V; the scanning mode is as follows: multiple Reaction Monitoring (MRM); MRM conditions: see table 1.
TABLE 1 Mass Spectrometry MRM conditions
Preferably, in step B), the internal standard curve method comprises the following steps:
A1) adding the stock solution of the 4 nitrosamine components into an internal standard solution, and preparing a series of standard solutions with different concentrations by adopting ammonium acetate aqueous solution to fix the volume;
A2) respectively carrying out UPCC-HPLC-MS/MS analysis on a series of standard solutions with different concentrations in the step A1), obtaining the linear relation between the chromatographic peak area ratio of various nitrosamine components/internal standards and the concentration ratio of the corresponding nitrosamine components/internal standards, drawing corresponding standard working curves, and calculating to obtain a regression equation of the standard working curves of the 4 nitrosamine components;
A3) carrying out UPCC-HPLC-MS/MS analysis on the sample solution, substituting the chromatographic peak area ratio of the obtained 4 kinds of nitrosamine components to the internal standard into the regression equation of the standard working curve of the corresponding nitrosamine component in the step 2), and calculating the content of the corresponding nitrosamine component in the sample solution according to the known concentration of the internal standard.
More preferably, in step a2) or A3), the standard working curve has the ratio of the chromatographic peak area of each nitrosamine component to the internal standard as ordinate (Y-axis) and the concentration ratio of the corresponding nitrosamine component to the internal standard as abscissa (X-axis).
The invention provides an online two-dimensional chromatography tandem mass spectrum system of tobacco-specific nitrosamine in cigarette mainstream smoke, which comprises a combined phase chromatography, a compensation pump, a Trap column, a communication interface, a switching valve, a liquid chromatography and a mass spectrum, wherein the combined phase chromatography comprises a first pump and a first separation column, and the liquid chromatography comprises a second pump and a second separation column; the first pump is connected with the first separation column, the sample outlet end of the second separation column is connected with the mass spectrum through a pipeline, the communication interface is connected with the sample outlet end of the first separation column, the compensating pump and the switching valve through pipelines respectively, and the switching valve is further connected with the sample inlet end of the second pump and the second separation column, the sample inlet end of the Trap column and the sample outlet end through pipelines respectively.
Preferably, the intercommunication interface is the tee bend interface, the intercommunication interface is equipped with 1 st interface, 2 nd interface, 3 rd interface, the 1 st interface links to each other through the appearance end of appearance of pipeline with first separation column, the 2 nd interface links to each other through pipeline and compensating pump, the 3 rd interface links to each other through pipeline and diverter valve.
Preferably, the diverter valve is two-position six-way valve, the diverter valve is equipped with 1 st interface, 2 nd interface, 3 rd interface, 4 th interface, 5 th interface, 6 th interface, 1 st interface links to each other with the intercommunication interface through the pipeline, 2 nd interface links to each other with the introduction port of Trap post through the pipeline, 3 rd interface links to each other with the second pump through the pipeline, 4 th interface links to each other with the introduction port of second separation column through the pipeline, 5 th interface links to each other with the appearance end of leading of Trap post through the pipeline, 6 th interface is the waste liquid mouth.
Preferably, the first pump is a pump dedicated to a conventionally used phase chromatograph. The first pump is a quaternary pump.
Preferably, the second pump is a pump dedicated to a liquid chromatograph that is conventionally used. The second pump is a quaternary pump.
The on-line two-dimensional chromatogram tandem mass spectrum system is matched with a combined chromatogram, a Trap column, a communication interface, a switching valve, a liquid chromatogram and a mass spectrum. The matching means that the components can effectively operate when being used as a whole after being communicated.
The third aspect of the invention provides a use method of an online two-dimensional chromatography tandem mass spectrometry system for tobacco-specific nitrosamines in cigarette mainstream smoke, which comprises the following steps:
a) opening a switching phase chromatograph, wherein a sample solution flows into a first separation column for primary separation under the drive of a first mobile phase introduced by a first pump to obtain a target fraction, the target fraction flows into a1 st interface of a communication interface, and meanwhile, a compensation mobile phase introduced by a compensation pump flows into a2 nd interface of the communication interface, so that the organic phase ratio in the target fraction is reduced after the target fraction and the compensation mobile phase are mixed;
b) opening the switching valve, allowing the target fraction obtained in the step a) to flow out through a3 rd interface of the communication interface, allowing the target fraction to enter from a1 st interface of the switching valve, allowing the target fraction to enter a Trap column through a2 nd interface of the switching valve for trapping, and allowing waste liquid after trapping the target fraction to pass through a 5 th interface of the switching valve and be discharged from a 6 th interface of the switching valve;
c) opening the liquid chromatogram, enabling a second mobile phase introduced by a second pump to flow in through a3 rd interface of the switching valve, enabling the second mobile phase to enter a Trap column through a2 nd interface of the switching valve to elute the trapped target fraction, and enabling the eluent to flow in a second separation column through a 5 th interface of the switching valve and a 4 th interface of the switching valve to perform secondary separation;
d) opening the mass spectrum and flowing the separation liquid obtained in step c) into the mass spectrum for measurement.
