CN112816283A - Preparation method of tobacco specific N-nitrosamine standard substance - Google Patents
Preparation method of tobacco specific N-nitrosamine standard substance Download PDFInfo
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- CN112816283A CN112816283A CN202011605216.2A CN202011605216A CN112816283A CN 112816283 A CN112816283 A CN 112816283A CN 202011605216 A CN202011605216 A CN 202011605216A CN 112816283 A CN112816283 A CN 112816283A
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- 241000208125 Nicotiana Species 0.000 title claims abstract description 146
- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 146
- 239000000126 substance Substances 0.000 title claims abstract description 141
- XKLJHFLUAHKGGU-UHFFFAOYSA-N nitrous amide Chemical compound ON=N XKLJHFLUAHKGGU-UHFFFAOYSA-N 0.000 title claims abstract description 121
- 238000002360 preparation method Methods 0.000 title claims abstract description 31
- XKABJYQDMJTNGQ-VIFPVBQESA-N n-nitrosonornicotine Chemical compound O=NN1CCC[C@H]1C1=CC=CN=C1 XKABJYQDMJTNGQ-VIFPVBQESA-N 0.000 claims abstract description 62
- BXYPVKMROLGXJI-JTQLQIEISA-N 3-[(2s)-1-nitrosopiperidin-2-yl]pyridine Chemical compound O=NN1CCCC[C@H]1C1=CC=CN=C1 BXYPVKMROLGXJI-JTQLQIEISA-N 0.000 claims abstract description 58
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- CIJBKNZDKBKMFU-UHFFFAOYSA-N N-nitrosomethanamine Chemical compound CNN=O CIJBKNZDKBKMFU-UHFFFAOYSA-N 0.000 claims description 10
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- 229920000435 poly(dimethylsiloxane) Polymers 0.000 claims description 9
- ZJOFAFWTOKDIFH-UHFFFAOYSA-N 3-(1-nitroso-3,6-dihydro-2h-pyridin-2-yl)pyridine Chemical compound O=NN1CC=CCC1C1=CC=CN=C1 ZJOFAFWTOKDIFH-UHFFFAOYSA-N 0.000 claims description 8
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 3
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 claims description 3
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G01—MEASURING; TESTING
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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Abstract
The invention provides a preparation method of a tobacco specific N-nitrosamine standard substance and the tobacco specific N-nitrosamine standard substance prepared by the same. The tobacco specific N-nitrosamine is at least one of N-nitrosonornicotine, 4- (methylnitroso) -1- (3-pyridyl) -1-butanone, N-nitrosonoranatine and N-nitrosoanabasine. The preparation method comprises the following steps: 1. the method comprises the following steps of candidate detection, 2 solvent verification, 3 tobacco specific N-nitrosamine standard substance preparation and subpackaging, 4 tobacco specific N-nitrosamine standard substance detection, 5 component valuing in the standard substance, and 6 uncertainty evaluation of the component content valuing result in the standard substance.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry, and particularly relates to a preparation method of a tobacco specific N-nitrosamine standard substance, the tobacco specific N-nitrosamine standard substance prepared by the method and application thereof.
Background
Tobacco-specific N-nitrosamines (TSNAs) are carcinogens widely existing in tobacco and cigarette smoke, and may be generated by nitrosation of tobacco alkaloids under the action of microorganisms, or may be generated by nitrite of nitrate under the action of microorganisms, and the nitrite and other nitrogen oxides react with the tobacco alkaloids to generate TSNAs. There are 8 TSNAs identified so far, and 4 of N-nitrosonornicotine (NNN), 4- (methylnitroso) -1- (3-pyridyl) -1-butanone (NNK), N-Nitrosonoranatabine (NAT), N-Nitrosoanabasine (NAB) and the like are generally concerned. N-nitrosonornicotine (NNN) and 4- (methylnitroso) -1- (3-pyridyl) -1-butanone (NNK) have been shown to have a clear mammalian carcinogenic potential. Epidemiological investigations have shown that humans are a susceptible population for cancer caused by TSNAs compounds. Thus, various methods for determining TSNAs in tobacco and smoke have emerged in the art, such as ultraviolet-visible spectrophotometry (UV-Vis), Thin Layer Chromatography (TLC), Capillary Electrophoresis (CE), Gas Chromatography (GC), High Performance Liquid Chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS); among them, liquid chromatography-tandem mass spectrometry is most commonly used and is also an analysis method specified in the ISO standard. Regardless of the assay method employed, the preparation of the standard solution is an important factor causing deviation in the results, in addition to the test environmental conditions, equipment status, and skill of the operator.
Currently, there is no registration of TSNAs standard substance on the International Data Bank on verified Reference Materials, COMAR, https:// www.comar.bam.de/home/logic. php) and the national standard substance resource sharing platform (https:// www.ncrm.org.cn /). When the domestic tobacco quality supervision and inspection departments at all levels and the middle tobacco technical center measure TSNAs, analytical standard substance preparation working curves provided by reagent companies are mainly adopted. The difficulty in obtaining the standard substance is solved by the commercial analysis standard substance, but the consistency of the final detection result is seriously influenced by the product purity difference of different reagent suppliers and the operation error of an inspector during weighing. More importantly, the current analysis standard does not evaluate TSNAs in a standard fixed value and uncertainty manner, cannot guarantee the value tracing during the test, cannot be used for the calibration evaluation of a standard method and equipment and the difference analysis between analysis systems, and seriously influences the daily detection and analysis work.
Therefore, in order to more conveniently measure the TSNAs content of tobacco and cigarette products and ensure the value traceability and comparability of analysis data, a preparation method of a tobacco-specific N-nitrosamine standard substance is needed to be developed, and the tobacco-specific N-nitrosamine standard substance is prepared and obtained.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a preparation method of a tobacco specific N-nitrosamine standard substance, and the tobacco specific N-nitrosamine standard substance prepared by the method.
Therefore, the invention adopts the following technical scheme:
a method for preparing a tobacco-specific N-nitrosamine standard substance selected from at least one of N-nitrosonornicotine (NNN), 4- (methylnitroso) -1- (3-pyridyl) -1-butanone (NNK), N-Nitrosonoranatabine (NAT), and N-Nitrosoanabasine (NAB); the preparation method comprises the following steps:
1. detection of candidates
1-1. Structure verification of candidates
Measuring the infrared spectrum, the ultraviolet spectrum, the mass spectrum, the nuclear magnetic resonance carbon spectrum and the nuclear magnetic resonance hydrogen spectrum of the candidate, and verifying the structure of the candidate according to the spectrum information; if the verification is passed, the following steps are carried out;
1-2. determination of candidate impurities
Determining the moisture content W of the candidateMoisture contentInorganic element content WInorganic substanceAnd residual solvent content WSolvent(s)；
1-3. determination of mutual impurity status of candidates
Determining whether each candidate contains other tobacco specific N-nitrosamines as candidates;
1-4. determination of purity of candidate
Determination of the purity P of each candidateMeasurement ofSubstituting the following formula to calculate the purity P of the candidatePurity of：
PPurity of＝(1-WMoisture content-WElement(s)-WSolvent(s))×PMeasurement of
If P isPurity ofWithin the range of purity and accuracy provided by the candidate analysis certificate, taking the purity of the analysis certificate as the purity of the candidate; if P isPurity ofOut of the range of purity and accuracy provided by the candidate certificate of analysis, then P is calculatedPurity ofIs the purity of the candidate;
2. solvent validation
The solvent is chromatographic grade methanol, and the solvent is analyzed by adopting liquid chromatography-tandem mass spectrometry to determine whether the candidate substance is contained; if the determination result shows that the candidate is not contained, the verification is passed;
3. preparation and subpackage of tobacco specific N-nitrosamine standard substance
Taking the candidate detected in the step 1, taking methanol verified in the step 2 as a solvent, and preparing a standard substance by adopting a weight-volume method according to the purity of the candidate determined in the step 1-4 under the environment that the temperature is 20 +/-2 ℃ and the relative humidity is 60% +/-5%; then subpackaging the standard substance, immediately sealing to obtain the subpackaged specific N-nitrosamine standard substance for the tobacco, and storing in a refrigerator at 0-4 ℃ in a dark place;
4. test of tobacco-specific N-nitrosamine standard substance
4-1. uniformity test of specific N-nitrosamine standard substance in tobacco
According to the requirement of extracting the number of units in the uniformity evaluation of JJF 1343-2012 'general principle and statistical principle of standard substance definite value', extracting a specified number of samples from the subpackaged tobacco specific N-nitrosamine standard substance obtained in the step 3, measuring the content of the tobacco specific N-nitrosamine in each sample by using a high performance liquid chromatography, performing statistical analysis by using an F test method, and if F is detected, performing statistical analysis by using an F test methodComputingLess than F0.05(the inter-group freedom degree and the intra-group freedom degree) shows that the significant difference does not exist between the measured samples under the confidence probability level of 95%, and the specific N-nitrosamine standard substance of the tobacco after subpackaging is uniform;
4-2. testing the stability of the specific N-nitrosamine standard substance in tobacco
The test for the stability of the specific N-nitrosamine standard substance in the tobacco comprises short-term stability test at 60 ℃ in a dark place for 14 days and long-term stability test at 0-4 ℃ in a dark place for 12 months;
5. definite value of each component in tobacco specific N-nitrosamine standard substance
Determining the content of each component in the specific N-nitrosamine standard substance of the tobacco by a single laboratory by adopting a single reference method;
6. uncertainty evaluation of quantitative result of content of each component in tobacco specific N-nitrosamine standard substance
Uncertainty components of the quantitative result of the content of each component in the specific N-nitrosamine standard substance of the tobacco come from uncertainty introduced by uniformity, uncertainty introduced by short-term stability, uncertainty introduced by long-term stability and uncertainty introduced in the quantitative process; the uncertainty components of each component are evaluated separately and the standard uncertainty is calculated.
Preferably, the tobacco specific N-nitrosamines are selected from all of N-nitrosonornicotine (NNN), 4- (methylnitroso) -1- (3-pyridyl) -1-butanone (NNK), N-Nitrosonoranatabine (NAT), and N-Nitrosoanabasine (NAB).
