CN106290627A - A kind of analysis method of nitrosamine burst size in cigarette smoke - Google Patents
A kind of analysis method of nitrosamine burst size in cigarette smoke Download PDFInfo
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- CN106290627A CN106290627A CN201610625685.8A CN201610625685A CN106290627A CN 106290627 A CN106290627 A CN 106290627A CN 201610625685 A CN201610625685 A CN 201610625685A CN 106290627 A CN106290627 A CN 106290627A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The present invention provides the analysis method of nitrosamine burst size in a kind of cigarette smoke, cigarette smoke is carried out sample pre-treatments, using multidimensional liquid chromatography mass combined system that pre-treatment gained testing sample carries out qualitative and quantitative analysis, described multidimensional liquid chromatography mass combined system includes the first dimension liquid-chromatography apparatus, two-dimensional liquid chromatography device, the mass spectrometric apparatus being sequentially connected with.The analysis method of nitrosamine burst size in a kind of cigarette smoke that the present invention provides, effectively remove and flue gas affects the interfering material of nitrosamine accurate quantitative analysis, make entrance mass spectrum sample interfering ion greatly reduce, there is advantage highly sensitive, that flux is high, be adapted to the batch detection of sample.
Description
Technical field
The invention belongs to the technical field of important chemical composition analysis in cigarette mainstream flue gas, relate in a kind of cigarette smoke
Nitrosamine release quantitative analysis method, is specifically related to a kind of employing multidimensional liquid chromatograph and analyzes cigarette smoke with mass spectrometric hyphenated technique
The method of middle nitrosamine compound burst size.
Background technology
Tobacco-specific nitrosamine (TSNAs, Tobacco-Specific Nitrosamines) is to find to exist only in Nicotiana tabacum L.
With the non-volatile nitrosamines in flue gas.It is generally believed that tobacco-specific nitrosamine is raw by Nicotiana tabacum L. in Nicotiana tabacum L. modulated process
Alkaloids Nitrification is formed.Tobacco-specific nitrosamine (TSNAs) mainly includes it being 4-(N-methyl nitrosamino group)-1-(3-pyrrole
Piperidinyl)-1-butanone (NNK), N-nitrosonornicotine (NNN), N-nitroso-group anabasine (NAB) and N-nitrosoanabasine
(NAT), its structure such as following formula.
Wherein, NNN and NNK is regarded as a class carcinogen by IARC (IARC), and NAB and NAT is proved
Animal there is carcinogenecity.Having more than 5,000 kinds of compositions due to cigarette smoke and composition is complicated, the method for current main flow mainly has height
Effect gas chromatogram-heat energy analysis (GC-TEA) method and liquid chromatography mass combination method (LC-MS).But, before GC-TEA method exists
Process the shortcomings such as step is loaded down with trivial details, sample analysis time length, detection sensitivity are relatively low, and LC-MS method has matrix interference serious, method
Sensitivity, the detection limit problem such as relatively low.
Multidimensional liquid chromatograph (MDLC), will be implanted sequentially color below at the eluent of first liquid-phase chromatographic column by sample
Spectrum post carries out the liquid chromatograph multiple techniques that separates, and it is complex sample to separate analysis at present be subject to most heavy with mass spectrum (MS) combination
Depending on one of means, therefore the assay method of nitrosamine burst size in cigarette smoke is necessary further to inquire into and
Research.
Summary of the invention
The shortcoming of prior art in view of the above, it is an object of the invention to provide nitrosamine in a kind of cigarette smoke and releases
Put quantitative analysis method, be used for solving in prior art lacking that high accuracy, favorable reproducibility, detection limit be low, sample after secondary separation
Product are difficult to pollute mass ion source, are adapted to the asking of analysis method of nitrosamine burst size in the cigarette smoke of sample batch detection
Topic.
For achieving the above object and other relevant purposes, the present invention provide nitrosamine burst size in a kind of cigarette smoke point
Analysis method, carries out sample pre-treatments to cigarette smoke, uses multidimensional liquid chromatography mass combined system to be measured to pre-treatment gained
Sample carries out qualitative and quantitative analysis, and described multidimensional liquid chromatography mass combined system includes the first dimension liquid chromatograph being sequentially connected with
Device, two-dimensional liquid chromatography device, mass spectrometric apparatus.
It is preferred that nitrosamine includes N-nitrosonornicotine (NNN), 4-(methyl nitrous in described cigarette smoke
Base amino)-l-(3-is than piperidinyl)-1-butanone (NNK), N-nitroso-group anabasine (NAB) and N-nitrosoanatabine
(NAT)。
It is preferred that described sample pre-treatments comprises the following steps:
1) choose Cigarette, after lighting, use filter disc Trapping ways trapping cigarette smoke；
2) stand after filter disc being carried out ultrasonic extraction, be centrifuged, after taking supernatant liquid filtering, obtain testing sample.
Preferably, in step 1) in, described filter disc is cambridge filter.A diameter of 44mm of described cambridge filter.
Preferably, in step 2) in, described filter disc is placed in EP pipe and extracts.
Preferably, in step 2) in, described extraction solution is 0.05-0.2mol/L ammonium acetate (NH4Ac) aqueous solution.More excellent
Selection of land, described extraction solution is 0.1mol/L ammonium acetate (NH4Ac) aqueous solution.Described ammonium acetate solution, by weighing acetic acid
Ammonium, after being completely dissolved with water, is transferred in volumetric flask, and the constant volume that adds water obtains.
Preferably, in step 2) in, the time of described ultrasonic extraction is 30-50min.It is highly preferred that described ultrasonic extraction
Time be 40min.
Preferably, in step 2) in, described time of repose is 4-6min.It is highly preferred that described time of repose is 5min.
Preferably, in step 2) in, described centrifugal condition is: centrifugation time: 4-6min；Centrifugal rotational speed: 2700-
3300rpm.It is highly preferred that described centrifugal condition is: centrifugation time: 5min；Centrifugal rotational speed: 3000rpm.
Preferably, in step 2) in, the mode of described filtration is aqueous phase membrane filtration mode.It is highly preferred that described aqueous phase
The aperture of filter membrane is 0.22 μm.
It is preferred that described employing multidimensional liquid chromatography mass combined system pre-treatment gained testing sample is carried out qualitative fixed
Component analysis, comprises the following steps:
A) preparation standard sample；
A1) weigh the standard substance of NAB, NAT, NNN, NNK respectively, add methanol constant volume, be made into hybrid standard deposit molten
Liquid；
Preferably, the concentration of the standard substance of described NAB, NAT, NNN, NNK is 0.5mg/ml.
Preferably, in described hybrid standard stock solution, the concentration of NAB, NAT, NNN, NNK is 50 μ g/ml.
A2) weigh d4-NNN, d4-NAT, d4-NAB, d4-NNK respectively, add methanol constant volume, be made into inner mark solution；
Preferably, in described inner mark solution, the concentration of d4-NNN, d4-NAT, d4-NAB, d4-NNK is 5 μ g/ml.
Preferably, described d4-NNN, d4-NAT, d4-NAB, d4-NNK are deuterated reagent.Concrete, described d4-NNN,
D4-NAT, d4-NAB, d4-NNK are respectively deuterated N-nitrosonornicotine, deuterated N-nitrosoanatabine, deuterated N-
Nitroso-group anabasine, deuterated 4-(methyl nitroso amino)-l-(3-is than piperidinyl)-1-butanone.
Described d4-NNN, d4-NAT, d4-NAB, d4-NNK are commercial reagents, its stable chemical nature, in being adapted as
Mark.
A3) pipette hybrid standard stock solution in step A1 of different volumes respectively, then be separately added into the step of certain volume
Inner mark solution in rapid A2, is formulated as the mixed standard solution of a series of variable concentrations by methanol constant volume.
Preferably, the concentration of described mixed standard solution is 0-10ng/ml.
Preferably, in described mixed standard solution, the concentration of d4-NNN, d4-NAT, d4-NAB, d4-NNK is 1ng/
ml。
B) sample qualitative detection: respectively by step A) standard sample prepared and after sample pre-treatments testing sample carry out
Multidimensional liquid chromatography mass combined system detects, after carrying out fraction by the first dimension liquid-chromatography apparatus, by described Two-dimensional Liquid
Phase chromatogram arrangement separates, then is measured by described mass spectrometric apparatus, compares retention time, scanning of the mass spectrum carries out qualitative, really
Determine 4 kinds of nitrosamine compositions in testing sample；
C) sample amounts detection: respectively by step A) standard sample prepared and after sample pre-treatments testing sample carry out
Multidimensional liquid chromatography mass combined system detects, after carrying out fraction by the first dimension liquid-chromatography apparatus, by described Two-dimensional Liquid
Phase chromatogram arrangement separates, then is measured by described mass spectrometric apparatus, uses Internal standard curve method to carry out MRM quantitatively, obtains
Obtain the content of 4 kinds of nitrosamine compositions in testing sample.