Preferably, in step b), the switching valve connects the Trap column and the first separation column for a cutting time of 3.1-5.1 min.
As mentioned above, the method for measuring the on-line two-dimensional chromatography tandem mass spectrum of the tobacco-specific nitrosamine in the mainstream smoke of the cigarette provided by the invention has the following beneficial effects:
(1) according to the method for measuring the on-line two-dimensional chromatography tandem mass spectrum of the tobacco-specific nitrosamine in the cigarette mainstream smoke, an on-line ultra-high performance combined chromatography-liquid chromatography-mass spectrum combined method is constructed according to the orthogonality of two separation principles of combined chromatography and liquid chromatography and is used for analyzing and detecting the tobacco-specific nitrosamine in the cigarette mainstream smoke, the separation of trace components to be measured and interfering substances in a complex matrix can be realized, the matrix effect is greatly reduced, the base line separation of all four TSNAs is realized, and the selectivity and the sensitivity of the separation are greatly improved.
(2) Compared with an off-line two-dimensional system, the method for measuring the on-line two-dimensional chromatography tandem mass spectrum of the tobacco-specific nitrosamine in the mainstream smoke of the cigarettes directly guides the target component eluted in one-dimensional combined phase into the two-dimensional chromatographic column for subsequent separation, and is very simple and convenient to operate. The organic phase proportion of the one-dimensional eluent is reduced through the compensation pump, and the complete cutting of the target substance is ensured through the Trap column trapping and the two-position six-way valve switching mode.
(3) According to the method for measuring the on-line two-dimensional chromatography tandem mass spectrum of the tobacco-specific nitrosamine in the mainstream smoke of the cigarettes, the ultra-high performance combined phase chromatographic column is used as the first-dimensional chromatographic column, and the supercritical carbon dioxide and a small amount of organic solvent are used as mobile phases, so that the organic solvent is saved, and the compatibility with the mobile phase of the second-dimensional liquid chromatography is very good along with the diffusion of the supercritical carbon dioxide in the one-dimensional eluent.
(4) Compared with the prior art, the method for measuring the tobacco-specific nitrosamine in the cigarette mainstream smoke by the online two-dimensional chromatography-tandem mass spectrometry has the advantages of simple sample pretreatment, accurate quantification, high sensitivity and low detection limit, and is suitable for analyzing and measuring the tobacco-specific nitrosamine in various types of cigarette mainstream smoke, particularly for accurately quantifying the trace nitrosamine in the medium-fine cigarette smoke.
Drawings
FIG. 1 is a schematic structural diagram 1A and 1B of an online two-dimensional chromatography tandem mass spectrometry system according to the present invention, wherein FIG. 1A is a Trapping mode; FIG. 1B shows a mass spectrometry mode.
In the figure, the position of the upper end of the main shaft,
11. a first pump for supplying a first fluid to the first pump,
12. the first separation column is provided with a first separation column,
2. the compensation pump is used for compensating the pressure of the air,
31. the first port 1 of the communication port is communicated with the second port,
32. the second port 2 of the connecting port is communicated,
33. the 3 rd port of the communication port is communicated,
41. the 1 st interface of the switching valve is provided,
42. the 2 nd interface of the switching valve,
43. the 3 rd interface of the switching valve,
44. the 4 th interface of the switching valve,
45. the 5 th interface of the switching valve,
46. the 6 th interface of the switching valve,
5. a Trap column is arranged on the upper surface of the substrate,
61. a second pump for supplying a second fluid to the chamber,
62. the second separation column is arranged on the first separation column,
7. and (4) mass spectrometry.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and not to limit the scope of the invention.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
The reagents and the test devices used in the following examples were all those conventionally used and commercially available. The reagents and instruments used were as follows:
1. reagent
Four tobacco-specific nitrosamine standards (NNN, NNK, NAT, NAB) and internal standards (NNN-d)4，NNK-d4，NAT-d4And NAB-d4) From Toronto Research Chemicals, Canada; methanol, acetonitrile, ammonium formate, ammonia, ammonium acetate are all chromatographic grade reagents (Merk company, germany); ultrapure water (self-made by a pure water instrument).
2. Instrument for measuring the position of a moving object
UPCC ultra performance phase chromatography (Waters, USA); 1-AA column (column length 100 mm. times. inner diameter 2.1mm, particle size 1.7 μm) (Waters corporation, USA); a compensation pump (Waters corporation, usa); agilent 1290 liquid chromatography (Agilent corporation, usa); poroshell 120EC-C18 column (column length 100 mm. times.inner diameter 3mm, particle size 2.7 μm) (Agilent, USA); an Agilent ZORBAX Eclipse Plus C8 column (column length 50 mm. times. inner diameter 4.6mm, particle size 5 μm) (Agilent Corp.); two-position, six-way valve (Agilent, usa); RM200A carousel smoking machine (Borwaldt, Germany); g6495 triple quadrupole mass spectrometer (Agilent, usa); sonicators (SONO, switzerland); Milli-Q water purifier (Millpore, USA).
