CN106053675B

CN106053675B - A kind of analysis method of twin columns liquid chromatography tandem mass spectrometry to nitrosamine burst size in cigarette smoke

Info

Publication number: CN106053675B
Application number: CN201610625682.4A
Authority: CN
Inventors: 陈敏; 王磊君; 吴达; 刘百战
Original assignee: Shanghai Tobacco Group Co Ltd
Current assignee: Shanghai Tobacco Group Co Ltd
Priority date: 2016-08-02
Filing date: 2016-08-02
Publication date: 2018-11-27
Anticipated expiration: 2036-08-02
Also published as: CN106053675A

Abstract

The present invention provides a kind of twin columns liquid chromatography tandem mass spectrometry to the analysis method of nitrosamine burst size in cigarette smoke, sample pre-treatments are carried out to cigarette smoke, qualitative and quantitative analysis is carried out to sample to be tested obtained by pre-treatment using twin columns Liquid Chromatography-Tandem Mass Spectrometry system, the twin columns Liquid Chromatography-Tandem Mass Spectrometry system includes the first dimension liquid chromatography pump, the first splitter, the second dimension liquid chromatography pump, the second splitter, mass spectrometric apparatus.Invention further provides a kind of twin columns Liquid Chromatography-Tandem Mass Spectrometry systems.The present invention provides a kind of twin columns liquid chromatography tandem mass spectrometry to the analysis method of nitrosamine burst size in cigarette smoke, sample pre-treatments are simple, and detection efficiency is high, can be effectively removed interfering substance in flue gas, excellent separating degree, sensitivity are obtained, testing result is accurate and reliable.

Description

A kind of twin columns liquid chromatography tandem mass spectrometry is to nitrosamine burst size in cigarette smoke Analysis method

Technical field

The invention belongs to the technical fields of chemical composition analysis important in cigarette mainstream flue gas, are related to a kind of twin columns liquid phase color Tandem mass spectrometry is composed to the analysis method of nitrosamine burst size in cigarette smoke.

Background technique

Tobacco-specific nitrosamine (tobacco-specific N-nitrosamines TSNAs) in cigarette smoke is Flue gas harmful substance in Hoffmann inventory and seven kinds of harmful components appraisement systems, be in flue gas most important compound it One, it is all the hot spot of tobacco chemistry research all the time.Tobacco-specific nitrosamine (TSNAs) mainly includes 4- (N- methyl nitrous Amido) -1- (3- pyridyl group) -1- butanone (NNK), N- nitrosonornicotine (NNN), N- nitroso anabasine (NAB) and N- Nitrosoanabasine (NAT), structure is as shown in Figure 1.

Since flue gas TSNAs burst size is lower and since smoke components backgrounds is complicated, matrix interference is serious, it is difficult to its standard Really measurement.It is worth noting that fire-cured tobacco type an order of magnitude lower than blended type cigarette or so in the case of same tar, thus to volume The accurate quantitative analysis of cigarette especially Virginian-type cigarette TSNAs burst size is always the key points and difficulties of tobacco chemistry analysis.

Currently, the analysis method of TSNAs mainly has gas-chromatography-thermal energy analyzer method (GC-TEA), gas-chromatography series connection Mass spectrography GC-MS/MS, LC-MS LC-MS/MS method.That there are pre-treatment steps is loaded down with trivial details for GC-TEA method, sample analysis time is long, The disadvantages of detection sensitivity is lower, and LC-MS method has that matrix interference is serious, the sensitivity of method, the problems such as detection limit is lower.Cause This, developing the applicable Multidimensional Liquid Chromatography Technology of such compound, to carry out separation analysis very necessary.

Multidimensional liquid chromatogram (MDLC) is that separating mechanism is different and mutually independent liquid phase separation mode is together in series The separation system of composition, sample enter subsequent chromatographic column in the target fraction of first liquid-phase chromatographic column and carry out secondary separation, It is to separate one of most important means of analysis to complex sample at present that it is combined with mass spectrum (MS).Multidimensional liquid chromatography-mass spectrography Combined system is used to analyze various complex matrices samples in tobacco and fume sample, although with high selectivity and sensitive Degree, but the compatibility of mobile phase, the switching of valve interface and the enrichment of object need to be comprehensively considered there are also mass spectrographic compatibility, so building Vertical online measuring technique difficulty is larger.