Preferably, described step B) or C) in, it is step A1 to be prepared that described first dimension liquid-chromatography apparatus carries out fraction
Hybrid standard stock solution and after sample pre-treatments testing sample solution by first dimension liquid-chromatography apparatus carry out fraction.
Preferably, described step B) or C) in, described two-dimensional liquid chromatography device separates, then is filled by described mass spectrum
Put the dilute solution being measured referring to mixed standard solution and testing sample step A3 prepared by the second dimension liquid phase color
Spectral apparatus separates, then is measured by described mass spectrometric apparatus.
Preferably, described step B) or C) in, the chromatographic condition of described first dimension liquid-chromatography apparatus is:
First dimension liquid chromatograph: positive liquid chromatograph；Chromatographic column: strong cation exchange chromatography post (SCX)；Detection wavelength:
220-240nm；Column temperature: 25-35 DEG C；Flow velocity: 0.5-1.5mL/min；Sample size: 5-15 μ l；Mobile phase A phase: 5-15mmol/L
KH2PO4+1-10mmol/L H3PO4+ 80-90%H2O+10-20%CH3CN (pH=2-4)；Mobile phase B phase: 5-15mmol/L
KH2PO4+1-10mmol/L H3PO4+ 0.1-1.0mol/L KCl+80-90%H2O+10-20%CH3CN (pH=2-4)；Gradient
Eluting.
It is highly preferred that the chromatographic condition of described first dimension liquid-chromatography apparatus is:
First dimension liquid chromatograph: Waters ACQUITY UPLC；Chromatographic column: Waters Spherisorb S5SCX
(4.6*150mm,5μm)；Detection wavelength: 230nm；Column temperature: 30 DEG C；Flow velocity: 1mL/min；Sample size: 10 μ l；Mobile phase A phase:
10mmol/L KH2PO4+5mmol/L H3PO4+ 85%H2O+15%CH3CN (pH=3)；Mobile phase B phase: 10mmol/L KH2PO4
+5mmol/L H3PO4+ 0.5mol/L KCl+85%H2O+15%CH3CN (pH=3)；Gradient elution.
Above-mentioned percentage ratio % is percent by volume.
Most preferably, as shown in table 1, the specific procedure of described gradient elution is:
0-5min, A phase: B phase volume ratio is 100:0-90:10；
5-5.1min, A phase: B phase volume ratio is 90:10-60:40；
5.1-15min, A phase: B phase volume ratio is 60:40-0:100；
15-15.1min, A phase: B phase volume ratio is 0:100-100:0；
15.1-40min, A phase: B phase volume ratio is 100:0-100:0.
The gradient elution of table 1 first dimension liquid-chromatography apparatus
| Retention time T (min) | Mobile phase A phase | Mobile phase B phase |
| 0 | 100 | 0 |
| 5 | 90 | 10 |
| 5.1 | 60 | 40 |
| 15 | 0 | 100 |
| 15.1 | 100 | 0 |
| 40 | 100 | 0 |
Preferably, described step B) or C) in, the condition of described two-dimensional liquid chromatography device is:
Two-dimensional liquid chromatography: reversed-phase liquid chromatography (RPLC)；Chromatographic column: C18 post；Column temperature: 25-35 DEG C；Flow velocity: 0.2-
0.3mL/min；Sample size: 5-15 μ l；Mobile phase A phase: 1-10mmol/L NH4Ac+92-98%H2O+2-8%CH3CN；Flowing
Phase B phase: 1-10mmol/L NH4Ac+92-98%CH3CN+2-8%H2O；Gradient elution.
It is highly preferred that the condition of described two-dimensional liquid chromatography device is:
Two-dimensional liquid chromatography: Agilent Technologies 1260Infinity reversed-phase liquid chromatography (RPLC)；Color
Spectrum post: Waters Cortecs C18 (2.1*150mm, 2.7 μm)；Column temperature: 30 DEG C；Flow velocity: 0.25mL/min；Sample size: 10 μ
l；Mobile phase A phase: 5mmol/L NH4Ac+95%H2O+5%CH3CN；Mobile phase B phase: 5mmol/L NH4Ac+95%CH3CN+
5%H2O；Gradient elution.Above-mentioned percentage ratio % is percent by volume.
Most preferably, as shown in table 2, the specific procedure of described gradient elution is:
0-10min, A phase: B phase volume ratio is 100:0-100:0；
10-20min, A phase: B phase volume ratio is 100:0-0:100；
20-22min, A phase: B phase volume ratio is 0:100-0:100；
22-22.1min, A phase: B phase volume ratio is 0:100-100:0；
22-25min, A phase: B phase volume ratio is 100:0-100:0.
The gradient elution of table 2 two-dimensional liquid chromatography device
| Retention time t/min | Mobile phase A phase | Mobile phase B phase |
| 0 | 100 | 0 |
| 10 | 100 | 0 |
| 20 | 0 | 100 |
| 22 | 0 | 100 |
| 22.1 | 100 | 0 |
| 25 | 100 | 0 |
Preferably, described step B) or C) in, the condition of described mass spectrometric apparatus is:
Mass spectrum: triple quadrupole bar mass spectrum；Ionization mode: ESI；Scan mode: multiple-reaction monitoring (MRM)；MRM condition: see
Table 3；Cleaning sample introduction needle program: needle wash 20-40s；Mass spectrum stream switches: start scanning, 26-30min from 8-12min
Terminate.
It is highly preferred that the condition of described mass spectrometric apparatus is:
Mass spectrum: Agilent Technologies 6460Triple Quad MS triple quadrupole bar mass spectrum；Ionization mode:
ESI；Scan mode: multiple-reaction monitoring (MRM)；MRM condition: be shown in Table 3；Cleaning sample introduction needle program: needle wash 30s；Matter
Spectrum stream switching: starting scanning from 10min, 28min terminates.
Table 3 MRM condition
| Parent ion (m/z) | Daughter ion (m/z) | Fragmentor | CE(eV) | |
| NNK | 208.1 | 122.1 | 60 | 8 |
| NAB | 192.1 | 162.1 | 60 | 4 |
| NAT | 190.1 | 160.1 | 60 | 4 |
| NNN | 178.1 | 148.1 | 50 | 5 |
Preferably, described step B) or C) in, described first dimension liquid-chromatography apparatus uses heartcut after carrying out fraction
Method, it is thus achieved that the fraction of 4 kinds of nitrosamine compositions.The fraction of described 4 kinds of nitrosamine compositions includes NNK, NNN, NAT, NAB fraction.
Described heartcut method is separation method conventional in multi-dimensional chromatograph, in the two-dimensional liquid chromatography of the present invention, i.e.
The cutting of the chromatographic peak of the first dimension liquid chromatograph isolated object fraction is completely transferred to two-dimensional liquid chromatography, other
Non-targeted thing fraction does not enter two-dimensional liquid chromatography.
It is highly preferred that the fraction of described 4 kinds of nitrosamine compositions is to be separated by two-dimensional liquid chromatography device, and through mass spectrum
Device determines, by Mass Spectrometer Method mass-to-charge ratio, the material that each chromatographic peak is corresponding.The mass-to-charge ratio numerical value of described 4 kinds of nitrosamine compositions
It is shown in Table 3.The parent ion mass-to-charge ratio of described NNK fraction is 208.1, and word ion mass-to-charge ratio is 122.1；The mother of described NAB fraction from
Sub-mass-to-charge ratio is 192.1, and word ion mass-to-charge ratio is 162.1；The parent ion mass-to-charge ratio of described NAT fraction is 190.1, word ion matter
Lotus ratio is 160.1；The parent ion mass-to-charge ratio of described NNN fraction is 178.1, and word ion mass-to-charge ratio is 148.1.
It is highly preferred that 6 fractions that the fraction of described 4 kinds of nitrosamine compositions is numbered 6,7,8,9,10,11.Such as table 4
Shown in, it is NNK that 6 fraction materials of described numbered 6,7,8,9,10,11 are respectively as follows: numbered 6 fraction materials；Numbered 7
Fraction material is NNK；Numbered 8 fraction materials are NNN；Numbered 9 fraction materials are NNN；Numbered 10 fraction materials are
NAT；Numbered 11 fraction materials are NAB.