Preferably, in the step 1-1, the candidate substance is subjected to gas chromatography-mass spectrometry to obtain a mass spectrogram; the gas chromatography-mass spectrometry conditions were:
the chromatographic column is
35ms (35% diphenyl/65% dimethylpolysiloxane), specification: 30m (length) × 0.25mm (inner diameter) × 0.25 μm (film thickness); temperature programming: maintaining at 80 deg.C for 1.0min, and then increasing to 280 deg.C at a rate of 10 deg.C/min and maintaining for 20 min; a split sample injection mode is adopted, and the split ratio is 50: 1; the sample injection amount is 1.0 mu L; the solvent cutting time is 10.0 min; high-purity helium (the purity is more than or equal to 99.999%) is taken as carrier gas; the flow rate is 1.0 mL/min; the temperatures of the sample inlet, the ion source and the interface are respectively 280 ℃, 230 and 280 ℃; the mass spectrum scanning mode is full scanning, and the scanning range is m/z, which is 40-250.
Preferably, in the step 1-2, the moisture of the candidate is measured by a Karl Fischer moisture meter.
Preferably, in the step 1-2, the inorganic elements in the candidate are determined by inductively coupled plasma mass spectrometry; the analysis parameters set were:
radio frequency voltage: 1500V; analysis mode: collision; rotation speed of peristaltic pump: 0.1 rps; helium gas is taken as collision gas flow, and the flow rate is as follows: 4.5 mL/min; argon gas is used as carrier gas, and the flow rate is as follows: 1.10 mL/min; collision cell stabilization time: 30 s; the quantitative method comprises the following steps: an internal standard method; integration time: 0.3 s; internal standard elements:45Sc、72Ge、115in and209Bi。
preferably, in the step 1-2, the solvent residue in each candidate substance is quantitatively analyzed by gas chromatography-mass spectrometry; the specific analysis conditions were:
a chromatographic column: a capillary column special for HP-VOC; specification: 60m (length) × 0.32mm (inner diameter) × 1.8 μm (film thickness); carrier gas: helium (He), constant flow mode, flow 2.0 mL/min; sample inlet temperature: 180 ℃; no shunt sampling; temperature programming: keeping at 40 deg.C for 2min, heating to 200 deg.C at 4 deg.C/min, and keeping for 10 min; mass spectrum interface temperature: 220 ℃; an ionization mode: an electron impact source (EI); ion source temperature: 230 ℃; ionization energy: 70 eV; temperature of the quadrupole rods: 150 ℃; firstly, analyzing a sample by using a full-scanning monitoring mode, and then accurately quantifying 26 typical alcohol, ketone, aromatic hydrocarbon and ester compounds by using a selective ion monitoring mode, wherein the scanning range is 29 amu-350 amu.
Further preferably, in the step 1-2, each candidate is prepared into a solution with the concentration of 1mg/mL by taking triacetin as a solvent, and the quantitative analysis is carried out by gas chromatography-mass spectrometry.
Preferably, in said steps 1-3, it is determined whether or not there are other tobacco specific N-nitrosamines as candidates by subjecting each candidate to gas chromatography-flame ionization (GC-FID) analysis.
Further preferably, in the steps 1-3, each candidate is prepared into a solution with the concentration of 1mg/mL by using methanol as a solvent, and gas chromatography-flame ionization analysis is carried out.
More preferably, in the steps 1 to 3, the analysis conditions of the gas chromatography-flame ionization analysis are as follows:
column DB-35MS elastic capillary chromatography (35% diphenyl/65% dimethyl polysiloxane), specification 30m (length) × 0.25mm (internal diameter) × 0.25 μm (film thickness); sample inlet temperature: 250 ℃; sample introduction amount: 1 mu L, split-flow sample injection, the split-flow ratio is 30: 1; carrier gas: nitrogen (purity is more than or equal to 99.999%), constant flow rate: 1.0 mL/min; temperature rising procedure: the initial temperature is 80 ℃, the temperature is kept for 1min, the temperature is increased to 280 ℃ at the speed of 10 ℃/min, and the temperature is kept for 20 min; a detector: a flame ionization detector; the flow rates of hydrogen, air and tail gas blowing of the detector are respectively 45, 350 and 3mL/min, and the tail gas blowing is nitrogen; the detector temperature was 250 ℃.
Preferably, in the steps 1 to 4, each candidate is prepared into a solution with the concentration of 1mg/mL by using methanol as a solvent, and gas chromatography-flame ionization analysis and high performance liquid chromatography-ultraviolet analysis are carried out.
More preferably, in the steps 1 to 4, the analysis conditions of the gas chromatography-flame ionization analysis are as follows:
column DB-35MS elastic capillary chromatography (35% diphenyl/65% dimethyl polysiloxane), specification 30m (length) × 0.25mm (internal diameter) × 0.25 μm (film thickness); sample inlet temperature: 250 ℃; sample introduction amount: 1 mu L, split-flow sample injection, the split-flow ratio is 30: 1; carrier gas: nitrogen (purity is more than or equal to 99.999%), constant flow rate: 1.0 mL/min; temperature rising procedure: the initial temperature is 80 ℃, the temperature is kept for 1min, the temperature is increased to 280 ℃ at the speed of 10 ℃/min, and the temperature is kept for 20 min; a detector: a flame ionization detector; the flow rates of hydrogen, air and tail gas blowing of the detector are respectively 45, 350 and 3mL/min, and the tail gas blowing is nitrogen; the detector temperature was 250 ℃.
Further preferably, in the steps 1 to 4, the chromatographic conditions of the high performance liquid chromatography-ultraviolet analysis are as follows: the analytical column is a Poroshell 120EC-C18 chromatographic column with specification of 100mm (length) x 4.6mm (inner diameter) and particle size of 4 μm; the column temperature was 40 ℃; the mobile phase consists of two phases of water and methanol, wherein the water is phase A, the methanol is phase B, a gradient elution mode is adopted, and the elution procedure is as follows: 30% (v/v) phase B at time t of 0min, keeping for 4 min, linearly increasing the volume ratio of phase B to 90% (v/v) within 3min, keeping for 5min, decreasing the volume of phase B to 30% (v/v) within 1min, and balancing for 5 min; the flow rate is 1.0 mL/min; the ultraviolet detection wavelength is as follows: n-nitrosonornicotine 357nm, 4- (methylnitrosamine) -1- (3-pyridyl) -1-butanone 339nm, N-nitrosoanatabine 361 nm; the amount of sample was 5. mu.L.
Preferably, in the step 2, the chromatographic conditions of the liquid chromatography-tandem mass spectrometry are as follows:
chromatographic column Poroshell 120EC-C18, specification 100mm (length) × 3.0mm (internal diameter), particle size 2.7 μm; the sample injection amount is 5 mu L; the column temperature is 40 ℃; mobile phase: solvent A was 10mM ammonium formate solution and solvent B was 0.1% formic acid (v/v) in acetonitrile, and the elution was performed in a gradient with the following procedure:
the flow rate was 0.4 mL/min.
Preferably, in the step 2, the multiple reaction monitoring ion pairs adopted by the liquid chromatography-tandem mass spectrometry are as follows: 178.1>148.2(15) for N-nitrosonornicotine (NNN), 208.1>122.1(16) for 4- (methylnitroso) -1- (3-pyridyl) -1-butanone (NNK), 190.1>160.1(15) for N-Nitrosonoranatabine (NAT), and 192.1>162.2(17) for N-Nitrosoanabasine (NAB).
Preferably, the specific operation of step 3 is:
weighing a certain amount of the candidate detected in the step 1 by using a calibrated first-grade balance in the environment with the temperature of 20 +/-2 ℃ and the relative humidity of 60% +/-5%, adding the methanol verified in the step 2 into the same calibrated A-grade 100mL volumetric flask, completely dissolving, fixing the volume to the scale by using the methanol, and shaking up to obtain the specific N-nitrosamine standard substance of the tobacco; transferring 0.5mL of the tobacco specific N-nitrosamine standard substance into a brown glass bottle with a threaded opening and a tapered groove at the bottom and a volume of 1.5mL, immediately sealing the bottle, and storing the bottle in a refrigerator at 0-4 ℃ in a dark place.
Preferably, in the step 4-1, the chromatographic conditions of the high performance liquid chromatography are as follows: the analytical column is a Poroshell 120EC-C18 chromatographic column with specification of 100mm (length) x 4.6mm (inner diameter) and particle size of 4 μm; the column temperature was 40 ℃; the mobile phase is methanol/water (40/60, v/v), and the flow rate is 1.0 mL/min; the detector is an ultraviolet detector, and the ultraviolet detection wavelength is 357nm for N-nitrosonornicotine, 339nm for 4- (methylnitrosamine) -1- (3-pyridyl) -1-butanone, and 361nm for N-nitrosoanabasine and N-nitrosoanabasine; the amount of sample was 5. mu.L.
Preferably, in the step 4-2, the specific operation of the short-term stability test at 60 ℃ in the dark for 14 days comprises:
A. taking out the subpackaged N-nitrosamine standard substances special for the tobacco, which are obtained in the step 3 and stored in a refrigerator, from 5 xn bottles, wherein N is 1-5, and N is 3 is more preferable; putting 4 Xn bottles in a drying oven with a set temperature of 60 ℃, measuring the content of the N-nitrosamine specific to the tobacco by the high performance liquid chromatography for the rest N bottles, and repeatedly measuring each bottle of sample for 3 times;
B. taking N bottles out of the oven on days 5, 7, 10 and 14 respectively, measuring the content of the tobacco-specific N-nitrosamine by high performance liquid chromatography, and repeating the measurement for 3 times for each bottle of sample;
C. the results of the short-term stability measurement of N-nitrosamines specific to each tobacco were fitted to a straight line with x as the horizontal axis representing time (day) and y as the measured content, and the equation of the straight line y was obtained as b + ax, and the standard deviation s of the straight line was calculated by the following equation2And uncertainty s of slope(a)，
Standard deviation of straight line:
uncertainty of slope:
if it is not
The slope was insignificant, indicating good short term stability.
Preferably, in the step 4-2, the specific operation of the long-term stability test at 0-4 ℃ in the dark for 12 months comprises:
A. taking out m bottles of the subpackaged N-nitrosamine standard substances, obtained in the step 3 and stored in a refrigerator, from 0 month, 2 months, 4 months, 7 months and 12 months respectively, wherein m is 1-5, and more preferably m is 3; measuring the content of the tobacco-specific N-nitrosamine by high performance liquid chromatography, and repeatedly measuring each bottle of sample for 3 times;
B. the long-term stability measurement results of N-nitrosamines specific to each tobacco were fitted to a straight line with x as the horizontal axis representing time (month) and y as the measured content, and the equation of the straight line y was obtained as b + ax, and the standard deviation s of the straight line was calculated by the following equation2And uncertainty s of slope(a)，
Standard deviation of straight line:
uncertainty of slope:
if it is not
The slope was insignificant, indicating good long-term stability.