Summary of the invention

In view of the foregoing deficiencies of prior art, the purpose of the present invention is to provide a kind of twin columns liquid chromatography tandem matter Spectrometry is used to solve in the prior art due to cigarette mainstream flue gas tobacco to the analysis method of nitrosamine burst size in cigarette smoke Unique nitrosamine (TSNAs) burst size is lower, and matrix interference is relatively serious, is difficult to the problem of Accurate Determining.

In order to achieve the above objects and other related objects, first aspect present invention provides a kind of twin columns liquid chromatography tandem matter Spectrometry carries out sample pre-treatments to cigarette smoke, using twin columns liquid phase to the analysis method of nitrosamine burst size in cigarette smoke Chromatographic tandem mass spectrometer system carries out qualitative and quantitative analysis, the twin columns Liquid Chromatography-Tandem Mass Spectrometry to sample to be tested obtained by pre-treatment System includes the first dimension liquid chromatogram, the first splitter, two-dimensional liquid chromatography, the second splitter, mass spectrometric apparatus.

Preferably, nitrosamine includes N- nitrosonornicotine (NNN), 4- (methyl nitrous in the cigarette smoke Base amino)-l- (3- is than piperidinyl) -1- butanone (NNK), N- nitroso anabasine (NAB) and N- nitrosoanatabine (NAT)。

Preferably, the sample pre-treatments include the following steps：

1) Cigarette is chosen, is aspirated after lighting, traps cigarette smoke using filter disc Trapping ways；

2) film is crossed after filter disc being carried out ultrasonic extraction to get sample to be tested.

Preferably, in step 1), the suction is carried out using multi-pore channel rotating disc type smoking machine.It is highly preferred that described more Duct rotating disc type smoking machine is 20 duct rotating disc type smoking machines.The 20 duct rotating disc type smoking machine can disposably aspirate 20 The flue gas of cigarette.

Preferably, in step 1), the filter disc is cambridge filter.The diameter of the cambridge filter is 44mm.

Preferably, in step 1), the trapping cigarette smoke is by prescriptive procedure in standard GB/T/T 19609-2004 It is trapped.

Preferably, in step 2), the extraction solution is ammonium acetate (NH₄Ac) aqueous solution, the extraction solution add Enter ammonium acetate aqueous solution/20 cigarette smoke that amount is 10-20mL 0.1mol/L.It is highly preferred that the addition of the extraction solution Amount is ammonium acetate aqueous solution/20 cigarette smoke of 20mL 0.1mol/L.The ammonium acetate aqueous solution, by weighing ammonium acetate, It after being completely dissolved with water, is transferred in volumetric flask, water constant volume is added to obtain.

Preferably, in step 2), inner mark solution is additionally added in the extraction solution, the additional amount of the inner mark solution is Inner mark solution/20 1-2mL400ng/mL cigarette smoke.It is highly preferred that the additional amount of the inner mark solution is 1mL 400ng/ Inner mark solution/20 mL cigarette smoke.

It is highly preferred that the inner mark solution by weighing NNN-d respectively₄、NNK-d₄、NAT-d₄、NAB-d₄, methanol is added It is obtained after constant volume.

It is further preferred that NNN-d in the inner mark solution₄、NNK-d₄、NAT-d₄、NAB-d₄Concentration be 400ng/ mL。

It is further preferred that the NNN-d₄、NNK-d₄、NAT-d₄、NAB-d₄For deuterated reagent.Specifically, the NNN- d₄、NNK-d₄、NAT-d₄、NAB-d₄Respectively deuterated N- nitrosonornicotine, deuterated 4- (methyl nitroso amino)-l- (3- is than piperidinyl) -1- butanone, deuterated N- nitrosoanatabine, deuterated N- nitroso anabasine.

The NNN-d₄、NNK-d₄、NAT-d₄、NAB-d₄For commercial reagents, chemical property is stablized, and is suitable for interior Mark.

Preferably, in step 2), the time of the ultrasonic extraction is 10-50min.It is highly preferred that the ultrasonic extraction Time be 40min.

Preferably, in step 2), the mode for crossing film is water phase membrane filtration mode.It is highly preferred that the water phase The aperture of filter membrane is 0.22 μm.