Table 4 fraction material
| Fraction | 6 | 7 | 8 | 9 | 10 | 11 |
| Material | NNK | NNK | NNN | NNN | NAT | NAB |
Preferably, described step B) or C) in, described first dimension liquid-chromatography apparatus and two-dimensional liquid chromatography device it
Between be additionally provided with the second dimension trapping column.
It is highly preferred that the condition of described second dimension trapping column is:
Chromatographic column: C18 post；Mobile phase A phase: 5-15mmol/L KH2PO4+1-10mmol/L H3PO4+ 80-90%H2O+
10-20%CH3CN (pH=2-4)；Mobile phase B phase: 1-10mmol/L NH4Ac+92-98%H2O+2-8%CH3CN；Gradient is washed
De-.
It is further preferred that the condition of described second dimension trapping column is:
Chromatographic column: Waters Cortecs C18 post (2.7 μm, 2.1 × 10mm i.d.)；Mobile phase A phase: 10mmol/L
KH2PO4+5mmol/L H3PO4+ 85%H2O+15%CH3CN (pH=3)；Mobile phase B phase: 5mmol/L NH4Ac+95%H2O+
5%CH3CN；Gradient elution.
Above-mentioned percentage ratio % is percent by volume.
Most preferably, the specific procedure of described gradient elution is: 0-30min, A phase: B phase volume ratio is 100:0-0:100.
Preferably, described step B) or C) in, described first dimension liquid-chromatography apparatus includes the first dimension being sequentially communicated
Liquid chromatography pump, two six-way valves of sample introduction, the first dimension chromatographic column, UV-detector.
It is highly preferred that two six-way valves of described sample introduction are provided with the 1st interface, the 2nd interface, interface 3, the 4th interface, the 5th connect
Mouthful, the 6th interface, described 4th interface is injection port, and described 4th interface is sequentially communicated the 5th interface, the 2nd interface through pipeline, the 3rd connects
Mouthful, interface 3 the sample introduction end of liquid chromatography pump is tieed up through pipeline connection first, described 1st interface is through pipeline connection the first dimension liquid
The sample outlet end of phase chromatogram pump, described 6th interface is through pipeline connection the first dimension chromatographic column, and described 1st interface and the 6th interface are connected
Logical.
Preferably, described step B) or C) in, described two-dimensional liquid chromatography device includes the second dimension liquid phase being connected
Chromatogram pump, the second dimension chromatographic column.
Preferably, described step B) or C) in, described multidimensional liquid chromatography mass combined system also includes communicating valve, institute
State communicating valve and tie up the UV-detector in liquid-chromatography apparatus, the Two-dimensional Liquid in two-dimensional liquid chromatography device respectively with first
Phase chromatogram pump, the second dimension chromatographic column are connected.
It is highly preferred that described communicating valve is two ten-way valves or two six-way valves.
It is further preferred that described two ten-way valves are provided with the 1st interface, the 2nd interface, interface 3, the 4th interface, the 5th connect
Mouth, the 8th interface, the 9th interface, the 10th interface, described 1st interface and the 10th interface is connected, described 1st interface through pipeline with purple
The sample outlet end of external detector is connected, and described 10th interface is connected through the sample introduction end of pipeline and the second dimension trapping column, and described the
The sample outlet end of two dimension trapping column is sequentially communicated interface 3, the 2nd interface, the sample introduction end of the second dimension liquid chromatography pump through pipeline, described
The sample outlet end of the second dimension liquid chromatography pump is sequentially communicated the 9th interface, the 8th interface, the 5th interface, the 4th interface, the second dimension through pipeline
Chromatographic column sample introduction end, the sample outlet end of described second dimension chromatographic column is connected with mass spectrograph through pipeline.Will in described two ten-way valves
After 8th interface and the 5th interface short circuit, the i.e. the 9th interface and the 4th interface are directly connected to, and its structure is identical with described two six-way valves,
But reserved 8th interface, the 5th interface can use when two trapping column switchings of needs.
It is further preferred that described two six-way valves are provided with the 1st interface, the 2nd interface, interface 3, the 4th interface, the 9th connect
Mouthful, the 10th interface, described 1st interface and the 10th interface is connected, and described 1st interface is through the sample outlet end of pipeline Yu UV-detector
Be connected, described 10th interface through pipeline and second dimension trapping column sample introduction end be connected, described second dimension trapping column go out sample
Hold and be sequentially communicated interface 3, the 2nd interface, the sample introduction end of the second dimension liquid chromatography pump, described second dimension liquid chromatography pump through pipeline
Sample outlet end be sequentially communicated the 9th interface, the 4th interface, the second dimension chromatographic column sample introduction end, described second dimension the going out of chromatographic column through pipeline
Sample end is connected with mass spectrograph through pipeline.
Most preferably, use multidimensional liquid chromatography mass combined system when being analyzed, the sample solution being analysed to by
4th interface of two six-way valves of sample introduction enters, and sample solution passes sequentially through the 5th interface, the 2nd interface, interface 3, enters first
The sample introduction end of dimension liquid chromatography pump, then entered the first dimension by the sample outlet end of the first dimension liquid chromatography pump through the 1st interface, the 6th interface
Chromatographic column, after effectively removing interfering material, by UV-detector, enters communicating valve, by two in the first dimension chromatographic column
1st interface of ten-way valve through the 10th interface enter second dimension trapping column, then from second dimension trapping column sample outlet end through interface 3,
2nd interface, the second dimension liquid chromatography pump, the 9th interface, the 8th interface, the 5th interface, the 4th interface enter the second dimension chromatographic column, the
TSNAs isolated by Two way chromatograms post, enters mass spectrograph and is measured.
Most preferably, use multidimensional liquid chromatography mass combined system when being analyzed, the sample solution being analysed to by
4th interface of two six-way valves of sample introduction enters, and sample solution passes sequentially through the 5th interface, the 2nd interface, interface 3, enters first
The sample introduction end of dimension liquid chromatography pump, then entered the first dimension by the sample outlet end of the first dimension liquid chromatography pump through the 1st interface, the 6th interface
Chromatographic column, after effectively removing interfering material, by UV-detector, enters communicating valve, by two in the first dimension chromatographic column
1st interface of six-way valve through the 10th interface enter second dimension trapping column, then from second dimension trapping column sample outlet end through interface 3,
2nd interface, the second dimension liquid chromatography pump, the 9th interface, the 4th interface enter the second dimension chromatographic column, isolate in the second dimension chromatographic column
TSNAs, enters mass spectrograph and is measured.
Preferably, described step C) in, described Internal standard curve method comprises the following steps:
Described Internal standard curve method refers to add internal standard material in standard solution, carries out Instrument measuring, it is thus achieved that standard
Working curve, and then the method measuring the actual sample concentration containing internal standard material.
, by step A) A3 in the mixed standard solution of a series of variable concentrations carry out multidimensional liquid chromatography mass respectively
Combined system detects, obtain respectively two grades of selection ion peak areas of 4 kinds of nitrosamine compositions/corresponding internal standard substance than with 4 kinds of nitrous
The linear relationship of the mass concentration ratio of amine component/corresponding internal standard substance, draws corresponding standard working curve, is calculated 4 respectively
Plant the regression equation of nitrosamine ingredient standard working curve.
Further, in described standard curve, with two grades of selection quasi-molecular ions faces of 4 kinds of nitrosamine compositions with corresponding internal standard substance
Long-pending is abscissa (X-axis) than the mass concentration ratio for vertical coordinate (Y-axis), its 4 kinds of nitrosamine compositions and corresponding internal standard substance.
, will after sample pre-treatments testing sample solution, carry out multidimensional liquid chromatography mass combined system detection, will obtain
Liquid to be measured in two grades of selection ion peak areas ratios of 4 kinds of nitrosamine compositions/corresponding internal standard substances, substitute in step corresponding 4
Plant the regression equation of nitrosamine ingredient standard working curve, and according to adding the known quality concentration of corresponding internal standard substance, calculate
The mass concentration of 4 kinds of nitrosamine compositions in testing sample solution.