As shown in fig. 1, the invention provides an online two-dimensional chromatography-tandem mass spectrometry system for tobacco-specific nitrosamines in cigarette mainstream smoke, which comprises a phase-combining chromatography, a compensation pump, a Trap column, a communication interface, a switching valve, a liquid chromatography and a mass spectrometry, wherein the phase-combining chromatography comprises a first pump and a first separation column, and the liquid chromatography comprises a second pump and a second separation column; the first pump is connected with the first separation column, the sample outlet end of the second separation column is connected with the mass spectrum through a pipeline, the communication interface is connected with the sample outlet end of the first separation column, the compensating pump and the switching valve through pipelines respectively, and the switching valve is further connected with the sample inlet end of the second pump and the second separation column, the sample inlet end of the Trap column and the sample outlet end through pipelines respectively.
In a preferred embodiment, as shown in fig. 1, the communication interface is a three-way interface, the communication interface is provided with a1 st interface, a2 nd interface and a3 rd interface, the 1 st interface is connected with the sample outlet end of the first separation column through a pipeline, the 2 nd interface is connected with the compensation pump through a pipeline, and the 3 rd interface is connected with the switching valve through a pipeline.
In a preferred embodiment, as shown in fig. 1, the switching valve is a two-position six-way valve, the switching valve is provided with a1 st interface, a2 nd interface, a3 rd interface, a 4 th interface, a 5 th interface and a 6 th interface, the 1 st interface is connected with the communicating interface through a pipeline, the 2 nd interface is connected with the sample injection end of the Trap column through a pipeline, the 3 rd interface is connected with the second pump through a pipeline, the 4 th interface is connected with the sample injection end of the second separation column through a pipeline, the 5 th interface is connected with the sample outlet end of the Trap column through a pipeline, and the 6 th interface is a waste liquid port.
As shown in figure 1, the invention provides a using method of an online two-dimensional chromatography tandem mass spectrometry system of tobacco-specific nitrosamines in cigarette mainstream smoke, which comprises the following steps:
a) as shown in fig. 1A, opening and closing the phase chromatography, wherein a sample solution flows into a first separation column for preliminary separation under the drive of a first mobile phase introduced by a first pump to obtain a target fraction, the target fraction flows into a1 st interface of a communication interface, and a compensation mobile phase introduced by a compensation pump flows into a2 nd interface of the communication interface, so that the organic phase ratio in the target fraction is reduced after the target fraction and the compensation mobile phase are mixed;
b) as shown in fig. 1A, the switching valve is opened, the target fraction obtained in step a) flows out through the 3 rd interface of the communication interface, then enters from the 1 st interface of the switching valve, enters into the Trap column through the 2 nd interface of the switching valve for trapping, and the waste liquid after trapping the target fraction passes through the 5 th interface of the switching valve and is discharged from the 6 th interface of the switching valve;
c) as shown in fig. 1B, the liquid chromatography is opened, the second mobile phase introduced by the second pump flows in through the 3 rd interface of the switching valve, enters the Trap column through the 2 nd interface of the switching valve to elute the trapped target fraction, and the eluent flows in the second separation column through the 5 th interface of the switching valve and the 4 th interface to perform secondary separation;
d) as shown in FIG. 1B, the mass spectrum is opened and the separated liquid obtained in step c) is introduced into the mass spectrum for measurement.
In the step b), the switching valve enables the cutting time of connecting the Trap column and the first separation column to be 3.1-5.1 min.
In a specific embodiment, the method for measuring the on-line two-dimensional chromatography tandem mass spectrum of the tobacco-specific nitrosamine in the mainstream smoke of the cigarette comprises the following detection process.
1. Sample pretreatment
Selecting 20 cigarettes, lighting the cigarettes, trapping smoke of the cigarettes by adopting a filter disc trapping mode, specifically, smoking the cigarettes by adopting a 20-channel turntable type smoking machine, and trapping the smoke according to a method specified in ISO 3308:2000 so that the smoke is adsorbed on a Cambridge filter disc with the diameter of 92 mm. Placing Cambridge filter in 100mL conical flask, adding 10-30mL 0.1mol/L ammonium acetate water solution and 50-150 μ L internal standard solution, placing in vortex mixing oscillator, extracting for 20-40min under oscillation, collecting extractive solution, and filtering with 0.22 μm PTFE filter membraneObtaining a sample solution. The internal standard solution is NNN-d4、NNK-d4、NAT-d4、NAB-d4Methanol solution of (4), NNN-d in internal standard solution4、NNK-d4、NAT-d4、NAB-d4The concentration of (B) is 1-3. mu.g/mL.