Preferably, described qualitative fixed to the progress of sample to be tested obtained by pre-treatment using twin columns Liquid Chromatography-Tandem Mass Spectrometry system Amount analysis, includes the following steps：

A standard sample) is prepared：Prepare hybrid standard stock solution, inner mark solution and mixed standard solution；

A1 the standard items of NAB, NAT, NNN, NNK) are weighed respectively, and methanol constant volume is added, is made into hybrid standard stock solution；

Preferably, the concentration of NAB, NAT, NNN, NNK are 10 μ g/ml in the hybrid standard stock solution.

A2 NNN-d4, NAT-d4, NAB-d4, NNK-d4) are weighed respectively, and methanol constant volume is added, is made into inner mark solution；

Preferably, the concentration of NNN-d4, NAT-d4, NAB-d4, NNK-d4 are 400ng/ml in the inner mark solution.

Preferably, described NNN-d4, NAT-d4, NAB-d4, NNK-d4 are deuterated reagent.Specifically, the NNN-d4, NAT-d4, NAB-d4, NNK-d4 are respectively deuterated N- nitrosonornicotine, deuterated 4- (methyl nitroso amino)-l- (3- Than piperidinyl) -1- butanone, deuterated N- nitrosoanatabine, deuterated N- nitroso anabasine.

Described NNN-d4, NAT-d4, NAB-d4, NNK-d4 are commercial reagents, and chemical property is stablized, and are suitable for interior Mark.

A3 hybrid standard stock solution in the step A1 of different volumes) is pipetted respectively, then is separately added into the step of certain volume Inner mark solution in rapid A2, a series of mixed standard solution of various concentrations is formulated as with methanol constant volume.

Preferably, the concentration of the mixed standard solution is 0-100ng/ml.

Preferably, in the mixed standard solution, the concentration of NNN-d4, NAT-d4, NAB-d4, NNK-d4 are 10ng/ ml。

B) sample qualitative detection：By the step A) standard sample prepared and after sample pre-treatments, sample to be tested is carried out respectively Twin columns Liquid Chromatography-Tandem Mass Spectrometry system detection, under the first dimension liquid chromatogram effect, after carrying out fraction by the first splitter, Under two-dimensional liquid chromatography effect, is separated, then be measured by the mass spectrometric apparatus, compared by second splitter Retention time, scanning of the mass spectrum progress are qualitative, determine 4 kinds of nitrous amine components in sample to be tested；

C) sample amounts detect：By the step A) standard sample prepared and after sample pre-treatments, sample to be tested is carried out respectively Twin columns Liquid Chromatography-Tandem Mass Spectrometry system detection, under the first dimension liquid chromatogram effect, after carrying out fraction by the first splitter, It under two-dimensional liquid chromatography effect, is separated, then be measured by the mass spectrometric apparatus, is used by second splitter Internal standard curve method carries out MRM and quantifies, and obtains the content of 4 kinds of nitrous amine components in sample to be tested.

Preferably, the step B) or C) in, it is described first dimension liquid chromatogram effect under, pass through the first splitter carry out Fraction be first dimension liquid chromatogram effect under, by the step A3 mixed standard solution prepared and after sample pre-treatments to test sample Product solution carries out fraction by the first splitter.

Preferably, the step B) or C) in, it is described two-dimensional liquid chromatography effect under, by second splitter into Row separation is under two-dimensional liquid chromatography effect, and the mixed standard solution and sample to be tested that step A3 is prepared are by second point It is separated from column.

Preferably, the step B) or C) in, it is described first dimension liquid chromatogram condition be：First dimension liquid chromatogram：Just Phase liquid chromatogram；Column temperature：25-35℃；Flow velocity：0.5-1.5mL/min；Sample volume：5-15μl；First dimension mobile phase A phase：5- 15mmol/L KH₂PO₄+1-10mmol/L H₃PO₄+ 80-90%H₂O+10-20%CH₃CN (pH=2-4)；First dimension 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 elution.

It is highly preferred that the condition of the first dimension liquid chromatogram is：First dimension liquid chromatogram：Agilent Technologies 1260Infinity positive liquid chromatogram；Column temperature：30℃；Flow velocity：1mL/min；Sample volume：10μl；First Tie up mobile phase A phase：10mmol/L KH₂PO₄+5mmol/L H₃PO₄+ 85%H₂O+15%CH₃CN (pH=2.8)；One-Dimensional flows Phase B phase：10mmol/L KH₂PO₄+5mmol/L H₃PO₄+ 0.5mol/L KCl+85%H₂O+15%CH₃CN (pH=2.8)；Ladder Degree elution.