As it has been described above, the analysis method of nitrosamine burst size in a kind of cigarette smoke of the present invention, fume sample is passed through
Loading after extraction, uses heartcut multidimensional liquid chromatography to carry out separating cutting to the first dimension liquid chromatograph (SCX) fraction, evaporates
Divide and enter Mass Spectrometer Method after entering two-dimensional HPLC separation.The method has the advantages that
(1) the analysis method of nitrosamine burst size in a kind of cigarette smoke that the present invention provides, by the first dimension liquid phase color
Spectrum separates rear center's cutting, effectively removes and affects the interfering material of nitrosamine accurate quantitative analysis in flue gas, reduces the interference of substrate.
(2) the analysis method of nitrosamine burst size in a kind of cigarette smoke that the present invention provides, by bidimensional liquid chromatograph
Separation, impurity removal, enters mass spectrum sample interfering ion and greatly reduces, and mass ion source is difficult to be contaminated.
(3) the analysis method of nitrosamine burst size, dividing by bidimensional liquid phase in a kind of cigarette smoke that the present invention provides
From being greatly improved the peak capacity of liquid chromatograph and detecting flux, method has advantage highly sensitive, that flux is high.
(4) the analysis method of nitrosamine burst size in a kind of cigarette smoke that the present invention provides, simple and efficient, accuracy
Height, favorable reproducibility, detection limit are low, are adapted to the batch detection of sample.
Accompanying drawing explanation
Fig. 1 is shown as the first dimension SCX liquid chromatogram analyzing nitrosamine in cigarette smoke of the present invention, wherein, 6,7
For NNK；8,9 is NNN；10 is NAT；11 is NAB.
Fig. 2 is shown as the canonical plotting of nitrosamine compound NNK in the cigarette smoke of the present invention.
Fig. 3 is shown as the canonical plotting of nitrosamine compound NAB in the cigarette smoke of the present invention.
Fig. 4 is shown as the canonical plotting of nitrosamine compound NAT in the cigarette smoke of the present invention.
Fig. 5 is shown as the canonical plotting of nitrosamine compound NNN in the cigarette smoke of the present invention.
Fig. 6 is shown as the overall structure schematic diagram of a kind of multidimensional liquid chromatography mass combined system of the present invention.
In figure,
1, the first dimension liquid chromatography pump,
2, two six-way valves of sample introduction,
21, the 1st interface,
22, the 2nd interface,
23, interface 3,
24, the 4th interface,
25, the 5th interface,
26, the 6th interface,
3, the first dimension chromatographic column,
4, UV-detector,
5, the second dimension trapping column,
6, communicating valve,
61, the 1st interface,
62, the 2nd interface,
63, interface 3,
64, the 4th interface,
65, the 5th interface,
66, the 8th interface,
67, the 9th interface,
68, the 10th interface,
7, the second dimension liquid chromatography pump,
8, the second dimension chromatographic column,
9, mass spectrograph.
Detailed description of the invention
The present invention is expanded on further, it should be appreciated that these embodiments are merely to illustrate the present invention below in conjunction with specific embodiment
Rather than limit the scope of the invention.
Below by way of specific instantiation, embodiments of the present invention being described, those skilled in the art can be by this specification
Disclosed content understands other advantages and effect of the present invention easily.The present invention can also be by the most different concrete realities
The mode of executing is carried out or applies, the every details in this specification can also based on different viewpoints and application, without departing from
Various modification or change is carried out under the spirit of the present invention.
The reagent and the experiment appliance that use in following example are conventional use of reagent and experiment appliance, Jun Kecong city
Acquisition is bought on Chang.Following example use instrument as follows: the first dimension chromatogram arrangement (Waters ACQUITY UPLC system,
Waters company), (Agilent Technologies 1260Infinity system, Agilent is public for the second dimension chromatogram arrangement
Department), mass spectrometric apparatus (Agilent Technologies 6460Triple Quad LC/MS system, Agilent company), first
Dimension chromatographic column (Waters Spherisorb S5SCX post (5 μm, 4.6 × 150mm i.d.), Waters company), the second dimension are caught
Clustered column (Waters Cortecs C18 post (2.7 μm, 2.1 × 10mm i.d.), Waters company), the second dimension chromatographic column
(Waters Cortecs C18 post (2.7 μm, 2.1 × 150mm i.d.), Waters company), UV-detector (Waters
ACQUITY UPLC system, Waters company).
Embodiment 1
1, sample pre-treatments
Choose Cigarette, use filter disc Trapping ways trapping cigarette smoke after lighting, make flue gas be adsorbed in cambridge filter
On.Cambridge filter is placed in EP pipe addition extraction solution, carries out ultrasonic extraction 30-50min in being placed in supersonic generator, extraction
Taking solution is 0.05-0.2mol/L NH4Ac aqueous solution, NH4Ac aqueous solution is by weighing ammonium acetate, after being completely dissolved with water,
Being transferred in volumetric flask, the constant volume that adds water obtains.Stand 5 ± 1min after extraction, take extract be placed on centrifuge centrifugal 5 ±
1min, centrifugal rotational speed is 3000 ± 300rpm, then takes supernatant after 0.22 μm aqueous phase membrane filtration, moves to chromatography
In Ping, obtain testing sample solution.
2, the preparation of standard solution
Pipette the standard substance of NAB, NAT, NNN, NNK that 10ml concentration is 0.5mg/ml respectively, be placed in 100mL volumetric flask
In, add methanol constant volume, be made into hybrid standard stock solution.The concentration of NAB, NAT, NNN, NNK in hybrid standard stock solution
It is 50 μ g/ml.Meanwhile, pipette d4-NNN, d4-NAT, d4-NAB, d4-NNK internal standard substance of certain volume respectively, add methanol
Constant volume, is made into inner mark solution, and in inner mark solution, the concentration of d4-NNN, d4-NAT, d4-NAB, d4-NNK is 5 μ g/ml.
The most accurately pipette different volumes hybrid standard stock solution, be placed in multiple 100mL volumetric flask, more accurately
Add the inner mark solution of certain volume, by methanol constant volume, be formulated as a series of mixed standard solution.The serial hybrid standard of preparation
Solution concentration is 0-10ng/ml, and wherein internal standard substance concentration is 1ng/ml.
3, the mensuration of sample size
Respectively testing sample solution in 2 standard solution and 1 prepared being carried out multidimensional liquid chromatography mass combination respectively is
System detection, will hybrid standard stock solution and testing sample solution tie up after liquid-chromatography apparatus carries out fraction by first, then
Mixed standard solution is separated by described two-dimensional liquid chromatography device with the dilute solution of testing sample solution, then by institute
State mass spectrometric apparatus to be measured, compare retention time, scanning of the mass spectrum carries out qualitative, determines 4 kinds of nitrosamine compositions in liquid to be measured；With
It is quantitative that Shi Caiyong Internal standard curve method carries out MRM, it is thus achieved that the content of 4 kinds of nitrosamine compositions in liquid to be measured.
Specifically, Internal standard curve method is first by the mixed standard solution of variable concentrations a series of in above-mentioned 2 respectively
Carry out the detection of multidimensional liquid chromatography mass combined system, obtain respectively two grades of selections of 4 kinds of nitrosamine compositions/corresponding internal standard substance from
The linear relationship of the mass concentration ratio of sub-peak area ratio and 4 kinds of nitrosamine composition/corresponding internal standard substances, draws the work of corresponding standard
Curve, with two grades of selection ion peak areas ratio (Y-axis) as vertical coordinate of 4 kinds of nitrosamine compositions with corresponding internal standard substance, its 4 kinds of nitrous
Amine component is abscissa (X-axis) with the mass concentration ratio of corresponding internal standard substance, is calculated 4 kinds of nitrosamine ingredient standard work respectively
The regression equation of curve.Liquid to be measured in above-mentioned 1 is carried out the detection of multidimensional liquid chromatography mass combined system again, to be measured by obtain
In liquid, two grades of selection ion peak areas ratios of 4 kinds of nitrosamine composition/corresponding internal standard substances, substitute into corresponding 4 kinds of nitrosamine and become minute mark
The regression equation of quasi-working curve, and according to adding the known quality concentration of corresponding internal standard substance, it is calculated 4 kinds of Asias in liquid to be measured
The mass concentration of nitramine composition.
Wherein, the chromatographic condition of described first dimension liquid-chromatography apparatus is:
First dimension liquid chromatograph: positive liquid chromatograph；Chromatographic column: strong cation exchange chromatography post (SCX)；Detection wavelength:
220-240nm；Column temperature: 25-35 DEG C；Flow velocity: 0.5-1.5mL/min；Sample size: 5-15 μ l；Mobile phase A phase: 5-15mmol/L
KH2PO4+1-10mmol/L H3PO4+ 80-90%H2O+10-20%CH3CN (pH=2-4)；Mobile phase B phase: 5-15mmol/L
KH2PO4+1-10mmol/L H3PO4+ 0.1-1.0mol/L KCl+80-90%H2O+10-20%CH3CN (pH=2-4)；Gradient
Eluting.