2. Qualitative and quantitative analysis
0.1mL of NNN, NNK, NAT and NAB standard substances with the concentration of 0.1mg/mL are respectively transferred and placed in a 10mL volumetric flask, and diluted by methanol to a constant volume to scale to prepare a stock solution with the concentration of 1 mug/mL.
And transferring the stock solutions with different volumes into different 10mL volumetric flasks, transferring 50 mu L internal standard solutions into the volumetric flasks, and metering the volume to a scale by using 0.1mol/L ammonium acetate aqueous solution to prepare standard solutions, wherein the concentration ranges of NAB, NAT, NNN and NNK in the standard solutions are all 0.1-100 ng/mL.
Respectively carrying out ultra-high performance combined chromatography-liquid chromatography-mass spectrometry on the sample solution and the standard solution, carrying out fraction by combined chromatography, then trapping by a Trap column, eluting Trap components by the Trap column, separating by liquid chromatography, carrying out mass spectrometry, comparing retention time and mass spectrometry scanning, carrying out MRM (mass spectrometry) qualitative determination, and determining 4 nitrosamine components in the sample solution.
And (4) performing MRM quantification by adopting an internal standard curve method to obtain the content of 4 nitrosamine components in the sample solution. Specifically, a series of standard solutions with different concentrations are subjected to UPCC-HPLC-MS/MS analysis respectively to obtain the linear relation between the chromatographic peak area ratio of various nitrosamine components/internal standards and the concentration ratio of the corresponding nitrosamine components/internal standards, corresponding standard working curves are drawn, and the regression equation of the standard working curves of the 4 nitrosamine components is calculated. Carrying out UPCC-HPLC-MS/MS analysis on the sample solution, substituting the chromatographic peak area ratio of the obtained 4 kinds of nitrosamine components to the internal standard into the regression equation of the standard working curve of the corresponding nitrosamine component in the step 2), and calculating the content of the corresponding nitrosamine component in the sample solution according to the known concentration of the internal standard. In the standard working curve, the chromatographic peak area ratio of various nitrosamine components to the internal standard is taken as the ordinate (Y axis), and the concentration ratio of the corresponding nitrosamine components to the internal standard is taken as the abscissa (X axis).
Wherein, the measuring conditions of the ultra-high performance phase-matching chromatography are as follows:
a chromatographic column: an amino chromatography column; column temperature: 30-50 ℃; back pressure control pressure: 1500-; sample introduction amount: 0.5-1.5 μ L; flow rate: 0.2-0.4 mL/min; mobile phase A: supercritical CO2(ii) a Mobile phase B: methanol containing 0.1-0.3% ammonia water; gradient elution; mobile phase of compensation pump: pure water; flow phase flow rate of compensation pump: 0.05-0.15 mL/min.
The specific procedure for gradient elution was:
0-1min, phase A: the volume ratio of the phase B is 95: 5-90: 10;
1-4min, phase A: the volume ratio of the phase B is 90: 10-70: 30, of a nitrogen-containing gas;
4-6min, phase A: the volume ratio of the phase B is 70: 30-70: 30, of a nitrogen-containing gas;
6-6.1min, phase A: the volume ratio of the phase B is 70: 30-95: 5.
the Trap column is a C8 chromatographic column.
The measurement conditions of the liquid chromatography are as follows:
a chromatographic column: a C18 chromatography column; column temperature: 30-50 ℃; flow rate: 0.3-0.5 mL/min; sample introduction amount: 0.5-10 μ l; mobile phase A: 5-15mmol/L ammonium formate aqueous solution; mobile phase B: is acetonitrile containing 5-15mmol/L ammonium formate; gradient elution.
The specific procedure for gradient elution was:
0-10min, phase A: the volume ratio of the phase B is 90: 10-90: 10;
10-12min, phase A: the volume ratio of the phase B is 90: 10-50: 50;
12-14min, phase A: the volume ratio of the phase B is 50: 50-30: 70;
14-16min, phase A: the volume ratio of the phase B is 30: 70-0: 100, respectively;
16-18min, phase A: the volume ratio of the phase B is 0: 100-0: 100, respectively;
18-18.1min, phase A: the volume ratio of the phase B is 0: 100-90: 10.
the mass spectrum measurement conditions were:
an ionization mode: electrospray ion source (H-ESI), positive ion mode; temperature of the drying gas: 190 ℃ to 210 ℃; flow rate of drying gas: 13-15L/min; temperature of sheath gas: 390 ℃ and 410 ℃; flow rate of sheath gas: 11-13L/min; sheath gas pressure: 45-55 psi; spraying voltage: 3000-4000V; taper hole voltage: 0V; the scanning mode is as follows: multiple Reaction Monitoring (MRM); MRM conditions: see table 1.