Above-mentioned percentage % is percent by volume.

Most preferably, as shown in table 1, the specific procedure of the 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 the dimension liquid chromatogram of table 1 first

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, the step B) or C) in, first splitter be strong cation exchange chromatography column (SCX).

It is highly preferred that the strong cation exchange chromatography column (SCX) is Waters Spherisorb S5 SCX column (4.6* 150mm,5μm)。

Preferably, the step B) or C) in, the condition of the two-dimensional liquid chromatography is：Two-dimensional liquid chromatography：Instead Phase liquid chromatogram (RPLC)；Column temperature：25-35℃；Flow velocity：0.2-0.3mL/min；Sample volume：5-15μl；Second dimension mobile phase A Phase：1-10mmol/L NH₄Ac+92-98%H₂O+2-8%CH₃CN；Second dimension Mobile phase B phase：1-10mmol/L NH₄Ac+92- 98%CH₃CN+2-8%H₂O；Gradient elution.

It is highly preferred that the condition of the two-dimensional liquid chromatography is：Two-dimensional liquid chromatography：Agilent Technologies 1260Infinity reversed-phase liquid chromatography (RPLC)；Column temperature：30℃；Flow velocity：0.25mL/min；Sample volume： 10μl；Second dimension mobile phase A phase：5mmol/LNH₄Ac+95%H₂O+5%CH₃CN；Second dimension Mobile phase B phase：5mmol/ LNH₄Ac+95%CH₃CN+5%H₂O；Gradient elution.

Above-mentioned percentage % is percent by volume.

Most preferably, as shown in table 2, the specific procedure of the 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 2 two-dimensional liquid chromatography of table

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, the step B) or C) in, second splitter be Trap trapping column.

It is highly preferred that the Trap trapping column is Thermo scientificAcclaim PolarAdvantage II C18 column (4.6*50mm, 3 μm).

Preferably, the step B) or C) in, the condition of the mass spectrometric apparatus is：

Mass spectrum：Agilent Technologies 6460Triple Quad MS triple quadrupole bar mass spectrum；Ionization mode： Electric spray ion source (ESI)；Scanning mode：Multiple-reaction monitoring (MRM)；MRM condition：It is shown in Table 3；(IS) voltage is by spraying： 5000V；Atomization gas (Gas1) pressure is：482.6kPa；Gas curtain gas (Curtain Gas) pressure is：206.7kPa；Assisted atomization Gas (Gas2) pressure is：482.6kPa；Ion source temperature (TEM) is：500℃；Clean sample introduction needle program：needle wash 30s；The switching of mass spectrum flow path：It is scanned since 10min, 28min terminates.

3 MRM condition of table

	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, the step B) or C) in, first splitter carries out the pH value tune of object fraction after fraction Section obtains the fraction of 4 kinds of nitrous amine components using heartcut method to neutrality.The fraction of 4 kinds of nitrous amine components includes NNK, NNN, NAT, NAB fraction.

It is highly preferred that the pH value being adjusted to neutrality is 6.8-7.2.

The heartcut method is common separation method in multi-dimensional chromatograph, in twin columns liquid chromatogram of the invention, i.e., Will be under the first dimension liquid chromatogram effect, the cutting of the chromatographic peak for the object fraction that the first splitter is isolated is complete, the Under two-dimensional liquid chromatography effect, it is transferred to the trapping of the second splitter and retains, other non-targeted object fractions are not into the second splitter.

It is highly preferred that the fraction of 4 kinds of nitrous amine components is to separate post separation by second, and pass through through mass spectrometric apparatus Mass Spectrometer Method mass-to-charge ratio determines the corresponding substance of each chromatographic peak.The mass-to-charge ratio numerical value of 4 kinds of nitrous amine components is shown in Table 3.Institute The mother ion mass-to-charge ratio for stating NNK fraction is 208.1, and daughter ion mass-to-charge ratio is 122.1；The mother ion mass-to-charge ratio of the NAB fraction It is 192.1, daughter ion mass-to-charge ratio is 162.1；The mother ion mass-to-charge ratio of the NAT fraction is 190.1, and daughter ion mass-to-charge ratio is 160.1；The mother ion mass-to-charge ratio of the NNN fraction is 178.1, and daughter ion mass-to-charge ratio is 148.1.