Preferably, in the step 5, the fixed value of each component in the tobacco specific N-nitrosamine standard substance is calculated by weighing weight, purity of the candidate substance and fixed volume; wherein the definite value of N-nitrosonornicotine (NNN) is 1.00mg/mL, the definite value of 4- (methylnitroso) -1- (3-pyridyl) -1-butanone (NNK) is 1.01mg/mL, the definite value of N-Nitrosonoranatabine (NAT) is 1.01mg/mL, and the definite value of N-Nitrosoanabasine (NAB) is 1.02 mg/mL.
Preferably, in step 6, the calculated uncertainty of relative expansion of each component is 3% (k ═ 2).
The invention also aims to provide a tobacco-specific N-nitrosamine standard substance in the methanol prepared by the preparation method; the tobacco specific N-nitrosamine is at least one selected from N-nitrosonornicotine (NNN), 4- (methylnitroso) -1- (3-pyridyl) -1-butanone (NNK), N-Nitrosonoranatabine (NAT) and N-Nitrosoanabasine (NAB).
Preferably, the tobacco specific N-nitrosamines are all N-nitrosonornicotine (NNN), 4- (methylnitroso) -1- (3-pyridyl) -1-butanone (NNK), N-Nitrosonoranatabine (NAT), and N-Nitrosoanabasine (NAB).
As a preferred embodiment, the present invention provides 4 tobacco-specific N-nitrosamine standard substances in methanol prepared by the above preparation method, wherein the tobacco-specific N-nitrosamines and their content fixed values are as follows:
the content definite value of N-nitrosonornicotine (NNN) is 1.00mg/mL, the content definite value of 4- (methylnitroso) -1- (3-pyridyl) -1-butanone (NNK) is 1.01mg/mL, the content definite value of N-Nitrosonoranatine (NAT) is 1.01mg/mL, and the content definite value of N-Nitrosoanabasine (NAB) is 1.02 mg/mL.
In addition, the third purpose of the invention is to provide the application of the tobacco-specific N-nitrosamine standard substance in the methanol in detecting the tobacco-specific N-nitrosamine in the tobacco and/or tobacco products.
In the present specification, the "candidate" refers to each tobacco-specific N-nitrosamine that is commercially available and is prepared for use in the preparation of the standard substance of the present invention.
The tobacco specific N-nitrosamine standard substance in the methanol fills up the blank of the tobacco specific N-nitrosamine standard substance, and improves the accuracy and the detection efficiency of detecting the content of the tobacco specific N-nitrosamine in the tobacco and the tobacco products.
Drawings
The invention will be further described with reference to the accompanying drawings.
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of N-nitrosonornicotine (NNN).
FIG. 2 is a nuclear magnetic resonance carbon spectrum of N-nitrosonornicotine (NNN).
FIG. 3 is a NMR spectrum of 4- (methylnitrosamine) -1- (3-pyridyl) -1-butanone (NNK).
FIG. 4 is a NMR carbon spectrum of 4- (methylnitrosamine) -1- (3-pyridyl) -1-butanone (NNK).
FIG. 5 is a nuclear magnetic resonance hydrogen spectrum of N-Nitrosoanatabine (NAT).
FIG. 6 is a nuclear magnetic resonance carbon spectrum of N-Nitrosoanatabine (NAT).
FIG. 7 is the nuclear magnetic resonance hydrogen spectrum of N-Nitrosoanabasine (NAB).
Figure 8 is the nuclear magnetic resonance carbon spectrum of N-nitrosoanabasine (NAB.
FIG. 9 is an infrared spectrum of N-nitrosonornicotine (NNN).
FIG. 10 is an infrared spectrum of 4- (methylnitrosamine) -1- (3-pyridyl) -1-butanone (NNK).
FIG. 11 is an infrared spectrum of N-Nitrosoanatabine (NAT).
FIG. 12 is the infrared spectrum of N-Nitrosoanabasine (NAB).
FIG. 13 is an ultraviolet spectrum of N-nitrosonornicotine (NNN).
FIG. 14 is an ultraviolet spectrum of 4- (methylnitrosamine) -1- (3-pyridyl) -1-butanone (NNK).
FIG. 15 is a UV spectrum of N-Nitrosoanatabine (NAT).
Figure 16 is the ultraviolet spectrum of N-Nitrosoanabasine (NAB).
FIG. 17 is a mass spectrum of N-nitrosonornicotine (NNN), where 17A is the mass spectrum of the N-nitrosonornicotine (NNN) tested and 17B is the mass spectrum of the NIST standard library.
FIG. 18 is a mass spectrum of 4- (methylnitrosamine) -1- (3-pyridyl) -1-butanone (NNK), where 18A is the mass spectrum of the subject 4- (methylnitrosamine) -1- (3-pyridyl) -1-butanone (NNK) and 18B is the mass spectrum in the NIST standard library.
FIG. 19 is a mass spectrum of N-Nitrosoanatabine (NAT), where 19A is the mass spectrum of the N-Nitrosoanatabine (NAT) tested and 19B is the mass spectrum from the NIST standard library.
FIG. 20 is a mass spectrum of N-Nitrosoanabasine (NAB), wherein 20A is the mass spectrum of the tested N-Nitrosoanabasine (NAB) and 20B is the mass spectrum in NIST standard spectral library.
FIG. 21 is a superposition of gas chromatography-flame ionization profiles of 4 TSNAs.
FIG. 22 is a liquid chromatography-tandem mass spectrometry spectrum of chromatographically pure methanol.
FIG. 23 is a graph of the short term stability of NNK in "4 tobacco specific N-nitrosamine standards in methanol".
FIG. 24 is a graph of the long-term and short-term stability of NNK in "4 tobacco specific N-nitrosamine standards in methanol".
Detailed Description
The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagent materials used in the following examples are all commercially available products unless otherwise specified. Wherein, the purchase conditions of partial reagents and equipment are as follows:
n-nitrosonornicotine (NNN): TRC, Canada, indicating 99.97 + -2.0% purity;
4- (methylnitroso) -1- (3-pyridyl) -1-butanone (NNK): TRC, Canada, indicating 99.96 + -2.0% purity;
N-Nitrosonoranatabine (NAT): TRC, Canada, indicating 99.82 + -2.0% purity;
N-Nitrosoanabasine (NAB): TRC, Canada, indicating 99.80. + -. 2.0% purity;
methanol: chromatographic grade, Merck, germany;
AQUARANAL coulomt AG (anolyte): shanghai Anpu experiment science and technology Co., Ltd;
nuclear magnetic resonance spectrometer: model Avance-III 300, Bruker;
fourier transform infrared spectrometer: model number Tensor 27, Bruker company;
uv-vis spectrometer: model 8453, Agilent corporation;
gas chromatography-mass spectrometer: model 7890A-5975C, Agilent corporation;
gas chromatography-mass spectrometer: model 7890B-5977A, Agilent corporation;
inductively coupled plasma mass spectrometer: model 7900, Agilent corporation;
gas chromatography-flame ionization combined instrument: model 7890A, Agilent corporation;
high performance liquid chromatography-ultraviolet analyzer: agilent 1260Infinity II, which comprises a G7111A1260 quaternary infusion pump, a G7114A1260 ultraviolet detector and a G7129A1260 autosampler;
liquid chromatography-tandem mass spectrometer: model number Agilent 1200-AB 5500, Agilent Corp;
karr fischer moisture meter: model C30, METTLERTODO.
EXAMPLE 1 preparation of 4 tobacco-specific N-nitrosamine Standard substances in methanol
4 tobacco-specific N-nitrosamine (hereinafter referred to as "TSNAs") candidates were N-nitrosonornicotine (hereinafter referred to as "NNN"), 4- (methylnitroso) -1- (3-pyridyl) -1-butanone (hereinafter referred to as "NNK"), N-nitrosonoranatabine (hereinafter referred to as "NAT"), and N-nitrosoanabasine (hereinafter referred to as "NAB"); 4 tobacco-specific N-nitrosamine standard substances in the methanol were prepared by the following steps:
1. detection of candidates
1-1. Structure verification of candidates
The infrared spectrum, ultraviolet spectrum, mass spectrum, nuclear magnetic resonance carbon spectrum and nuclear magnetic resonance hydrogen spectrum of the candidate are measured, and compared with data in domestic and foreign Standard spectrum libraries such as a national Standard Reference Database (NIST Standard Reference Database), a Japanese Organic compound Spectrum Database (SDBS) and a national chemical professional Database (the institute of Organic chemistry in Shanghai of Chinese academy of sciences undertakes), and the like, the structure of the candidate is confirmed.
1-1-1 nuclear magnetic resonance spectroscopy
The results of the analysis of 4TSNAs candidates are shown in FIGS. 1 to 8, and tables 1 to 4. The results show that the chemical shift and intensity ratio of the characteristic peaks in the nuclear magnetic resonance carbon spectrum and the hydrogen spectrum of the 4TSNAs are consistent with the type and number ratio of the chemical environment of carbon and hydrogen atoms in candidate molecules.
TABLE 1 NMR spectra of NNN candidates
TABLE 2 NMR spectra of NNK candidates
TABLE 3 NMR spectra of NAT candidates
TABLE 4 NMR spectra of NAB candidates
1-1-2 Infrared Spectroscopy
Infrared spectroscopy (coating) was performed on the 4TSNAs candidate using a Fourier transform infrared spectrometer. The ir spectra of the 4TSNAs candidates are shown in fig. 9-12.
FIG. 9 is an infrared spectrum of NNN candidates, where: the absorption peak related to the pyridine ring is 3075-3020cm-1C-H stretching vibration absorption peak, 1620 and 1590cm-1At the position of the heteroaromatic ring stretching vibration absorption peak, 920-720cm-1The out-of-plane bending vibration absorption peak of the aromatic hydrogen is positioned; the absorption peak related to N-NO is 1500--1The absorption peak of the stretching vibration of N ═ O; the absorption peak related to the pyrrolidinyl group is 1360-1310cm-1The C-N stretching vibration absorption peak at position, 2925-2850cm-1Of (C is a-CH)2-absorption peak of stretching vibration, 1470cm-1Of (C is a-CH)2In-plane bending stretching vibration absorption peak, 725--1Of (C is a-CH)2-out-of-plane bending stretching vibration absorption peak.