Example 1
1. Sample pretreatment
Selecting 20 cigarettes, lighting the cigarettes, trapping smoke of the cigarettes by adopting a filter disc trapping mode, specifically, smoking the cigarettes by adopting a 20-channel turntable type smoking machine, and trapping the smoke according to a method specified in ISO 3308:2000 so that the smoke is adsorbed on a Cambridge filter disc with the diameter of 92 mm. Placing Cambridge filter in 100mL conical flask, adding 20mL of 0.1mol/L ammonium acetate aqueous solution and 100 μ L of internal standard solution, placing in vortex mixing oscillator, oscillating for 30min, and filtering the extractive solution with 0.22 μm PTEF filter to obtain sample solution. The internal standard solution is NNN-d4、NNK-d4、NAT-d4、NAB-d4Methanol solution of (4), NNN-d in internal standard solution4、NNK-d4、NAT-d4、NAB-d4The concentrations of (A) and (B) were all 2. mu.g/mL.
2. Qualitative and quantitative analysis
0.1mL of NNN, NNK, NAT and NAB standard substances with the concentration of 0.1mg/mL are respectively transferred and placed in a 10mL volumetric flask, and diluted by methanol to a constant volume to scale to prepare a stock solution with the concentration of 1 mug/mL.
Then transferring 1 muL, 2 muL, 5 muL, 10 muL, 20 muL, 50 muL, 100 muL, 200 muL and 1mL of stock solutions into different 10mL volumetric flasks, transferring 50 muL of internal standard solution into different volumetric flasks, and using 0.1mol/L of ammonium acetate aqueous solution to fix the volume to the scale to prepare a series of standard solutions with the concentrations of 0.1, 0.2, 0.5, 1, 2, 5, 10, 20 and 100ng/mL respectively, wherein the concentration of the internal standard solution is 10 ng/mL.
Respectively carrying out ultra-high performance combined chromatography-liquid chromatography-mass spectrometry on the sample solution and the standard solution, carrying out fraction by combined chromatography, then trapping by a Trap column, eluting Trap components by the Trap column, separating by liquid chromatography, carrying out mass spectrometry, comparing retention time and mass spectrometry scanning, carrying out MRM (mass spectrometry) qualitative determination, and determining 4 nitrosamine components in the sample solution.
And (4) performing MRM quantification by adopting an internal standard curve method to obtain the content of 4 nitrosamine components in the sample solution. Specifically, a series of standard solutions with different concentrations are subjected to UPCC-HPLC-MS/MS analysis respectively to obtain the linear relation between the chromatographic peak area ratio of various nitrosamine components/internal standards and the concentration ratio of the corresponding nitrosamine components/internal standards, corresponding standard working curves are drawn, and the regression equation of the standard working curves of the 4 nitrosamine components is calculated. Carrying out UPCC-HPLC-MS/MS analysis on the sample solution, substituting the chromatographic peak area ratio of the obtained 4 kinds of nitrosamine components to the internal standard into the regression equation of the standard working curve of the corresponding nitrosamine component in the step 2), and calculating the content of the corresponding nitrosamine component in the sample solution according to the known concentration of the internal standard. In the standard working curve, the chromatographic peak area ratio of various nitrosamine components to the internal standard is taken as the ordinate (Y axis), and the concentration ratio of the corresponding nitrosamine components to the internal standard is taken as the abscissa (X axis).
Wherein, the measuring conditions of the ultra-high performance phase-matching chromatography are as follows:
a chromatographic column: 1-amino anthracene chromatographic column (1-AA) (column length 100mm X inner diameter 2.1mm, particle size 1.7 μm); column temperature: 40 ℃; back pressure control pressure: 2000 psi; sample introduction amount: 1 mu L of the solution; flow rate: 0.3 mL/min; mobile phase A: supercritical CO2(ii) a Mobile phase B: methanol containing 0.2% ammonia; gradient elution; mobile phase of compensation pump: pure water; flow phase flow rate of compensation pump: 0.1 mL/min.
The specific procedure for gradient elution was:
0-1min, phase A: the volume ratio of the phase B is 95: 5-90: 10;
1-4min, phase A: the volume ratio of the phase B is 90: 10-70: 30, of a nitrogen-containing gas;
4-6min, phase A: the volume ratio of the phase B is 70: 30-70: 30, of a nitrogen-containing gas;
6-6.1min, phase A: the volume ratio of the phase B is 70: 30-95: 5.
the Trap column was an Agilent ZORBAX Eclipse Plus C8 column (column length 50 mm. times. inner diameter 4.6mm, particle size 5 μm).