As shown in table 1, the specific procedure of described gradient elution is:
0-5min, A phase: B phase volume ratio is 100:0-90:10；
5-5.1min, A phase: B phase volume ratio is 90:10-60:40；
5.1-15min, A phase: B phase volume ratio is 60:40-0:100；
15-15.1min, A phase: B phase volume ratio is 0:100-100:0；
15.1-40min, A phase: B phase volume ratio is 100:0-100:0.
First dimension liquid-chromatography apparatus carries out fraction, as it is shown in figure 1, in spectrogram six peaks are collected fraction respectively, from a left side
To right number consecutively 6,7,8,9,10,11, thus obtain 6 fractions of numbered 6,7,8,9,10,11.Use heartcut again
Method, is completely transferred to two-dimensional liquid chromatography by the cutting of the chromatographic peak of the first dimension liquid chromatograph isolated object fraction,
Other non-targeted thing fraction does not enter two-dimensional liquid chromatography.
The condition of described two-dimensional liquid chromatography device is:
Two-dimensional liquid chromatography: reversed-phase liquid chromatography (RPLC)；Chromatographic column: C18 post；Column temperature:: 25-35 DEG C；Flow velocity:
0.2-0.3mL/min；Sample size: 5-15 μ l；Mobile phase A phase: 1-10mmol/L NH4Ac+92-98%H2O+2-8%CH3CN；
Mobile phase B phase: 1-10mmol/L NH4Ac+92-98%CH3CN+2-8%H2O；Gradient elution.
As shown in table 3, the specific procedure of described gradient elution is:
0-10min, A phase: B phase volume ratio is 100:0-100:0；
10-20min, A phase: B phase volume ratio is 100:0-0:100；
20-22min, A phase: B phase volume ratio is 0:100-0:100；
22-22.1min, A phase: B phase volume ratio is 0:100-100:0；
22-25min, A phase: B phase volume ratio is 100:0-100:0.
The condition of described mass spectrometric apparatus is:
Mass spectrum: triple quadrupole bar mass spectrum；Ionization mode: ESI；Scan mode: multiple-reaction monitoring (MRM)；MRM condition: see
Table 4；Cleaning sample introduction needle program: needle wash 20-40s；Mass spectrum stream switches: start scanning, 26-30min from 8-12min
Terminate.
After 6 fraction dilutions of obtain after the first dimension liquid-chromatography apparatus fraction numbered 6,7,8,9,10,11,
Separated by two-dimensional liquid chromatography device, and determine that each chromatographic peak is corresponding through mass spectrometric apparatus by Mass Spectrometer Method mass-to-charge ratio
Material.In first dimension liquid-chromatography apparatus, TSNAs peak sequence is NNK, NNK, NNN, NNN, NAT, NAB, the second dimension liquid phase color
In spectral apparatus, TSNAs peak sequence is NNN, NNK, NAT, NAB.
It is additionally provided with the second dimension trapping column between first dimension liquid-chromatography apparatus and two-dimensional liquid chromatography device.Described second
The condition of dimension trapping column is:
Chromatographic column: C18 post；Mobile phase A phase: 5-15mmol/L KH2PO4+1-10mmol/L H3PO4+ 80-90%H2O+
10-20%CH3CN (pH=2-4)；Mobile phase B phase: 1-10mmol/L NH4Ac+92-98%H2O+2-8%CH3CN；Gradient is washed
De-.
The specific procedure of described gradient elution is: 0-30min, A phase: B phase volume ratio is 100:0-0:100.
It addition, the multidimensional liquid chromatography mass combined system of the present invention is when specifically measuring, as shown in Figure 6, described first
Dimension liquid-chromatography apparatus includes first be sequentially communicated and ties up liquid chromatography pump, two six-way valves of sample introduction, the first dimension chromatographic column, purple
External detector.
As shown in Figure 6, two six-way valves of described sample introduction are provided with the 1st interface, the 2nd interface, interface 3, the 4th interface, the 5th connect
Mouthful, the 6th interface, described 4th interface is injection port, and described 4th interface is sequentially communicated the 5th interface, the 2nd interface through pipeline, the 3rd connects
Mouthful, interface 3 the sample introduction end of liquid chromatography pump is tieed up through pipeline connection first, described 1st interface is through pipeline connection the first dimension liquid
The sample outlet end of phase chromatogram pump, described 6th interface is through pipeline connection the first dimension chromatographic column, and described 1st interface and the 6th interface are connected
Logical.
As shown in Figure 6, described multidimensional liquid chromatography mass combined system also includes communicating valve, described communicating valve respectively with
UV-detector in first dimension liquid-chromatography apparatus, the second dimension liquid chromatography pump in two-dimensional liquid chromatography device, second
Dimension chromatographic column is connected.Described communicating valve is two ten-way valves or two six-way valves.
Described two ten-way valves be provided with the 1st interface, the 2nd interface, interface 3, the 4th interface, the 5th interface, the 8th interface, the 9th
Interface, the 10th interface, described 1st interface and the 10th interface is connected, and described 1st interface goes out sample through pipeline and UV-detector
End is connected, and described 10th interface is connected through the sample introduction end of pipeline and the second dimension trapping column, going out of described second dimension trapping column
Sample end is sequentially communicated interface 3, the 2nd interface, the sample introduction end of the second dimension liquid chromatography pump, described two-dimensional liquid chromatography through pipeline
The sample outlet end of pump is sequentially communicated the 9th interface, the 8th interface, the 5th interface, the 4th interface, the second dimension chromatographic column sample introduction end, institute through pipeline
The sample outlet end stating the second dimension chromatographic column is connected with mass spectrograph through pipeline.
Described two six-way valves are provided with the 1st interface, the 2nd interface, interface 3, the 4th interface, the 9th interface, the 10th interface, institute
Stating the 1st interface and the 10th interface is connected, described 1st interface is connected with the sample outlet end of UV-detector through pipeline, and described
10 interfaces are connected through the sample introduction end of pipeline and the second dimension trapping column, and the sample outlet end of described second dimension trapping column connects successively through pipeline
Logical interface 3, the 2nd interface, the sample introduction end of the second dimension liquid chromatography pump, the sample outlet end of described second dimension liquid chromatography pump is through pipeline
Being sequentially communicated the 9th interface, the 4th interface, the second dimension chromatographic column sample introduction end, the sample outlet end of described second dimension chromatographic column is through pipeline and matter
Spectrometer is connected.
When using multidimensional liquid chromatography mass combined system to be analyzed, the sample solution being analysed to is by sample introduction two six
4th interface of logical valve enters, and sample solution passes sequentially through the 5th interface, the 2nd interface, interface 3, enters the first dimension liquid chromatograph
The sample introduction end of pump, then entered the first dimension chromatographic column, the by the sample outlet end of the first dimension liquid chromatography pump through the 1st interface, the 6th interface
After one-dimensional chromatographic column effectively removes interfering material, by UV-detector, enter communicating valve, by the 1st of two ten-way valves the
Interface through the 10th interface enter second dimension trapping column, then from second dimension trapping column sample outlet end through interface 3, the 2nd interface, second
Dimension liquid chromatography pump, the 9th interface, the 8th interface, the 5th interface, the 4th interface enter the second dimension chromatographic column, divide in the second dimension chromatographic column
Separate out TSNAs, enter mass spectrograph and be measured；Or enter the second dimension by the 1st interface of two six-way valves through the 10th interface
Trapping column, then from second dimension trapping column sample outlet end through interface 3, the 2nd interface, second dimension liquid chromatography pump, the 9th interface, the 4th
Interface enters the second dimension chromatographic column, isolates TSNAs in the second dimension chromatographic column, enters mass spectrograph and be measured.
Embodiment 2
1, sample pre-treatments
Choose Cigarette, use filter disc Trapping ways trapping cigarette smoke after lighting, make flue gas be adsorbed in cambridge filter
On.Cambridge filter is placed in 50ml EP pipe addition extraction solution, in being placed in supersonic generator, carries out ultrasonic extraction 40min,
Extraction solution is 50.0ml 0.1mol/L NH4Ac aqueous solution, 0.1mol/L NH4Ac aqueous solution is by weighing 3.85g acetic acid
Ammonium, after being completely dissolved with water, is transferred in 500ml volumetric flask, and the constant volume that adds water obtains.Stand 5min after extraction, take 2ml extract
Being placed on centrifuge centrifugal 5min, centrifugal rotational speed is 3000rpm, then takes 1ml supernatant after 0.22 μm aqueous phase membrane filtration,
Move to, in chromatography bottle, obtain testing sample solution.