FIG. 10 is an infrared spectrum of an NNK candidate, wherein: the absorption peak related to the pyridine ring is 3075-3020cm-1C-H stretching vibration absorption peak, 1620 and 1590cm-1The heteroaromatic ring stretching vibration absorption peak at 1360-1310cm-1The C-N stretching vibration absorption peak at position 920--1The out-of-plane bending vibration absorption peak of the aromatic hydrogen is positioned; the absorption peak related to N-NO is 1500--1The absorption peak of the stretching vibration of N ═ O; the absorption peak related to carbonyl is 1700-1600cm-1C ═ O strong stretching vibration absorption peak; and- (CH)2)3The relevant absorption peak is 2925-2850cm-1Of (C is a-CH)2-absorption peak of stretching vibration, 1470cm-1Of (C is a-CH)2In-plane bending stretching vibration absorption peak, 725--1Of (C is a-CH)2-an out-of-plane bending stretching vibration absorption peak; and-CH3The relevant absorption peak is 2960-2870cm-1At 1460-1380cm-1And the in-plane bending stretching vibration absorption peak is formed.
Fig. 11 is an infrared spectrum of NAT candidates, in which: the absorption peak related to the pyridine ring is 3075-3020cm-1C-H stretching vibration absorption peak, 1620 and 1590cm-1At the position of the heteroaromatic ring stretching vibration absorption peak, 920-720cm-1The out-of-plane bending vibration absorption peak of the aromatic hydrogen is positioned; the absorption peak related to N-NO is 1500--1The absorption peak of the stretching vibration of N ═ O; the absorption peak related to the tetrahydropyridine ring is 1660cm-1C ═ C stretching vibration absorption peak, 2925--1Of (C is a-CH)2-absorption peak of stretching vibration, 1470cm-1Of (C is a-CH)2In-plane bending stretching vibration absorption peak, 1360--1The C-N stretching vibration absorption peak at position, 725-720cm-1Of (C is a-CH)2-out-of-plane bending stretching vibration absorption peak.
Fig. 12 is an infrared spectrum of NAB candidates, where: the absorption peak related to the pyridine ring is 3075-3020cm-1C-H stretching vibration absorption peak, 1620 and 1590cm-1At the position of the heteroaromatic ring stretching vibration absorption peak, 920-720cm-1The out-of-plane bending vibration absorption peak of the aromatic hydrogen is positioned; the absorption peak related to N-NO is 1500--1The absorption peak of the stretching vibration of N ═ O; the absorption peak related to the piperidine ring is 2925-2850cm-1Of (C is a-CH)2-absorption peak of stretching vibration, 1470cm-1Of (C is a-CH)2In-plane bending stretching vibration absorption peak, 1360--1The C-N stretching vibration absorption peak at position, 725-720cm-1Of (C is a-CH)2-out-of-plane bending stretching vibration absorption peak.
1-1-3. ultraviolet spectral analysis
Ultraviolet spectrum analysis (methanol as solvent) was performed on 4TSNAs candidates using an ultraviolet-visible spectrometer, and the ultraviolet spectra are shown in FIGS. 13-16, respectively.
The maximum ultraviolet absorption wavelengths of the 4TSNAs candidates are 357nm (NNN), 339nm (NNK), 361Nm (NAT) and 361Nm (NAB) respectively according to ultraviolet spectrum analysis. The uv absorption spectra of the 4TSNAs candidates were consistent with their characteristic functional groups.
1-1-4. Mass Spectrometry
Gas chromatography-mass spectrometry analysis was performed on 4TSNAs candidates using a gas chromatography-mass spectrometer. The specific gas chromatography-mass spectrometry analysis conditions are as follows: gas chromatography-Mass spectrometer model 7890A-5975C (Agilent). The MassHunter workstation is adopted to complete the control of the instrument and the data acquisition and processing; chromatography-mass spectrometry conditions: the chromatographic column is
35ms (35% diphenyl/65% dimethylpolysiloxane), specification: 30m (length) × 0.25mm (inner diameter) × 0.25 μm (film thickness); temperature programming: maintaining at 80 deg.C for 1.0min, and then increasing to 280 deg.C at a rate of 10 deg.C/min and maintaining for 20 min; adopting a split sample injection mode, wherein the split ratio is 50:1, the sample injection amount is 1.0 mu L, and the solvent cutting time is 10.0 min; taking high-purity He (the purity is more than or equal to 99.999%) as a carrier gas, and controlling the flow rate to be 1.0 mL/min; the temperatures of the sample inlet, the ion source and the interface are respectively 280 ℃, 230 and 280 ℃; the mass spectrum scanning mode is full scanning, and the scanning range is m/z, which is 40-250. The mass spectra of the 4TSNAs candidates are shown in FIGS. 17-20.
As can be seen in FIG. 17, the NNN candidates and the mass spectra in the NIST standard library all have characteristic ions (m/z)177, 147, 118, 105, and 78, and the relative intensities of the characteristic ions are consistent. In addition, other fragment ion information of the two mass spectrograms is consistent.
As can be seen from FIG. 18, the NNK candidates and the mass spectra in the NIST standard library all have characteristic ions (m/z)177, 146, 106 and 78, and the relative intensities of the characteristic ions are consistent. In addition, other fragment ion information of the two mass spectrograms is consistent.
As can be seen from fig. 19, the mass spectra of the NAT candidate and the NIST standard library all have characteristic ions (m/z)159, 144, 130, 117, 105, 92 and 78, and the relative intensities of the characteristic ions are consistent. In addition, other fragment ion information of the two mass spectrograms is consistent.
As can be seen from fig. 20, the NAB candidates and the mass spectra in the NIST standard library all have characteristic ions (m/z)161, 132, 118, 105, 92 and 78, and the relative intensities of the characteristic ions are consistent. In addition, other fragment ion information of the two mass spectrograms is consistent.
Thus, the structure of the commercially available 4TSNAs candidates is consistent with that stated in the certificate of analysis.
Small knot
Through nuclear magnetism, infrared, ultraviolet and mass spectrometry, the information of functional group information, relative molecular mass, structural formula and the like of 4 commercially available TSNAs candidates is verified to be consistent with the statement in the certificate of analysis.
1-2. determination of candidate impurities
Determination of moisture content W in 4TSNAs candidatesMoisture contentInorganic element content WInorganic substanceAnd residual solvent content WSolvent(s)。
1-2-1. moisture content WMoisture contentMeasurement of (2)
Moisture content was measured in 4TSNAs candidates using a Karl Fischer moisture meter (METTLERTOCO model C30). A pyridine-free Karl Fischer reagent (AQUARANAL Coulomat AG) is used as a titration reagent, and a reaction solvent in a reaction tank is titrated to be anhydrous. After dissolving a certain weight of the candidate in the reaction solvent, the end point is titrated with the karl fischer reagent. Each candidate was analyzed 6 times repeatedly, and the moisture content data obtained after averaging were:
NNN：Wmoisture content＝1.6％，
NNK：WMoisture content＝1.0％，
NAT：WMoisture content＝0.3％，
NAB：WMoisture content＝0.7％。
1-2-2. inorganic element content WInorganic substanceMeasurement of (2)
4TSNAs candidate solutions with a concentration of 0.2mg/mL were prepared with ultrapure water, and the inorganic elements in the candidates were measured by inductively coupled plasma mass spectrometry (Agilent 7900). Instrumental analysis parameters: radio frequency voltage, 1500V; analysis mode, collision; peristaltic pump speed, 0.1 rps; collision gas flow rate (He), 4.5 mL/min; carrier gas flow rate (Ar), 1.10 mL-min; collision cell settling time, 30 s; quantitative method, internal standard method; integration time, 0.3 s; an internal standard element, wherein the internal standard element,45Sc、72Ge、115in and209Bi。
the detection result shows that the tested candidate contains different inorganic elements with different types and contents, mainly contains trace elements such as Na, Cr, Mg, Ni and the like, has the total amount of 139.00-3274.16ng/g, and has small proportion. Therefore, the inorganic element content is considered to be extremely low, which does not affect the calculation of the purity of the TSNAs candidate and is therefore ignored.
1-2-3. residual solvent content WSolvent(s)Measurement of (2)
Using glyceryl triacetate as a solvent, preparing each candidate into a solution with the concentration of 1mg/mL, and quantitatively analyzing the solvent residue in each candidate by using a gas chromatography-mass spectrometer (Agilent 7890B-5977A). Specific analytical conditions, column: the specification of the capillary column special for HP-VOC is 60m (length) multiplied by 0.32mm (inner diameter) multiplied by 1.8 mu m (film thickness); carrier gas: helium (He), constant flow mode, flow 2.0 mL/min; sample inlet temperature: 180 ℃; no shunt sampling; temperature programming: keeping at 40 deg.C for 2min, heating to 200 deg.C at 4 deg.C/min, and keeping for 10 min; mass spectrum interface-assisting temperature: 220 ℃; an ionization mode: an electron impact source (EI); ion source temperature: 230 ℃; ionization energy: 70 eV; temperature of the quadrupole rods: 150 ℃; firstly, analyzing a sample by using a full-scanning monitoring mode (the scanning range is 29 amu-350 amu); then, accurately quantifying 26 typical alcohol, ketone, aromatic hydrocarbon and ester compounds in a selected ion monitoring mode; ion selection parameter principle: selecting ions with higher specificity and response from mass spectrum ion fragments of each solvent residue as quantitative ions; while another 1 fragment ion was selected as the auxiliary qualitative ion. Retention times and ion selection parameters for typical solvent residues are shown in table 5.
TABLE 5 retention time and ion selection parameters for typical solvent residues
The measurement results are shown in Table 6.
TABLE 6 measurement of solvent residue in candidates (unit: μ g/g)
The results in table 6 show: 1) the 4 candidates only contain ethanol and isopropanol with different contents, and the candidates are probably introduced in the artificial synthesis process; 2) the total solvent residue in the 4 candidates is 64.3-296.3 mug/g, i.e. the mass percent of the solvent residue is 0.0064-0.030%. Therefore, the residual solvent content is considered to be low, and the residual solvent content does not affect the calculation of the purity of the candidate, so that the residual solvent content is ignored.
1-3. determination of mutual impurity status of candidates
Determining whether each candidate contains other tobacco specific N-nitrosamines as candidates.
Gas chromatography-flame ionization (GC-FID) analysis was performed on the 4 candidates. Each candidate was formulated in a 1mg/mL solution in methanol and each candidate solution was analyzed in triplicate. Specific analysis conditions were as follows: the instrument model is Agilent 7890A in the United states; column DB-35MS elastic capillary chromatography column [ 35% diphenyl/65% dimethylpolysiloxane, 30m (length) × 0.25mm (inner diameter) × 0.25 μm (film thickness) ]; sample inlet temperature: 250 ℃; sample introduction amount: 1 mu L, split-flow sample injection (split-flow ratio is 30: 1); carrier gas: nitrogen (purity is more than or equal to 99.999%), constant flow rate: 1.0 mL/min; temperature rising procedure: the initial temperature is 80 ℃, the temperature is kept for 1min, the temperature is increased to 280 ℃ at the speed of 10 ℃/min, and the temperature is kept for 20 min; the detector is a flame ionization detector; the detector hydrogen, air and tail blow (nitrogen) flow rates were 45, 350 and 3mL/min, respectively; the detector temperature was 250 ℃.