The measurement conditions of the liquid chromatography are as follows:
a chromatographic column: poroshell 120EC-C18 column (column length 100 mm. times.inner diameter 3mm, particle size 2.7 μm); column temperature: 40 ℃; flow rate: 0.4 mL/min; sample introduction amount: 1 mul; mobile phase A: 10mmol/L ammonium formate aqueous solution; mobile phase B: is acetonitrile containing 10mmol/L ammonium formate; gradient elution.
The specific procedure for gradient elution was:
0-10min, phase A: the volume ratio of the phase B is 90: 10-90: 10;
10-12min, phase A: the volume ratio of the phase B is 90: 10-50: 50;
12-14min, phase A: the volume ratio of the phase B is 50: 50-30: 70;
14-16min, phase A: the volume ratio of the phase B is 30: 70-0: 100, respectively;
16-18min, phase A: the volume ratio of the phase B is 0: 100-0: 100, respectively;
18-18.1min, phase A: the volume ratio of the phase B is 0: 100-90: 10.
the mass spectrum measurement conditions were:
an ionization mode: electrospray ion source (H-ESI), positive ion mode; temperature of the drying gas: 200 ℃; flow rate of drying gas: 14L/min; temperature of sheath gas: 400 ℃; flow rate of sheath gas: 12L/min; sheath gas pressure: 50 psi; spraying voltage: 3500V; taper hole voltage: 0V; the scanning mode is as follows: multiple Reaction Monitoring (MRM); MRM conditions: see table 1.
Example 2
As shown in step 2 of the above example 1, the stock solutions were accurately removed, 50. mu.L of the internal standard solution was added, and the volume was scaled up with 0.1mol/L ammonium acetate aqueous solution to prepare a series of standard solutions with concentrations of 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, and 100ng/mL, respectively, wherein the internal standard concentration was 10 ng/mL.
Performing UPCC-HPLC-MS/MS analysis on the prepared series of standard solutions with different concentrations respectively, taking the chromatographic peak area ratio of 4 nitrosamine components to an internal standard as a ordinate (Y axis), taking the concentration ratio of the corresponding nitrosamine components to the internal standard as an abscissa (X axis), performing regression analysis, and obtaining a good linear relation of a regression equation, wherein the correlation coefficient R of the regression equation of the 4 nitrosamine components is greater than 0.999 within a linear concentration range of 0.1-100ng/mL, and the obtained good dynamic linear range is provided, and specific data are shown in Table 2.
TABLE 2
Figure BDA0002217229550000131
Example 3
A ramuscule tobacco sample is selected as an object, and the day repeatability, the recovery rate and the detection limit of the method are examined. As described above in example 1, the pretreatment and analysis were carried out in parallel 9 times under the same conditions in one day and in the middle of the day, respectively, and the reproducibility of the method was examined as the relative standard deviation of the measurement results. Adding high, medium and low concentration TSNAs standard solution in the extraction process of the fine smoke sample, and calculating the standard recovery rate of different concentrations. Meanwhile, internal standards with different concentrations are added in the process of extracting the ramuscule smoke samples, the 10-time signal-to-noise ratio is taken as the limit of quantitation (LOQ) of the method, and specific data are shown in Table 3. As can be seen from Table 3, the method has high recovery rate, good accuracy and repeatability, and high sensitivity, and can meet the quantitative requirements.
Table 3 quantitation limit, repeatability and recovery results for the methods
Figure BDA0002217229550000141
Example 4
The tobacco-specific nitrosamines in the mainstream smoke of 10 different types of finished cigarettes were analyzed and detected in the pretreatment and analysis processes as described in example 1, and the results are shown in table 4. Wherein the content (ng/count) of the sample is determined by measuring the concentration (ng/mL) of the sample by 20mL/20 counts. As can be seen from Table 4, the method of the present invention can accurately determine the content of nitrosamine in the smoke of a cigarette, especially the trace amount of nitrosamine in the smoke of a fine-cut flue-cured cigarette.
TABLE 410 tobacco-specific nitrosamine content in the mainstream smoke of finished cigarettes
Figure BDA0002217229550000142
Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims (15)

1. A method for measuring tobacco-specific nitrosamines in cigarette mainstream smoke by online two-dimensional chromatography tandem mass spectrometry comprises the following steps: the method comprises the steps of collecting and extracting cigarette mainstream smoke to obtain a sample solution, and then performing qualitative and quantitative analysis on nitrosamine components in the sample solution by adopting an ultra-high performance combined chromatography-liquid chromatography-mass spectrometry combined method.
2. The method for determining tobacco-specific nitrosamines in cigarette mainstream smoke according to claim 1, wherein said tobacco-specific nitrosamines in cigarette mainstream smoke comprise N-nitrosonornicotine, 4- (methylnitrosoamino) -l- (3-pyridinylmethyl) -1-butanone, N-nitrosoanabasine and N-nitrosoanatabine.