2, the preparation of standard solution
Pipette the standard substance of NAB, NAT, NNN, NNK that 10ml concentration is 0.5mg/ml respectively, be placed in 100mL volumetric flask
In, add methanol constant volume, be made into hybrid standard stock solution.The concentration of NAB, NAT, NNN, NNK in hybrid standard stock solution
It is 50 μ g/ml.Meanwhile, pipette d4-NNN, d4-NAT, d4-NAB, d4-NNK internal standard substance of certain volume respectively, add methanol
Constant volume, is made into inner mark solution, and in inner mark solution, the concentration of d4-NNN, d4-NAT, d4-NAB, d4-NNK is 5 μ g/ml.
The most accurately pipette different volumes hybrid standard stock solution, be placed in multiple 100mL volumetric flask, more accurately
Add the inner mark solution of certain volume, by methanol constant volume, be formulated as a series of mixed standard solution.The serial hybrid standard of preparation
Solution concentration is 0-10ng/ml, and wherein internal standard substance concentration is 1ng/ml.
3, the mensuration of sample size
Respectively testing sample solution in 2 standard solution and 1 prepared being carried out multidimensional liquid chromatography mass combination respectively is
System detection, will hybrid standard stock solution and testing sample solution tie up after liquid-chromatography apparatus carries out fraction by first, then
By the dilute solution of mixed standard solution Yu testing sample solution, (fraction of numbering 6,7,8,9,10,11 uses CH respectively3CN is dilute
Release 100 times, each fraction equal sample introduction 10 μ L) separated by described two-dimensional liquid chromatography device, then by described mass spectrometric apparatus
It is measured, compares retention time, scanning of the mass spectrum carries out qualitative, determines 4 kinds of nitrosamine compositions in liquid to be measured；Use internal standard simultaneously
It is quantitative that standard curve method carries out MRM, it is thus achieved that the content of 4 kinds of nitrosamine compositions in liquid to be measured.
Specifically, Internal standard curve method is first by the mixed standard solution of variable concentrations a series of in above-mentioned 2 respectively
Carry out the detection of multidimensional liquid chromatography mass combined system, obtain respectively two grades of selections of 4 kinds of nitrosamine compositions/corresponding internal standard substance from
The linear relationship of the mass concentration ratio of sub-peak area ratio and 4 kinds of nitrosamine composition/corresponding internal standard substances, draws the work of corresponding standard
Curve, with two grades of selection ion peak areas ratio (Y-axis) as vertical coordinate of 4 kinds of nitrosamine compositions with corresponding internal standard substance, its 4 kinds of nitrous
Amine component is abscissa (X-axis) with the mass concentration ratio of corresponding internal standard substance, is calculated 4 kinds of nitrosamine ingredient standard work respectively
The regression equation of curve.Liquid to be measured in above-mentioned 1 is carried out the detection of multidimensional liquid chromatography mass combined system again, to be measured by obtain
In liquid, two grades of selection ion peak areas ratios of 4 kinds of nitrosamine composition/corresponding internal standard substances, substitute into corresponding 4 kinds of nitrosamine and become minute mark
The regression equation of quasi-working curve, and according to adding the known quality concentration of corresponding internal standard substance, it is calculated 4 kinds of Asias in liquid to be measured
The mass concentration of nitramine composition.
Wherein, the chromatographic condition of described first dimension liquid-chromatography apparatus is:
First dimension liquid chromatograph: Waters ACQUITY UPLC；Chromatographic column: Waters Spherisorb S5SCX
(4.6*150mm,5μm)；Detection wavelength: 230nm；Column temperature: 30 DEG C；Flow velocity: 1mL/min；Sample size: 10 μ l；Mobile phase A phase:
10mmol/L KH2PO4+5mmol/L H3PO4+ 85%H2O+15%CH3CN (pH=3)；Mobile phase B phase: 10mmol/L KH2PO4
+5mmol/L H3PO4+ 0.5mol/L KCl+85%H2O+15%CH3CN (pH=3)；Gradient elution.
As shown in table 1, the specific procedure of described gradient elution is:
0-5min, A phase: B phase volume ratio is 100:0-90:10；
5-5.1min, A phase: B phase volume ratio is 90:10-60:40；
5.1-15min, A phase: B phase volume ratio is 60:40-0:100；
15-15.1min, A phase: B phase volume ratio is 0:100-100:0；
15.1-40min, A phase: B phase volume ratio is 100:0-100:0.
First dimension liquid-chromatography apparatus carries out fraction, as it is shown in figure 1, in spectrogram six peaks are collected fraction respectively, from a left side
To right number consecutively 6,7,8,9,10,11, thus obtain 6 fractions of numbered 6,7,8,9,10,11.Use heartcut again
Method, is completely transferred to two-dimensional liquid chromatography by the cutting of the chromatographic peak of the first dimension liquid chromatograph isolated object fraction,
Other non-targeted thing fraction does not enter two-dimensional liquid chromatography.
The condition of described two-dimensional liquid chromatography device is:
Two-dimensional liquid chromatography: Agilent Technologies 1260Infinity reversed-phase liquid chromatography (RPLC)；Color
Spectrum post: Waters Cortecs C18 (2.1*150mm, 2.7 μm)；Column temperature: 30 DEG C；Flow velocity: 0.25mL/min；Sample size: 10
μl；Mobile phase A phase: 5mmol/L NH4Ac+95%H2O+5%CH3CN；Mobile phase B phase: 5mmol/L NH4Ac+95%CH3CN+
5%H2O；Gradient elution.
As shown in table 3, the specific procedure of described gradient elution is:
0-10min, A phase: B phase volume ratio is 100:0-100:0；
10-20min, A phase: B phase volume ratio is 100:0-0:100；
20-22min, A phase: B phase volume ratio is 0:100-0:100；
22-22.1min, A phase: B phase volume ratio is 0:100-100:0；
22-25min, A phase: B phase volume ratio is 100:0-100:0.
The condition of described mass spectrometric apparatus is:
Mass spectrum: Agilent Technologies 6460Triple Quad MS；Ionization mode: ESI；Scan mode: many
Reaction monitoring (MRM)；MRM condition: be shown in Table 4；Cleaning sample introduction needle program: needle wash 30s；Mass spectrum stream switches: from
10min starts scanning, and 28min terminates.
After 6 fraction dilutions of obtain after the first dimension liquid-chromatography apparatus fraction numbered 6,7,8,9,10,11,
Separated by two-dimensional liquid chromatography device, and determine that each chromatographic peak is corresponding through mass spectrometric apparatus by Mass Spectrometer Method mass-to-charge ratio
Material.In first dimension liquid-chromatography apparatus, TSNAs peak sequence is NNK, NNK, NNN, NNN, NAT, NAB, the second dimension liquid phase color
In spectral apparatus, TSNAs peak sequence is NNN, NNK, NAT, NAB.
It is additionally provided with the second dimension trapping column between first dimension liquid-chromatography apparatus and two-dimensional liquid chromatography device.Described second
The condition of dimension trapping column is:
Chromatographic column: Waters Cortecs C18 post (2.7 μm, 2.1 × 10mm i.d.)；Mobile phase A phase: 10mmol/L
KH2PO4+5mmol/L H3PO4+ 85%H2O+15%CH3CN (pH=3)；Mobile phase B phase: 5mmol/L NH4Ac+95%H2O+
5%CH3CN；Gradient elution.
The specific procedure of described gradient elution is: 0-30min, A phase: B phase volume ratio is 100:0-0:100.
It addition, the multidimensional liquid chromatography mass combined system of the present invention is when specifically measuring, as shown in Figure 6, described first
Dimension liquid-chromatography apparatus includes first be sequentially communicated and ties up liquid chromatography pump, two six-way valves of sample introduction, the first dimension chromatographic column, purple
External detector.