The results are shown in FIG. 21 and Table 7. Fig. 21 shows: none of the other three candidates were found in each candidate.
TABLE 7.4 Table of the mutual impurities of the candidates
1-4. determination of purity of candidate
Quantitative determination of purity P of each candidateMeasurement ofSubstituting the following formula to calculate the purity P of the candidatePurity of：
PPurity of＝(1-WMoisture content-WElement(s)-WSolvent(s))×PMeasurement of
1-4-1 gas chromatography-flame ionization quantitative determination
Gas chromatography-flame ionization analysis (GC-FID) was performed on the Agilent 7890A system, usa. Each candidate was formulated as a 1mg/mL solution in methanol. Specific analysis conditions were as follows: column DB-35MS elastic capillary chromatography column [ 35% diphenyl/65% dimethylpolysiloxane, 30m (length) × 0.25mm (inner diameter) × 0.25 μm (film thickness) ]; sample inlet temperature: 250 ℃; sample introduction amount: 1 mu L, split-flow sample injection (split-flow ratio is 30: 1); carrier gas: nitrogen (purity is more than or equal to 99.999%), constant flow rate: 1.0 mL/min; temperature rising procedure: the initial temperature is 80 ℃, the temperature is kept for 1min, the temperature is increased to 280 ℃ at the speed of 10 ℃/min, and the temperature is kept for 20 min; the detector is a flame ionization detector; the detector hydrogen, air and tail blow (nitrogen) flow rates were 45, 350 and 3mL/min, respectively; the detector temperature was 250 ℃.
Repeatedly analyzing each candidate solution for 6 times by area normalization method, and averaging to obtain the purity (P) of NNN, NNK, NAT and NABMeasurement of) 99.84%, 99.79%, 99.52% and 99.44%, respectively.
The actual purity of the 4 candidates should be the result after deduction of all impurities (moisture, inorganic elements and solvent residues), i.e. PPurity of＝(1-WMoisture content-WElement(s)-WSolvent(s))×PMeasurement ofThe effect on the purity calculation due to inorganic elements and solvent residues was negligible and the moisture did not respond under GC-FID analysis conditions. Therefore, 4 kindsThe purity of the candidate can be expressed as the result after moisture subtraction and the data is shown in table 8. It can be found that the purity test result of the gas chromatography is within the range of purity and accuracy provided by the candidate analysis certificate, which preliminarily indicates that the purity value and accuracy value given by the purchased candidate analysis report are accurate and reliable.
TABLE 8 gas chromatography area normalization purity test results (%)
1-4-2 high performance liquid chromatography-ultraviolet (HPLC-UV) quantitative determination
High performance liquid chromatography-ultraviolet (HPLC-UV) analysis was done on Agilent 1260Infinity II, usa. The whole set of analysis system comprises a G7111A1260 quaternary infusion pump, a G7114A1260 ultraviolet detector and a G7129A1260 autosampler. Each candidate was formulated as a 1mg/mL solution in methanol. Chromatographic conditions are as follows: the analytical column is a Poroshell 120EC-C18 chromatographic column with specification of 100mm (length) x 4.6mm (inner diameter) and particle size of 4 μm; the column temperature was 40 ℃; the mobile phase is methanol (B phase) -water (A phase), a gradient elution mode is adopted, 30% (v/v) B is kept for 4 minutes when t is 0min, then the linear rise is carried out to 90% (v/v) B within 3min, the maintenance is carried out for 5min, and the mobile phase is returned to the original proportion within 1min and is balanced for 5 min; the flow rate was 1.0 mL/min. The ultraviolet detection wavelength is 357nm (NNN)/339nm (NNK)/361nm (NAT and NAB), and the sample injection amount is 5 mu L.
Repeatedly analyzing each candidate solution for 6 times by area normalization method, and averaging to obtain the purity (P) of NNN, NNK, NAT and NABMeasurement of) 99.70%, 99.76%, 99.57% and 99.43%, respectively. The actual purity of the 4 candidates should be the result after deduction of all impurities (moisture, inorganic elements and solvent residues), i.e. PPurity of＝(1-WMoisture content-WElement(s)-WSolvent(s))×PMeasurement of. The influence of inorganic elements and solvent residues on the purity calculation is negligible, and the moisture does not respond under the condition of HPLC-UV analysis; therefore, the purity of the 4 candidates can be determined by subtracting the water contentThe results are shown in Table 9. It can be found that the liquid chromatography purity test result is within the range of purity and accuracy provided by the candidate analysis certificate, which indicates that the purity value and accuracy value marked by the purchased candidate analysis certificate are accurate and reliable.
TABLE 9 liquid chromatography area normalization method purity test results (%)
The purity of each of the commercially available candidates can be determined by the purity indicated by the certificate of analysis, combining the results of the tests 1-4-1 and 1-4-2.
2. Solvent validation
And (4) analyzing the chromatographic grade methanol by using a liquid chromatography-tandem mass spectrum, and if the determination result shows that the candidate substance is not contained in the chromatographic grade methanol, the verification is passed.
Specific conditions of liquid chromatography-tandem mass spectrometry: the instrument model is Agilent 1200-AB 5500; chromatographic column Poroshell 120EC-C18, specification 100mm (length) × 3.0mm (internal diameter), particle size 2.7 μm; the sample injection amount is 5 mu L; the column temperature is 40 ℃; mobile phase: 10mM ammonium formate solution (solvent A), acetonitrile (0.1% formic acid, v/v, solvent B); the flow rate is 0.4 mL/min; gradient of mobile phase:
multiple reaction monitoring parameters for 4 candidates: NNN 178.1>148.2(15), NNK 208.1>122.1(16), NAT 190.1>160.1(15), NAB 192.1>162.2 (17).
The liquid chromatography-tandem mass spectrometry spectrum is shown in FIG. 22. The figures show that: the chromatogram of this chromatographically pure methanol contains no such 4TSNAs, indicating that the selected solvent is non-interfering with the target.
3. Preparation and subpackage of tobacco specific N-nitrosamine standard substance
Taking the candidate detected in the step 1, preparing a standard substance by a weight-volume method by taking methanol verified in the step 2 as a solvent and taking the purity of a commercial candidate marker as a standard under the environment of the temperature of 20 +/-2 ℃ and the relative humidity of 60 +/-5%; and then subpackaging the standard substance, immediately sealing to obtain the subpackaged specific N-nitrosamine mixed solution standard substance for the tobacco, and storing in a refrigerator at 0-4 ℃ in a dark place.
Specifically, a certain amount of the candidate detected in step 1 is weighed by a calibrated (r) grade balance and transferred to the same calibrated A grade 100mL volumetric flask, and the specific weighed mass is shown in Table 10. And (3) adding the chromatographic pure methanol verified in the step (2) to completely dissolve the N-nitrosamine, and fixing the volume to a scale with the methanol and shaking up to obtain 4 specific N-nitrosamine standard substances in the methanol for the tobacco. Considering the amount of the standard solution required by the preparation of the series of standard solutions when measuring TSNAs in the mainstream smoke of tobaccos, tobacco products and cigarettes, 0.5mL of the '4 tobacco-specific N-nitrosamine standard substances in methanol' is transferred into a threaded-mouth brown glass bottle (the volume is 1.5-mL) with a conical groove at the bottom, the bottle is immediately sealed, and 192 bottles of '4 tobacco-specific N-nitrosamine standard substances in methanol' samples are obtained in total and are stored in a refrigerator at 0-4 ℃ in a dark place.
TABLE 10.4 candidate weighing masses
4. Test of tobacco-specific N-nitrosamine standard substance
4-1. Standard substance homogeneity test
According to the requirement of extracting the number of units (when N is less than or equal to 200, the number of the extracting units is not less than 11) in the uniformity evaluation of JJF 1343-2012 'general principle and statistical principle of standard substance definite value', in the well-packaged '4 tobacco specific N-nitrosamine standard substances in methanol' sample obtained in step 3, respectively extracting 4 bottles, 4 bottles and 4 bottles of samples before, during and after the sample is packaged according to the packaging sequence. 3 samples were prepared for each vial, for a total of 36 samples, and each sample was numbered 1#, 2#, 3#, … …, 34#, 35#, and 36 #. In order to avoid the influence of instrument system errors on the result, 36 samples are randomly arranged, the random sequence is 6#, 7#, 17#, 10#, 9#, 14#, 27#, 13#, 26#, 11#, 29#, 1#, 18#, 30#, 3#, 31#, 33#, 12#, 34#, 21#, 16#, 24#, 8#, 28#, 23#, 15#, 22#, 5#, 2#, 32#, 4#, 25#, 20#, 19# and 36#, and the samples are measured according to the sequence.
The single-factor variance analysis specified by the general principle and statistical principle of standard substance valuation of JJF 1343-2012 serves as a mathematical statistical method for the uniformity test of the application.
A standard working solution was prepared by the following method:
taking methanol verified in the step 2 as a solvent, weighing 20.0mg of the candidate detected in the step 1 (TRC company, Canada) into a 10mL volumetric flask by using a ten-thousandth balance to prepare a mixed stock solution, and preparing standard working curve solutions by using a stepwise dilution method, wherein the concentrations of the standard working curve solutions are respectively 2.00mg/mL, 1.50mg/mL, 1.00mg/mL, 0.75mg/mL and 0.50 mg/mL.
At each measurement, the standard working solution was reconstituted and a standard working curve was prepared according to this method.
The contents of 4 components in a sample of 4 tobacco-specific N-nitrosamine standard substances in methanol were determined by using the standard working curve and the HPLC-UV detection method established in section 1-4-2, and the results of the uniformity test are shown in tables 11 to 14. Statistical analysis was performed by the F-test method according to JJF 1343-. Since 12 sets of samples were tested in total, 3 data per set, the inter-set degree of freedom for all data was 12-1-11 and the intra-set degree of freedom for all data was 12 × 2-24. Looking up F distribution table, when the confidence probability is 95%, F0.052.2163 (11, 24). Due to FComputing＜F0.05(11,24), showing that no significant difference exists between the samples under the confidence probability of 95%, and the divided samples of the '4 tobacco specific N-nitrosamine standard substances in methanol' are uniform.
TABLE 11 homogeneity test results of NNN in "4 tobacco specific N-nitrosamine standards in methanol" samples
Note: in the table, the number of the first and second,
the same applies below.