3. The method for measuring the on-line two-dimensional chromatography tandem mass spectrometry of the tobacco-specific nitrosamines in the cigarette mainstream smoke according to claim 1, wherein the method for obtaining the sample solution after the cigarette mainstream smoke is captured and extracted comprises the following steps:
1) selecting cigarette, igniting and then smoking, and trapping cigarette smoke by adopting a filter disc trapping mode;
2) and adding an ammonium acetate aqueous solution and an internal standard solution into the filter disc, oscillating, extracting and filtering to obtain a sample solution.
4. The method for measuring the on-line two-dimensional chromatographic tandem mass spectrometry of the tobacco-specific nitrosamines in the mainstream smoke of the cigarette according to claim 3, wherein the step 1) comprises any one or more of the following conditions:
A1) the selected number of the cigarettes is 20;
A2) the suction is carried out by adopting a multi-channel rotating disc type smoking machine;
A3) the filter disc is a Cambridge filter disc, and the diameter of the Cambridge filter disc is 92 mm.
5. The method for measuring the on-line two-dimensional chromatographic tandem mass spectrometry of the tobacco-specific nitrosamines in the mainstream smoke of the cigarette according to claim 3, wherein the step 1) comprises any one or more of the following conditions:
B1) the ammonium acetate (NH)4Ac) aqueous solution with the concentration of 0.05-0.15 mol/L;
B2) the adding amount of the ammonium acetate aqueous solution is 10-30mL per 20 cigarettes;
B3) the adding amount of the internal standard solution is 50-150 mu L per 20 cigarettes;
B4) the internal standard solution is NNN-d4、NNK-d4、NAT-d4、NAB-d4The methanol solution of (4);
B5) the time of the oscillation extraction is 20-40 min;
B6) the filtration is membrane filtration.
6. The method for determining the on-line two-dimensional chromatography tandem mass spectrometry of the tobacco-specific nitrosamines in the mainstream smoke of the cigarette according to claim 1, wherein the qualitative and quantitative analysis of the nitrosamine components in the sample solution is performed by adopting an ultra-performance convergence chromatography-liquid chromatography-mass spectrometry combined method, and the method comprises the following steps:
A) preparing a standard solution: respectively taking standards of NAB, NAT, NNN and NNK, adding methanol to prepare stock solution, adding internal standard solution into the stock solution, and adopting ammonium acetate aqueous solution to fix volume to prepare standard solution;
B) and (3) qualitative and quantitative analysis: respectively carrying out ultra-high performance combined chromatography-liquid chromatography-mass spectrometry on the sample solution and the standard solution prepared in the step A), carrying out fraction through combined chromatography, then trapping through a Trap column, eluting Trap column trapping components, then separating through liquid chromatography, carrying out mass spectrometry, comparing retention time and mass spectrometry scanning to carry out MRM (MRM qualitative determination), determining 4 nitrosamine components in the sample solution, and carrying out MRM quantitative determination by adopting an internal standard curve method to obtain the content of the 4 nitrosamine components in the sample solution.
7. The method for determining the on-line two-dimensional chromatography tandem mass spectrometry of the tobacco-specific nitrosamines in the mainstream smoke of the cigarette according to claim 6, wherein in the step B), the determination conditions of the ultra-high performance affinity chromatography are as follows:
a chromatographic column: an amino chromatography column; column temperature: 30-50 ℃; back pressure control pressure: 1500-; sample introduction amount: 0.5-1.5 μ L; flow rate: 0.2-0.4 mL/min; mobile phase A: supercritical CO2(ii) a Mobile phase B: methanol containing 0.1-0.3% ammonia water; gradient elution; mobile phase of compensation pump: pure water; flow phase flow rate of compensation pump: 0.05-0.15 mL/min.
8. The method for determining the on-line two-dimensional chromatography tandem mass spectrometry of the tobacco-specific nitrosamines in the mainstream smoke of the cigarette according to claim 7, wherein the specific procedure of gradient elution is as follows:
0-1min, phase A: the volume ratio of the phase B is 95: 5-90: 10;
1-4min, phase A: the volume ratio of the phase B is 90: 10-70: 30, of a nitrogen-containing gas;
4-6min, phase A: the volume ratio of the phase B is 70: 30-70: 30, of a nitrogen-containing gas;
6-6.1min, phase A: the volume ratio of the phase B is 70: 30-95: 5.
9. the method for measuring the on-line two-dimensional chromatographic tandem mass spectrometry of the tobacco-specific nitrosamines in the mainstream smoke of the cigarette according to claim 6, wherein in the step B), the measuring conditions of the liquid chromatogram are as follows:
a chromatographic column: a C18 chromatography column; column temperature: 30-50 ℃; flow rate: 0.3-0.5 mL/min; sample introduction amount: 0.5-10 μ l; mobile phase A: 5-15mmol/L ammonium formate aqueous solution; mobile phase B: is acetonitrile containing 5-15mmol/L ammonium formate; gradient elution.