As shown in Figure 6, two six-way valves of described sample introduction are provided with the 1st interface, the 2nd interface, interface 3, the 4th interface, the 5th connect
Mouthful, the 6th interface, described 4th interface is injection port, and described 4th interface is sequentially communicated the 5th interface, the 2nd interface through pipeline, the 3rd connects
Mouthful, interface 3 the sample introduction end of liquid chromatography pump is tieed up through pipeline connection first, described 1st interface is through pipeline connection the first dimension liquid
The sample outlet end of phase chromatogram pump, described 6th interface is through pipeline connection the first dimension chromatographic column, and described 1st interface and the 6th interface are connected
Logical.
As shown in Figure 6, described multidimensional liquid chromatography mass combined system also includes communicating valve, described communicating valve respectively with
UV-detector in first dimension liquid-chromatography apparatus, the second dimension liquid chromatography pump in two-dimensional liquid chromatography device, second
Dimension chromatographic column is connected.Described communicating valve is two ten-way valves or two six-way valves.
Described two ten-way valves be provided with the 1st interface, the 2nd interface, interface 3, the 4th interface, the 5th interface, the 8th interface, the 9th
Interface, the 10th interface, described 1st interface and the 10th interface is connected, and described 1st interface goes out sample through pipeline and UV-detector
End is connected, and described 10th interface is connected through the sample introduction end of pipeline and the second dimension trapping column, going out of described second dimension trapping column
Sample end is sequentially communicated interface 3, the 2nd interface, the sample introduction end of the second dimension liquid chromatography pump, described two-dimensional liquid chromatography through pipeline
The sample outlet end of pump is sequentially communicated the 9th interface, the 8th interface, the 5th interface, the 4th interface, the second dimension chromatographic column sample introduction end, institute through pipeline
The sample outlet end stating the second dimension chromatographic column is connected with mass spectrograph through pipeline.
Described two six-way valves are provided with the 1st interface, the 2nd interface, interface 3, the 4th interface, the 9th interface, the 10th interface, institute
Stating the 1st interface and the 10th interface is connected, described 1st interface is connected with the sample outlet end of UV-detector through pipeline, and described
10 interfaces are connected through the sample introduction end of pipeline and the second dimension trapping column, and the sample outlet end of described second dimension trapping column connects successively through pipeline
Logical interface 3, the 2nd interface, the sample introduction end of the second dimension liquid chromatography pump, the sample outlet end of described second dimension liquid chromatography pump is through pipeline
Being sequentially communicated the 9th interface, the 4th interface, the second dimension chromatographic column sample introduction end, the sample outlet end of described second dimension chromatographic column is through pipeline and matter
Spectrometer is connected.
When using multidimensional liquid chromatography mass combined system to be analyzed, the sample solution being analysed to is by sample introduction two six
4th interface of logical valve enters, and sample solution passes sequentially through the 5th interface, the 2nd interface, interface 3, enters the first dimension liquid chromatograph
The sample introduction end of pump, then entered the first dimension chromatographic column, the by the sample outlet end of the first dimension liquid chromatography pump through the 1st interface, the 6th interface
After one-dimensional chromatographic column effectively removes interfering material, by UV-detector, enter communicating valve, by the 1st of two ten-way valves the
Interface through the 10th interface enter second dimension trapping column, then from second dimension trapping column sample outlet end through interface 3, the 2nd interface, second
Dimension liquid chromatography pump, the 9th interface, the 8th interface, the 5th interface, the 4th interface enter the second dimension chromatographic column, divide in the second dimension chromatographic column
Separate out TSNAs, enter mass spectrograph and be measured；Or enter the second dimension by the 1st interface of two six-way valves through the 10th interface
Trapping column, then from second dimension trapping column sample outlet end through interface 3, the 2nd interface, second dimension liquid chromatography pump, the 9th interface, the 4th
Interface enters the second dimension chromatographic column, isolates TSNAs in the second dimension chromatographic column, enters mass spectrograph and be measured.
Embodiment 3
As shown in above-described embodiment 22, the most accurately pipette hybrid standard stock solution, use methanol constant volume, prepare dense
Degree is 0.25,0.5,1.0,2.5,25,50 and a series of mixed standard solutions of 100ng/mL, and wherein internal standard substance concentration is
10ng/ml。
The mixed standard solution of the above-mentioned a series of variable concentrations prepared is carried out multidimensional liquid chromatography mass connection respectively
Detecting by system, with two grades of selection ion peak areas ratio (Y-axis) as vertical coordinate of 4 kinds of nitrosamine compositions with corresponding internal standard substance, they are 4 years old
Kind of nitrosamine composition is abscissa (X-axis) with the mass concentration ratio of corresponding internal standard substance, carries out regression analysis, obtain regression equation and
Its correlation coefficient, as shown in table 5, Fig. 2-5.From table 5, Fig. 2-5, the linear relationship of regression equation is good, coefficient R2
=0.9817～0.9997.Object in mixed standard solution is responded signal, uses least concentration standard specimen to repeat sample introduction 10
Secondary, carry out multidimensional liquid chromatography mass combined system analysis, the detection limit (LOD) with 3 times of signal to noise ratios as method, 10 times of signal to noise ratios
For quantitative limit (LOQ), after being scaled sample size, show that the method detection of object is limited to 0.023～0.045ng/ml, quantitatively
It is limited to 0.076～0.151ng/ml, there is higher sensitivity.
Table 5 working curve and detection limit
Y: ion peak areas ratio；X: concentration ratio
Embodiment 4
Choose the cigarette smoke sample of known nitrosamine concentration, add the mixed standard solution of concentration known, then carry out
The pre-treatment of 1 in above-described embodiment 2, and carry out multidimensional liquid chromatography mass combined system analysis, according to sample with add scalar=
1:1/2 or 1:1 or 1:2, calculates its response rate.Same testing sample solution is carried out parallel assay 5 times (n=5) simultaneously, obtains
The precision determination data of 4 kinds of nitrosamine compositions, the results are shown in Table 6.
The response rate of 64 kinds of nitrosamine compositions of table and precision
As can be seen from Table 6, the response rate of object between 93.4～106.2%, in a few days relative standard deviation (RSD)
Less than 6%, relative standard deviation (RSD) is less than 9% in the daytime, and response rate height, accuracy and the repeatability of the inventive method are described
Good, quantitative needs can be met.
Embodiment 5
In the analysis method mensuration conventional cigarette fume sample of the employing such as present invention in embodiment 2,4 kinds of nitrosamine contains
Amount, concrete outcome is shown in Table 7.As shown in Table 7, it is thus achieved that TSNAs content: NNK 5.37ng/cig, NAB 2.89ng/cig, NAT
5.52ng/cig, NNN 7.68ng/cig, consistent with the contents level of classical way sample, the method has the most accurately
Property, can apply in the analysis mensuration of high-volume actual sample.Wherein, the content (ng/cig) of sample is dividing the present invention
Obtain after sample concentration (ng/mL) × 20mL/20cig that analysis method measures.
The content of 4 kinds of nitrosamine in table 7 actual cigarette smoke sample
| Sequence number | Components Name | The content (ng/cig) of sample |
| 1 | NNK | 5.37 |
| 2 | NAB | 2.89 |
| 3 | NAT | 5.52 |
| 4 | NNN | 7.68 |
So, the present invention effectively overcomes various shortcoming of the prior art and has high industrial utilization.
The principle of above-described embodiment only illustrative present invention and effect thereof, not for limiting the present invention.Any ripe
Above-described embodiment all can be modified under the spirit and the scope of the present invention or change by the personage knowing this technology.Cause
This, have usually intellectual such as complete with institute under technological thought without departing from disclosed spirit in art
All equivalences become are modified or change, and must be contained by the claim of the present invention.
Claims (11)
1. an analysis method for nitrosamine burst size in cigarette smoke, carries out sample pre-treatments to cigarette smoke, uses multidimensional
Liquid chromatography mass combined system carries out qualitative and quantitative analysis, described multidimensional liquid chromatography mass to pre-treatment gained testing sample
Combined system includes the first dimension liquid-chromatography apparatus, two-dimensional liquid chromatography device, the mass spectrometric apparatus being sequentially connected with.
The analysis method of nitrosamine burst size in cigarette smoke the most according to claim 1, it is characterised in that described Medicated cigarette
In flue gas nitrosamine include N-nitrosonornicotine, 4-(methyl nitroso amino)-l-(3-is than piperidinyl)-1-butanone,
N-nitroso-group anabasine and N-nitrosoanatabine.
The analysis method of nitrosamine burst size in cigarette smoke the most according to claim 1, it is characterised in that described sample
Pre-treatment comprises the following steps:
1) choose Cigarette, after lighting, use filter disc Trapping ways trapping cigarette smoke；
2) stand after filter disc being carried out ultrasonic extraction, be centrifuged, after taking supernatant liquid filtering, obtain testing sample.