TABLE 12 homogeneity test results of NNK in "4 tobacco specific N-nitrosamine Standard in methanol" samples
TABLE 13 homogeneity test results for NAT in "4 tobacco-specific N-nitrosamine Standard in methanol" samples
TABLE 14 homogeneity test results for NAB in "4 tobacco specific N-nitrosamine standards in methanol" samples
Small knot
From the above test results, it can be seen that the uniformity of each component in the "4 tobacco specific N-nitrosamine standard substances in methanol" samples after subpackaging is good.
4-2. Standard substance stability test
4-2-1. short term stability of Standard substance
According to the regulations of JJF 1343-.
Considering that a sample of 4 tobacco specific N-nitrosamine standard substances in methanol is stored in a refrigerator, the environment is high humidity (the relative humidity is more than 95 percent) and is protected from light, a sample of 4 tobacco specific N-nitrosamine standard substances in methanol is packaged in a brown bottle, and the sample is transported in a dark state by adopting the outer package of a black kraft paper bag, so that the short-term stability of the sample of 4 tobacco specific N-nitrosamine standard substances in methanol is only influenced by high temperature on the characteristic values of all components. Namely, the content change conditions of each component of 4 tobacco specific N-nitrosamine standard substances in methanol are examined on 0 day, 5 days, 7 days, 10 days and 14 days at the temperature of 60 ℃.
15 bottles of samples of "4 tobacco-specific N-nitrosamine standards in methanol" were taken out of the refrigerator, 12 bottles of which were placed in an oven set at 60 ℃ and the remaining 3 bottles were tested directly, and 3 bottles of samples were taken out of the oven on days 5, 7, 10 and 14, respectively, and the test was repeated 3 times for each bottle. Standard working solutions were reconstituted and standard working curves were prepared for each measurement, see homogeneity test section. The results of the NNK experiments are shown in Table 15 and FIG. 23.
TABLE 15 short term stability test data for NNK in samples
According to the regulation of JJF 1343-2012, because an accurate model does not reflect the real change rule of the NNK content in the solution, a straight line is adopted as an empirical model in the experiment.
The equation of a straight line: y is b + ax.
Standard deviation of straight line:
uncertainty of slope:
when in use
The slope was not significant and the stability was good.
With a 95% confidence probability, the degree of freedom is 3 and the bin point value of the two-tailed test t-distribution is 3.18.
The data in table 15, with x representing time (day) and y representing the value of the measurement result, are fitted to a straight line according to the specification of JJF 1343-: y 1.0789E-05x +1.0121, where the slope a 1.0789E-05 and the intercept b 1.0121.
The standard deviation of the straight line can be calculated by:
slope uncertainty is calculated using the following equation:
the degree of freedom was n-2-5-2-3 and p-0.95 (95% confidence interval), and the table was found to be 3.18.
Due to the fact that
Namely 1.0789E-05 < 3.18X 3.9365E-05 ═ 0.00013. The slope was not significant and no effect on stability was observed. The stability of NNK of the sample of the 4 tobacco specific N-nitrosamine mixed solution standard substances in methanol is good within 14 days under the specified storage condition (60 ℃).
The short-term stability test results of the remaining 3 components in the dispensed "4 tobacco-specific N-nitrosamine mixed solution standard substances in methanol" samples are shown in table 16.
TABLE 16 stability test results for the remaining 3 components in "4 tobacco specific N-nitrosamine standards in methanol" samples
Small knot
From the above test results, it can be seen that the short-term stability of each component in the "4 tobacco-specific N-nitrosamine standard substances in methanol" samples after being dispensed is good.
4-2-2. long term stability of Standard substance
According to the regulations of JJF 1343-2012 'general principle and statistical principle of standard substance fixed value', the long-term stability of the national secondary standard substance is required to be 6 months or more. Considering the stability of the sample of the 4 tobacco specific N-nitrosamine standard substances in methanol in a low-temperature state, the packaged standard substance sample is stored at 0-4 ℃ in the test, and the content change conditions of the components of the sample in 0 month, 2 months, 4 months, 7 months and 12 months are respectively inspected according to the principle of first densification and then combing. At each time point, 3 random vials were drawn and the assay repeated 3 times for each vial. For each measurement, the standard working solution was reconstituted and a standard working curve was prepared according to the method described in "preparation of standard working curve" in homogeneity test, and the measurement method is described in homogeneity test section.
The results of the long-term stability experiments on NNK in the "4 tobacco specific N-nitrosamine standard in methanol" samples are shown in Table 17 and FIG. 24.
TABLE 17 "NNK Long-term stability test data in samples of 4 tobacco-specific N-nitrosamine standards in methanol
According to the regulation of JJF 1343-2012, because an accurate model does not reflect the real change rule of the NNK content in the solution, a straight line is adopted as an empirical model in the experiment.
The equation of a straight line: y is b + ax.
Standard deviation of straight line:
uncertainty in slopeDegree determination:
when in use
The slope was not significant and the stability was good.
With a 95% confidence probability, the degree of freedom is 3 and the bin point value of the two-tailed test t-distribution is 3.18.
The data in table 17, with x representing time (month) and y representing the value of the measurement result, were fitted to a straight line as specified in JJF 1343-: y-1.449E-04 x +1.0137, where slope a-1.449E-04 and intercept b-1.0137.
The standard deviation of the straight line can be calculated by:
slope uncertainty is calculated using the following equation:
the degree of freedom was n-2-5-2-3 and p-0.95 (95% confidence interval), and the table was found to be 3.18.
Since 1.449E-04 < 3.18 × 0.000158 ═ 0.000503, i.e. 0.000503
The slope was not significant and no effect on stability was observed. The NNK in the sample of "4 tobacco specific N-nitrosamine standard substances in methanol" was found to have good stability over 12 months under the specified storage conditions.
The results of the long-term stability tests of the remaining 3 components of the "4 tobacco-specific N-nitrosamine standard in methanol" sample are shown in Table 18.
TABLE 18 Long-term stability test results for the remaining 3 components in the "4 tobacco-specific N-nitrosamine standards in methanol" sample
Small knot
From the above test results, it can be seen that the long-term stability of each component in the "4 tobacco specific N-nitrosamine standard substances in methanol" samples after being subpackaged is good.
5. Constant value of each component content in standard substance
The method is characterized in that a single laboratory is used for setting a value by adopting a single reference method, namely a balance is used for weighing a certain mass of detected candidate, and methanol with verified purity is used for fixing the volume to a volumetric flask with a certain volume. The contents of all components in 4 specific tobacco N-nitrosamine standard substances in methanol are traced to candidates and scales and volumetric flasks for metrological verification.
The concentration standard value c of each component in the prepared standard substance is calculated according to the following formula:
wherein: c-standard value of concentration of component in prepared standard substance; m-weighing the mass of the candidate; v-volume of formulation; ρ -purity of the candidate.
The quantitative values (i.e., concentration standard values c) of the contents of the respective components in "4 tobacco-specific N-nitrosamine standard substances in methanol" are shown in Table 19.
TABLE 19 quantitative values of the contents of the respective components in the reference substances
Uncertainty evaluation of quantitative results of contents of components in 4 tobacco-specific N-nitrosamine standard substances in methanol
The uncertainty of the quantitative result of the content of each component in 4 specific N-nitrosamine standard substances in the tobacco in the methanol is composed of 3 parts: the uncertainty introduced by the homogeneity of the standard substance, the uncertainty introduced by the stability (short and long term) of the standard substance, the uncertainty introduced by the valuing (purity, weighing, volume) process of the standard substance. Relative synthesis standard uncertainty:
wherein u isc(m)、uc(ρ)、uc(v)、uc(H)、uc(t) and uc(T) represents the uncertainty introduced by weighing, purity, volume, homogeneity, short term stability and long term stability, respectively.
6-1 weighing the incoming uncertainty
(1) Class a uncertainty. The validated NNK candidate 0.1000g was repeatedly weighed 8 times by a balance, with actual weighing results of 0.1003g, 0.1005g, 0.1007g, 0.1002g, 0.0998g, 0.1006g, 0.0996g, and 0.0998g, respectively, and standard deviation of Bessel's formula of
Therefore, the standard caused by weighing during the preparation of the standard substance was not determined to be 0.0004121 g.
(2) Class B uncertainty. The electronic balance used in the experiment is calibrated to a grade I balance by Guangzhou Guangdong electric metering detection GmbH, and the standard uncertainty caused by balance calibration in the preparation process of the standard substance
(3) Standard uncertainty. Synthesizing the weighed uncertainty of A class and B class into weighing uncertainty,
(4) relative standardUncertainty. By weighing the mass of the NNK candidate,
similarly, the relative standard uncertainties caused during weighing of candidate NNN, NAT and NAB were 0.004373, 0.004347 and 0.004304, respectively.
6-2. degree of uncertainty introduced by purity
Since the purity of the candidate NNK was the purity indicated on the certificate, and therefore it had only one class B uncertainty, the uncertainty introduced by the purity in the weighing is shown in table 20.
TABLE 20 purity introduction uncertainty of candidate NNK
Similarly, the relative standard uncertainties caused by the purities of the candidates NNN, NAT and NAB were 0.01001, 0.01002 and 0.01003, respectively.
6-3. uncertainty of volume introduction
(1) Class a uncertainty. The volumetric flask was repeatedly subjected to constant volume 8 times, weighed with a balance, and examined for group A uncertainty due to volume, and the results obtained 8 times were 99.96mL, 99.93mL, 99.87mL, 100.02mL, 99.94mL, 100.01mL, 100.00mL, and 100.02 mL. The standard deviation was 0.05357mL by bessel's formula, so the volume-induced uncertainty in the standard substance preparation process was 0.05357mL, and the relative standard deviation indicated a relative standard uncertainty of 0.0005357.
(2) Class B uncertainty. The uncertainty of B type comes from the two aspects of volumetric flask calibration and the inconsistency of the calibration temperature and the experimental temperature. The volumetric flask for the experiment is calibrated to be A grade by the institute of measurement and science in Henan province. The standard capacity tolerance of the A-grade 100mL volumetric flask at 20 ℃ is +/-0.10 mL, and the standard uncertainty is
The volumetric flask is calibrated at 20 ℃ and the laboratory temperature is between (20 +/-2) DEG CThe uncertainty of the change in volume it causes can be estimated from the temperature variation range and the volume expansion coefficient. Since the volume expansion of methanol is significantly greater than that of the volumetric flask, only the former need be considered. The volume expansion coefficient of methanol is 1.20X 10-3·℃-1The resulting change in volume is ± (100 × 2 × 1.20 × 10)-3) When the standard uncertainty is +/-0.24 mL
Class B synthetic uncertainty
Relative standard uncertainty
(3) Synthetic relative standard uncertainty
Similarly, the relative standard uncertainty caused by volume during weighing of candidate NNN, NAT and NAB is also 0.001407.