10. The method for determining the on-line two-dimensional chromatography tandem mass spectrometry of the tobacco-specific nitrosamines in the mainstream smoke of the cigarette according to claim 9, wherein the specific procedure of gradient elution is as follows:
0-10min, phase A: the volume ratio of the phase B is 90: 10-90: 10;
10-12min, phase A: the volume ratio of the phase B is 90: 10-50: 50;
12-14min, phase A: the volume ratio of the phase B is 50: 50-30: 70;
14-16min, phase A: the volume ratio of the phase B is 30: 70-0: 100, respectively;
16-18min, phase A: the volume ratio of the phase B is 0: 100-0: 100, respectively;
18-18.1min, phase A: the volume ratio of the phase B is 0: 100-90: 10.
11. the method for measuring the on-line two-dimensional chromatographic tandem mass spectrum of the tobacco-specific nitrosamines in the mainstream smoke of the cigarette according to claim 6, wherein in the step B), the measuring conditions of the mass spectrum are as follows:
an ionization mode: electrospray ion source H-ESI, positive ion mode; temperature of the drying gas: 190 ℃ to 210 ℃; flow rate of drying gas: 13-15L/min; temperature of sheath gas: 390 ℃ and 410 ℃; flow rate of sheath gas: 11-13L/min; sheath gas pressure: 45-55 psi; spraying voltage: 3000-4000V; taper hole voltage: 0V; the scanning mode is as follows: multiple reactions monitor MRM.
12. An online two-dimensional chromatography tandem mass spectrum system of tobacco-specific nitrosamines in cigarette mainstream smoke is characterized by comprising a combined phase chromatography, a compensation pump, a Trap column, a communication interface, a switching valve, a liquid chromatography and a mass spectrum, wherein the combined phase chromatography comprises a first pump and a first separation column, and the liquid chromatography comprises a second pump and a second separation column; the first pump is connected with the first separation column, the sample outlet end of the second separation column is connected with the mass spectrum through a pipeline, the communication interface is connected with the sample outlet end of the first separation column, the compensating pump and the switching valve through pipelines respectively, and the switching valve is further connected with the sample inlet end of the second pump and the second separation column, the sample inlet end of the Trap column and the sample outlet end through pipelines respectively.
13. The on-line two-dimensional chromatography-tandem mass spectrometry system for tobacco-specific nitrosamines in cigarette mainstream smoke according to claim 12, wherein the communication port is a three-way port, the communication port is provided with a1 st port, a2 nd port and a3 rd port, the 1 st port is connected with the sample outlet end of the first separation column through a pipeline, the 2 nd port is connected with the compensation pump through a pipeline, and the 3 rd port is connected with the switching valve through a pipeline.
14. The on-line two-dimensional chromatography tandem mass spectrometry system for tobacco specific nitrosamines in cigarette mainstream smoke according to claim 12, wherein the switching valve is a two-position six-way valve, the switching valve is provided with a1 st interface, a2 nd interface, a3 rd interface, a 4 th interface, a 5 th interface and a 6 th interface, the 1 st interface is connected with the communicating interface through a pipeline, the 2 nd interface is connected with the sample introduction end of the Trap column through a pipeline, the 3 rd interface is connected with the second pump through a pipeline, the 4 th interface is connected with the sample introduction end of the second separation column through a pipeline, the 5 th interface is connected with the sample discharge end of the Trap column through a pipeline, and the 6 th interface is a waste liquid port.
15. A use method of an online two-dimensional chromatography tandem mass spectrometry system for tobacco-specific nitrosamine in cigarette mainstream smoke comprises the following steps:
a) opening a switching phase chromatograph, wherein a sample solution flows into a first separation column for primary separation under the drive of a first mobile phase introduced by a first pump to obtain a target fraction, the target fraction flows into a1 st interface of a communication interface, and meanwhile, a compensation mobile phase introduced by a compensation pump flows into a2 nd interface of the communication interface, so that the organic phase ratio in the target fraction is reduced after the target fraction and the compensation mobile phase are mixed;
b) opening the switching valve, allowing the target fraction obtained in the step a) to flow out through a3 rd interface of the communication interface, allowing the target fraction to enter from a1 st interface of the switching valve, allowing the target fraction to enter a Trap column through a2 nd interface of the switching valve for trapping, and allowing waste liquid after trapping the target fraction to pass through a 5 th interface of the switching valve and be discharged from a 6 th interface of the switching valve;
c) opening the liquid chromatogram, enabling a second mobile phase introduced by a second pump to flow in through a3 rd interface of the switching valve, enabling the second mobile phase to enter a Trap column through a2 nd interface of the switching valve to elute the trapped target fraction, and enabling the eluent to flow in a second separation column through a 5 th interface of the switching valve and a 4 th interface of the switching valve to perform secondary separation;
d) opening the mass spectrum and flowing the separation liquid obtained in step c) into the mass spectrum for measurement.
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