The analysis method of nitrosamine burst size in cigarette smoke the most according to claim 1, it is characterised in that described employing
Multidimensional liquid chromatography mass combined system carries out qualitative and quantitative analysis to pre-treatment gained testing sample, comprises the following steps:
A) preparation standard sample: preparation hybrid standard stock solution, inner mark solution and mixed standard solution；
B) sample qualitative detection: respectively by step A) standard sample prepared and after sample pre-treatments testing sample carry out multidimensional
Liquid chromatography mass combined system detects, after carrying out fraction by the first dimension liquid-chromatography apparatus, by described second dimension liquid phase color
Spectral apparatus separates, then is measured by described mass spectrometric apparatus, compares retention time, scanning of the mass spectrum carries out qualitative, determines and treats
4 kinds of nitrosamine compositions in test sample product；
C) sample amounts detection: respectively by step A) standard sample prepared and after sample pre-treatments testing sample carry out multidimensional
Liquid chromatography mass combined system detects, after carrying out fraction by the first dimension liquid-chromatography apparatus, by described second dimension liquid phase color
Spectral apparatus separates, then is measured by described mass spectrometric apparatus, uses Internal standard curve method to carry out MRM quantitative, it is thus achieved that to treat
The content of 4 kinds of nitrosamine compositions in test sample product.
The analysis method of nitrosamine burst size in cigarette smoke the most according to claim 4, it is characterised in that described step
B), in or C), the chromatographic condition of described first dimension liquid-chromatography apparatus is:
First dimension liquid chromatograph: positive liquid chromatograph；Chromatographic column: strong cation exchange chromatography post；Detection wavelength: 220-240nm；
Column temperature: 25-35 DEG C；Flow velocity: 0.5-1.5mL/min；Sample size: 5-15 μ l；Mobile phase A phase: 5-15mmol/LKH2PO4+1-
10mmol/L H3PO4+ 80-90%H2O+10-20%CH3CN, pH=2-4；Mobile phase B phase: 5-15mmol/LKH2PO4+1-
10mmol/L H3PO4+ 0.1-1.0mol/L KCl+80-90%H2O+10-20%CH3CN, pH=2-4；Gradient elution.
The analysis method of nitrosamine burst size in cigarette smoke the most according to claim 5, it is characterised in that described gradient
The specific procedure of eluting is:
0-5min, A phase: B phase volume ratio is 100:0-90:10；
5-5.1min, A phase: B phase volume ratio is 90:10-60:40；
5.1-15min, A phase: B phase volume ratio is 60:40-0:100；
15-15.1min, A phase: B phase volume ratio is 0:100-100:0；
15.1-40min, A phase: B phase volume ratio is 100:0-100:0.
The analysis method of nitrosamine burst size in cigarette smoke the most according to claim 4, it is characterised in that described step
B), in or C), the condition of described two-dimensional liquid chromatography device is:
Two-dimensional liquid chromatography: reversed-phase liquid chromatography；Chromatographic column: C18 post；Column temperature: 25-35 DEG C；Flow velocity: 0.2-0.3mL/min；
Sample size: 5-15 μ l；Mobile phase A phase: 1-10mmol/L NH4Ac+92-98%H2O+2-8%CH3CN；Mobile phase B phase: 1-
10mmol/L NH4Ac+92-98%CH3CN+2-8%H2O；Gradient elution.
The analysis method of nitrosamine burst size in cigarette smoke the most according to claim 7, it is characterised in that described gradient
The specific procedure of eluting is:
0-10min, A phase: B phase volume ratio is 100:0-100:0；
10-20min, A phase: B phase volume ratio is 100:0-0:100；
20-22min, A phase: B phase volume ratio is 0:100-0:100；
22-22.1min, A phase: B phase volume ratio is 0:100-100:0；
22-25min, A phase: B phase volume ratio is 100:0-100:0.
The analysis method of nitrosamine burst size in cigarette smoke the most according to claim 4, it is characterised in that described step
B), in or C), the condition of described mass spectrometric apparatus is:
Mass spectrum: triple quadrupole bar mass spectrum；Ionization mode: ESI；Scan mode: multiple-reaction monitoring MRM；Cleaning sample introduction needle program:
needle wash 20-40s；Mass spectrum stream switches: starting scanning from 8-12min, 26-30min terminates.
The analysis method of nitrosamine burst size in cigarette smoke the most according to claim 4, it is characterised in that described step
Rapid B) or C) in, be additionally provided with the second dimension trapping column between described first dimension liquid-chromatography apparatus and two-dimensional liquid chromatography device；
The condition of described second dimension trapping column is:
Chromatographic column: C18 post；Mobile phase A phase: 5-15mmol/L KH2PO4+1-10mmol/L H3PO4+ 80-90%H2O+10-
20%CH3CN, pH=2-4；Mobile phase B phase: 1-10mmol/L NH4Ac+92-98%H2O+2-8%CH3CN；Gradient elution.
The analysis method of nitrosamine burst size in 11. cigarette smokes according to claim 10, it is characterised in that described ladder
The specific procedure of degree eluting is: 0-30min, A phase: B phase volume ratio is 100:0-0:100.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111257490A (en) * | 2020-02-17 | 2020-06-09 | 沈阳农业大学 | Method for simultaneously detecting contents of 13 substances in tobacco leaves |
| CN113970601A (en) * | 2020-07-23 | 2022-01-25 | 湖南中烟工业有限责任公司 | On-line analysis and detection device and method for smoke of heated cigarette |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104535694A (en) * | 2015-01-21 | 2015-04-22 | 中国烟草总公司郑州烟草研究院 | Method for detecting four tobacco-specific nitrosamines (TSNAs) in lateral exhaust gas of cigarettes by virtue of gas chromatography-tandem mass spectrometry |
| US20150198574A1 (en) * | 2014-01-15 | 2015-07-16 | King Abdulaziz City For Science And Technology | Automated microextraction technique for the analysis of n-nitrosoamines in water |
| CN105510489A (en) * | 2015-12-22 | 2016-04-20 | 云南中烟工业有限责任公司 | Liquid phase and gaseous phase two-dimensional chromatogram for liquid chromatogram fraction online continuous cutting detection and application thereof |
| CN105510462A (en) * | 2015-12-09 | 2016-04-20 | 中国烟草总公司郑州烟草研究院 | Improved method for analyzing and detecting TSNAs (Tobacco-Specific Nitrosamines) in tobacco liquid of electronic cigarette |
-
2016
- 2016-08-02 CN CN201610625685.8A patent/CN106290627B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150198574A1 (en) * | 2014-01-15 | 2015-07-16 | King Abdulaziz City For Science And Technology | Automated microextraction technique for the analysis of n-nitrosoamines in water |
| CN104535694A (en) * | 2015-01-21 | 2015-04-22 | 中国烟草总公司郑州烟草研究院 | Method for detecting four tobacco-specific nitrosamines (TSNAs) in lateral exhaust gas of cigarettes by virtue of gas chromatography-tandem mass spectrometry |
| CN105510462A (en) * | 2015-12-09 | 2016-04-20 | 中国烟草总公司郑州烟草研究院 | Improved method for analyzing and detecting TSNAs (Tobacco-Specific Nitrosamines) in tobacco liquid of electronic cigarette |
| CN105510489A (en) * | 2015-12-22 | 2016-04-20 | 云南中烟工业有限责任公司 | Liquid phase and gaseous phase two-dimensional chromatogram for liquid chromatogram fraction online continuous cutting detection and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| JIE ZHANG等: "Fully automated analysis of four tobacco-specific N-nitrosamines in mainstream cigarette smoke using two-dimensional online solid phase extraction combined with liquid chromatography–tandem mass spectrometry", 《TALANTA》 * |
| 万文亚等: "LC-MS-MS法测定卷烟侧流烟气中的亚硝胺", 《分析试验室》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111257490A (en) * | 2020-02-17 | 2020-06-09 | 沈阳农业大学 | Method for simultaneously detecting contents of 13 substances in tobacco leaves |
| CN111257490B (en) * | 2020-02-17 | 2022-09-20 | 沈阳农业大学 | Method for simultaneously detecting contents of 13 substances in tobacco leaves |
| CN113970601A (en) * | 2020-07-23 | 2022-01-25 | 湖南中烟工业有限责任公司 | On-line analysis and detection device and method for smoke of heated cigarette |
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