6-4. uncertainty introduced by uniformity
As shown in tables 11-14, the relative standard uncertainties, expressed as relative standard deviations of uniformity, are:
NNN：0.00070；NNK：0.00158；NAT：0.00102；NAB：0.00099。
6-5. short term stability induced uncertainty
As shown in tables 15 and 16, the relative standard uncertainty, expressed as the relative standard deviation of short term stability, is:
NNN：0.00122；NNK：0.00055；NAT：0.00097；NAB：0.00084。
6-6. uncertainty introduced by Long term stability
As shown in tables 17 and 18, the relative standard uncertainty, expressed as the relative standard deviation of long-term stability, is:
NNN：0.00156；NNK：0.00188；NAT：0.00127；NAB：0.00135。
6-7. Total synthetic relative Standard uncertainty
The total synthesis relative standard uncertainty was calculated according to the following formula, and the uncertainty of the components in the standard material is shown in Table 21.
Wherein u isc(m)、uc(ρ)、uc(v)、uc(H)、uc(t) and uc(T) represents the uncertainty introduced by weighing, purity, volume, homogeneity, short term stability and long term stability, respectively.
TABLE 21.4 candidate relative Standard uncertainty Components and Synthesis
Synthesis Standard uncertainty Total Synthesis Standard uncertainty was calculated according to the following formula, and the results are shown in tables 6-3.
TABLE 22 Standard uncertainty
When the confidence probability is 95%, the expansion factor is taken as 2, and the expansion uncertainty is up＝k×uc(c) The results of the calculations are shown in Table 23.
TABLE 23. extended uncertainty and reduction
7. Conclusion
In this example, samples of "4 tobacco-specific N-nitrosamine standards in methanol" were prepared for the purpose of actual testing requirements. The standard value is calculated by weighing weight, candidate purity and constant volume, and the uncertainty of the components in the standard substance is evaluated at the same time, so that the content fixed values are NNN1.00mg/mL, NNK1.01 mg/mL, NAT1.01 mg/mL and NAB1.02 mg/mL, and the relative expansion uncertainty is 3% (k is 2). The standard substance is inquired by a national standard substance information service platform, the standard substance is not available, and the blank of the specific N-nitrosamine standard substance of the tobacco is filled.
Example 2 use of 4 tobacco specific N-nitrosamine standard substances in methanol prepared in example 1
"4 tobacco-specific N-nitrosamine standards in methanol" can be used as standards for TSNAs testing.
According to the method specified in the high performance liquid chromatography-tandem mass spectrometry combined usage (YQ/T17-2012) for determining the specific N-nitrosamine in the total particulate matter of the main stream smoke of the cigarettes and the high performance liquid chromatography-tandem mass spectrometry combined usage (YQ/T29-2013) for determining the specific N-nitrosamine in the tobaccos and tobacco products, the TSNAs content in the main stream smoke and the tobaccos of different types of cigarettes is determined by taking the specific N-nitrosamine standard substances in 4 types of tobaccos in methanol prepared in example 1 as standard reference substances. At the same time, four TSNAs standards were used to prepare standard working curves, and the same sample was tested in parallel, with the results shown in Table 24. The results show that the use of the "4 tobacco-specific N-nitrosamine standard substances in methanol" prepared in example 1 is in agreement with the results obtained using the validated candidates as standard controls, respectively. Further proves the feasibility of the tobacco-specific N-nitrosamine standard substance provided by the invention as a standard substance. Meanwhile, the standard substance provided by the invention can complete the determination of the content of 4TSNAs at one time, and improve the working efficiency of TSNAs detection.
TABLE 24 determination of TSNAs content in tobacco (ng/g) and cigarette mainstream Smoke (ng/cig)
aThe data is the measurement result of taking '4 tobacco specific N-nitrosamine standard substances in methanol' prepared in the invention example 1 as a standard reference substance;
bthe column data is the assay result with the corresponding candidate verified as the standard control.
Claims (14)
1. A method for preparing tobacco specific N-nitrosamine standard substance, the tobacco specific N-nitrosamine is selected from at least one of N-nitrosonornicotine, 4- (methylnitroso) -1- (3-pyridyl) -1-butanone, N-nitrosonoranatabine and N-nitrosoanabasine; the preparation method comprises the following steps:
1. detection of candidates
1-1. Structure verification of candidates
Measuring the infrared spectrum, the ultraviolet spectrum, the mass spectrum, the nuclear magnetic resonance carbon spectrum and the nuclear magnetic resonance hydrogen spectrum of the candidate, and verifying the structure of the candidate according to the spectrum information; if the verification is passed, the following steps are carried out;
1-2. determination of candidate impurities
Determining the moisture content W of the candidateMoisture contentInorganic element content WInorganic substanceAnd residual solvent content WSolvent(s)；
1-3. determination of mutual impurity status of candidates
Determining whether each candidate contains other tobacco specific N-nitrosamines as candidates;
1-4. determination of purity of candidate
Determination of the purity P of each candidateMeasurement ofSubstituting the following formula to calculate the purity P of the candidatePurity of：
PPurity of＝(1-WMoisture content-WElement(s)-WSolvent(s))×PMeasurement of
If P isPurity ofWithin the range of purity and accuracy provided by the candidate certificate of analysis, the certificate of analysis is usedThe purity of the book label is the purity of the candidate; if P isPurity ofOut of the range of purity and accuracy provided by the candidate certificate of analysis, then P is calculatedPurity ofIs the purity of the candidate;
2. solvent validation
The solvent is chromatographic grade methanol, and the solvent is analyzed by adopting liquid chromatography-tandem mass spectrometry to determine whether the candidate substance is contained; if the determination result shows that the candidate is not contained, the verification is passed;
3. preparation and subpackage of tobacco specific N-nitrosamine standard substance
Taking the candidate detected in the step 1, taking methanol verified in the step 2 as a solvent, and preparing a standard substance by adopting a weight-volume method according to the purity of the candidate determined in the step 1-4 under the environment that the temperature is 20 +/-2 ℃ and the relative humidity is 60% +/-5%; then subpackaging the standard substance, immediately sealing to obtain the subpackaged specific N-nitrosamine standard substance for the tobacco, and storing in a refrigerator at 0-4 ℃ in a dark place;
4. test of tobacco-specific N-nitrosamine standard substance
4-1. uniformity test of specific N-nitrosamine standard substance in tobacco
According to the requirement of extracting the number of units in the uniformity evaluation of JJF 1343-2012 'general principle and statistical principle of standard substance definite value', extracting a specified number of samples from the subpackaged tobacco specific N-nitrosamine standard substance obtained in the step 3, measuring the content of the tobacco specific N-nitrosamine in each sample by using a high performance liquid chromatography, performing statistical analysis by using an F test method, and if F is detected, performing statistical analysis by using an F test methodComputingLess than F0.05(the inter-group freedom degree and the intra-group freedom degree) shows that the significant difference does not exist between the measured samples under the confidence probability level of 95%, and the specific N-nitrosamine standard substance of the tobacco after subpackaging is uniform;
4-2. testing the stability of the specific N-nitrosamine standard substance in tobacco
The test for the stability of the specific N-nitrosamine standard substance in the tobacco comprises short-term stability test at 60 ℃ in a dark place for 14 days and long-term stability test at 0-4 ℃ in a dark place for 12 months;
5. definite value of each component content in tobacco specific N-nitrosamine standard substance
Determining the content of each component in the specific N-nitrosamine standard substance of the tobacco by a single laboratory by adopting a single reference method;
6. uncertainty evaluation of quantitative result of content of each component in tobacco specific N-nitrosamine standard substance
Uncertainty components of the quantitative result of the content of each component in the specific N-nitrosamine standard substance of the tobacco come from uncertainty introduced by uniformity, uncertainty introduced by short-term stability, uncertainty introduced by long-term stability and uncertainty introduced in the quantitative process; the uncertainty components of each component are evaluated separately and the standard uncertainty is calculated.
2. The method of manufacturing according to claim 1, wherein said tobacco-specific N-nitrosamines are selected from all of N-nitrosonornicotine, 4- (methylnitroso) -1- (3-pyridyl) -1-butanone, N-nitrosonoranatabine, and N-nitrosoanabasine.
3. The method according to claim 1, wherein in step 1-1, the candidate substance is subjected to gas chromatography-mass spectrometry to obtain a mass spectrum; the gas chromatography-mass spectrometry conditions were:
the chromatographic column is
35ms (35% diphenyl/65% dimethylpolysiloxane), specification: 30m × 0.25mm, film thickness 0.25 μm; temperature programming: maintaining at 80 deg.C for 1.0min, and then increasing to 280 deg.C at a rate of 10 deg.C/min and maintaining for 20 min; a split sample injection mode is adopted, and the split ratio is 50: 1; the sample injection amount is 1.0 mu L; the solvent cutting time is 10.0 min; high-purity helium with the purity of more than or equal to 99.999 percent is taken as carrier gas; the flow rate is 1.0 mL/min; the temperatures of the sample inlet, the ion source and the interface are respectively 280 ℃, 230 and 280 ℃; the mass spectrum scanning mode is full scanning, and the scanning range is m/z, which is 40-250.
4. The method according to claim 1, wherein in the step 1-2, the moisture of the candidate is measured by a Karl Fischer moisture meter;
preferably, in the step 1-2, the inorganic elements in the candidate are determined by inductively coupled plasma mass spectrometry; the analysis parameters set were:
radio frequency voltage: 1500V; analysis mode: collision; rotation speed of peristaltic pump: 0.1 rps; helium gas is taken as collision gas flow, and the flow rate is as follows: 4.5 mL/min; argon gas is used as carrier gas, and the flow rate is as follows: 1.10 mL/min; collision cell stabilization time: 30 s; the quantitative method comprises the following steps: an internal standard method; integration time: 0.3 s; internal standard elements:45Sc、72Ge、115in and209Bi；
also preferably, in the step 1-2, the solvent residue in each candidate substance is quantitatively analyzed by gas chromatography-mass spectrometry; the specific analysis conditions were:
a chromatographic column: a capillary column special for HP-VOC; specification: 60m × 0.32mm, and the film thickness is 1.8 μm; carrier gas: helium, constant flow mode, flow 2.0 mL/min; sample inlet temperature: 180 ℃; no shunt sampling; temperature programming: keeping at 40 deg.C for 2min, heating to 200 deg.C at 4 deg.C/min, and keeping for 10 min; mass spectrum interface temperature: 220 ℃; an ionization mode: an electron bombardment source; ion source temperature: 230 ℃; ionization energy: 70 eV; temperature of the quadrupole rods: 150 ℃; firstly, analyzing a sample by using a full-scanning monitoring mode, and then accurately quantifying 26 typical alcohol, ketone, aromatic hydrocarbon and ester compounds by using a selective ion monitoring mode, wherein the scanning range is 29 amu-350 amu;
further preferably, in the step 1-2, each candidate is prepared into a solution with the concentration of 1mg/mL by taking triacetin as a solvent, and the quantitative analysis is carried out by gas chromatography-mass spectrometry.
5. The method according to claim 1, wherein in the steps 1 to 3, it is determined whether or not there are other tobacco-specific N-nitrosamines as candidates by subjecting each candidate to gas chromatography-flame ionization analysis;
further preferably, in the step 1-3, each candidate is prepared into a solution with the concentration of 1mg/mL by using methanol as a solvent, and gas chromatography-flame ionization analysis is carried out;
more preferably, in the steps 1 to 3, the analysis conditions of the gas chromatography-flame ionization analysis are as follows:
chromatographic column DB-35MS elastic capillary chromatographic column (35% diphenyl/65% dimethyl polysiloxane), specification 30m × 0.25mm, film thickness 0.25 μm; sample inlet temperature: 250 ℃; sample introduction amount: 1 mu L, split-flow sample injection, the split-flow ratio is 30: 1; carrier gas: nitrogen with purity more than or equal to 99.999%, constant flow rate: 1.0 mL/min; temperature rising procedure: the initial temperature is 80 ℃, the temperature is kept for 1min, the temperature is increased to 280 ℃ at the speed of 10 ℃/min, and the temperature is kept for 20 min; a detector: a flame ionization detector; the flow rates of hydrogen, air and tail gas blowing of the detector are respectively 45, 350 and 3mL/min, and the tail gas blowing is nitrogen; the detector temperature was 250 ℃.
6. The method according to claim 1, wherein in the steps 1 to 4, each candidate is prepared into a solution with a concentration of 1mg/mL by using methanol as a solvent, and gas chromatography-flame ionization analysis and high performance liquid chromatography-ultraviolet analysis are carried out;
more preferably, in the steps 1 to 4, the analysis conditions of the gas chromatography-flame ionization analysis are as follows:
chromatographic column DB-35MS elastic capillary chromatographic column (35% diphenyl/65% dimethyl polysiloxane), specification 30m × 0.25mm, film thickness 0.25 μm; sample inlet temperature: 250 ℃; sample introduction amount: 1 mu L, split-flow sample injection, the split-flow ratio is 30: 1; carrier gas: nitrogen with purity more than or equal to 99.999%, constant flow rate: 1.0 mL/min; temperature rising procedure: the initial temperature is 80 ℃, the temperature is kept for 1min, the temperature is increased to 280 ℃ at the speed of 10 ℃/min, and the temperature is kept for 20 min; a detector: a flame ionization detector; the flow rates of hydrogen, air and tail gas blowing of the detector are respectively 45, 350 and 3mL/min, and the tail gas blowing is nitrogen; the temperature of the detector is 250 ℃;
further preferably, in the steps 1 to 4, the chromatographic conditions of the high performance liquid chromatography-ultraviolet analysis are as follows: the analytical column is a Poroshell 120EC-C18 chromatographic column with specification of 100mm multiplied by 4.6mm and particle size of 4 μm; the column temperature was 40 ℃; the mobile phase consists of two phases of water and methanol, wherein the water is phase A, the methanol is phase B, a gradient elution mode is adopted, and the elution procedure is as follows: 30% (v/v) phase B at time t of 0min, keeping for 4 min, linearly increasing the volume ratio of phase B to 90% (v/v) within 3min, keeping for 5min, decreasing the volume of phase B to 30% (v/v) within 1min, and balancing for 5 min; the flow rate is 1.0 mL/min; the ultraviolet detection wavelength is as follows: n-nitrosonornicotine 357nm, 4- (methylnitrosamine) -1- (3-pyridyl) -1-butanone 339nm, N-nitrosoanatabine 361 nm; the amount of sample was 5. mu.L.
7. The preparation method according to claim 1, wherein in the step 2, the chromatographic conditions of the liquid chromatography-tandem mass spectrometry are as follows:
chromatographic column Poroshell 120EC-C18 with specification of 100mm × 3.0mm and particle size of 2.7 μm; the sample injection amount is 5 mu L; the column temperature is 40 ℃; mobile phase: solvent A was 10mM ammonium formate solution and solvent B was 0.1% formic acid (v/v) in acetonitrile, and the elution was performed in a gradient with the following procedure:
the flow rate is 0.4 mL/min;
also preferably, in the step 2, the multiple reaction monitoring ion pairs adopted by the liquid chromatography-tandem mass spectrometry are: 178.1>148.2(15) for N-nitrosonornicotine, 208.1>122.1(16) for 4- (methylnitroso) -1- (3-pyridyl) -1-butanone, 190.1>160.1(15) for N-nitrosonoranatabine and 192.1>162.2(17) for N-nitrosoanabasine.
8. The preparation method according to claim 1, wherein the specific operation of step 3 is:
weighing a certain amount of the candidate detected in the step 1 by using a calibrated first-grade balance in the environment with the temperature of 20 +/-2 ℃ and the relative humidity of 60% +/-5%, adding the methanol verified in the step 2 into the same calibrated A-grade 100mL volumetric flask, completely dissolving, fixing the volume to the scale by using the methanol, and shaking up to obtain the specific N-nitrosamine standard substance of the tobacco; transferring 0.5mL of the tobacco specific N-nitrosamine standard substance into a brown glass bottle with a threaded opening and a tapered groove at the bottom and a volume of 1.5mL, immediately sealing the bottle, and storing the bottle in a refrigerator at 0-4 ℃ in a dark place.
9. The preparation method according to claim 1, wherein in the step 4-1, the chromatographic conditions of the high performance liquid chromatography are as follows: the analytical column is a Poroshell 120EC-C18 chromatographic column with specification of 100mm multiplied by 4.6mm, particle size of 4 μm and column temperature of 40 ℃; the mobile phase is methanol/water (40/60, v/v), and the flow rate is 1.0 mL/min; the detector is an ultraviolet detector, and the ultraviolet detection wavelength is 357nm for N-nitrosonornicotine, 339nm for 4- (methylnitrosamine) -1- (3-pyridyl) -1-butanone, and 361nm for N-nitrosoanabasine and N-nitrosoanabasine; the sample injection amount is 5 mu L;
also preferably, in the step 4-2, the specific operation of the short-term stability test at 60 ℃ in the dark for 14 days comprises:
A. taking out the subpackaged N-nitrosamine standard substances special for the tobacco, which are obtained in the step 3 and stored in a refrigerator, from 5 xn bottles, wherein N is 1-5, and N is 3 is more preferable; putting 4 Xn bottles in a drying oven with a set temperature of 60 ℃, measuring the content of the N-nitrosamine specific to the tobacco by the high performance liquid chromatography for the rest N bottles, and repeatedly measuring each bottle of sample for 3 times;
B. taking N bottles out of the oven on days 5, 7, 10 and 14 respectively, measuring the content of the tobacco-specific N-nitrosamine by high performance liquid chromatography, and repeating the measurement for 3 times for each bottle of sample;
C. the results of the short-term stability measurement of N-nitrosamines specific to each tobacco were fitted to a straight line with x as the horizontal axis representing time (day) and y as the measured content, and the equation of the straight line y was obtained as b + ax, and the standard deviation s of the straight line was calculated by the following equation2And uncertainty s of slope(a)，
Standard deviation of straight line:
uncertainty of slope:
if it is not
The slope was not significant, indicating good short term stability;
still preferably, in the step 4-2, the specific operation of the long-term stability test at 0-4 ℃ in the dark for 12 months comprises:
A. taking out m bottles of the subpackaged N-nitrosamine standard substances, obtained in the step 3 and stored in a refrigerator, from 0 month, 2 months, 4 months, 7 months and 12 months respectively, wherein m is 1-5, and more preferably m is 3; measuring the content of the tobacco-specific N-nitrosamine by high performance liquid chromatography, and repeatedly measuring each bottle of sample for 3 times;
B. the long-term stability measurement results of N-nitrosamines specific to each tobacco were fitted to a straight line with x as the horizontal axis representing time (month) and y as the measured content, and the equation of the straight line y was obtained as b + ax, and the standard deviation s of the straight line was calculated by the following equation2And uncertainty s of slope(a)，
Standard deviation of straight line:
uncertainty of slope:
if it is not
The slope was insignificant, indicating good long-term stability.
10. The preparation method according to claim 1, wherein in the step 5, the fixed value of each component in the tobacco specific N-nitrosamine standard substance is calculated by weighing weight, candidate purity and fixed volume; wherein the definite value of N-nitrosonornicotine is 1.00mg/mL, the definite value of 4- (methylnitroso) -1- (3-pyridyl) -1-butanone is 1.01mg/mL, the definite value of N-nitrosonoranatabine is 1.01mg/mL, and the definite value of N-nitrosoanabasine is 1.02 mg/mL.
11. The method according to claim 1, wherein in step 6, the calculated relative expansion uncertainty of each component is 3% (k-2).
12. A tobacco-specific N-nitrosamine standard substance in methanol, prepared by the preparation method of any one of claims 1 to 11; the tobacco specific N-nitrosamine is at least one of N-nitrosonornicotine, 4- (methylnitroso) -1- (3-pyridyl) -1-butanone, N-nitrosonoranatine and N-nitrosoanabasine;
preferably, the tobacco specific N-nitrosamines are all N-nitrosonornicotine, 4- (methylnitroso) -1- (3-pyridyl) -1-butanone, N-nitrosonoranatine and N-nitrosoanabasine.
13. 4 tobacco-specific N-nitrosamine standard substances in methanol, prepared by the preparation method according to any one of claims 1 to 11, at a content rating of:
the content of N-nitrosonornicotine is 1.00mg/mL, the content of 4- (methylnitroso) -1- (3-pyridyl) -1-butanone is 1.01mg/mL, the content of N-nitrosonoranatabine is 1.01mg/mL, and the content of N-nitrosoanabasine is 1.02 mg/mL.
14. Use of a tobacco specific N-nitrosamine standard in methanol according to claim 12 or 13, or in methanol prepared by a method of preparation according to any one of claims 1 to 11, for the detection of tobacco specific N-nitrosamines in tobacco and/or tobacco products.
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