Preferably, the step B) or C) in, it is described first dimension liquid chromatogram and the first splitter between be equipped with sampling valve.

It is highly preferred that the sampling valve is two six-way valves, the sampling valve connects equipped with the 1st interface, the 2nd interface, the 3rd Mouth, the 4th interface, the 5th interface, the 6th interface, the 4th interface are injection port, and the interface 3 is waste discharge mouth, and the described 1st connects Mouth is connected through pipeline with the first dimension liquid chromatogram, and the 6th interface is connected through pipeline with the first splitter.

Preferably, the step B) or C) in, between first splitter and two-dimensional liquid chromatography be equipped with communicating valve, Between the communicating valve and the first splitter be equipped with mixer, the mixer respectively with first splitter, communicating valve phase Connection.

It is highly preferred that the communicating valve is two six-way valves, the communicating valve connects equipped with the 1st interface, the 2nd interface, the 3rd Mouthful, the 4th interface, the 5th interface, the 6th interface, the 1st interface is connected through pipeline with the sample introduction end of the second splitter, described the 4 interfaces are connected through pipeline with the sample outlet end of the second splitter, and the 2nd interface is connected through pipeline with two-dimensional liquid chromatography Logical, the interface 3 is connected through pipeline with mass spectrometric apparatus, and the 5th interface is waste discharge mouth, and the 6th interface is through pipeline and mixes Clutch is connected.

It is further preferred that the 5th interface is also connected with UV detector, the UV detector is through pipeline and the 5th Interface is connected.

It is further preferred that the Detection wavelength of the UV detector is 220-240nm.

Most preferably, the Detection wavelength of the UV detector is 230nm.

It is highly preferred that the mixer is also connected with compensation pump, the compensation pump is connected through pipeline with mixer.It is described For adjusting the pH value of One-Dimensional flows phase and diluting acetonitrile concentration in One-Dimensional flows phase, the compensation pump can add compensation pump It does not add.

It is further preferred that the compensation pump is that unitary pumps.

It is further preferred that the condition of the compensation pump is：Compensating mobile phase is：1-10mmol/L K₂HPO₄；Flow velocity： 0.6-1.0mL/min。

Most preferably, the condition of the compensation pump is：Compensating mobile phase is：5mmol/L K₂HPO₄；Flow velocity：0.8mL/ min。

Preferably, the step C) in, the Internal standard curve method includes the following steps：

The Internal standard curve method refers to is added internal standard substance in standard solution, carries out Instrument measuring, obtains standard Working curve, and then the method for measuring the actual sample concentration containing internal standard substance.

The mixed standard solution of various concentrations a series of in the A3 of step A) is carried out twin columns liquid chromatography mass by I respectively Combined system detection obtains 4 kinds of nitrous amine components/corresponding internal standard compound second level selection ion peak areas ratio and 4 kinds of nitrous respectively The linear relationship of amine component/corresponding internal standard compound mass concentration ratio, draws corresponding standard working curve, calculates separately to obtain 4 The regression equation of kind nitrosamine ingredient standard working curve.

Further, in the standard curve, quasi-molecular ions face is selected with the second level of 4 kinds of nitrous amine components and corresponding internal standard compound For product than being ordinate (Y-axis), the mass concentration ratio of 4 kinds of nitrous amine components and corresponding internal standard compound is abscissa (X-axis).

II will will obtain in testing sample solution, progress twin columns liquid chromatography mass combined system detection after sample pre-treatments 4 kinds of nitrous amine components/corresponding internal standard compound second level selects ion peak areas ratio in the prepare liquid obtained, substitutes into corresponding 4 in step I The regression equation of kind nitrosamine ingredient standard working curve, and according to the known quality concentration that corresponding internal standard compound is added, it calculates The mass concentration of 4 kinds of nitrous amine components into testing sample solution.

Second aspect of the present invention provides a kind of twin columns Liquid Chromatography-Tandem Mass Spectrometry system, include the first dimension liquid chromatogram, Sampling valve, the first splitter, mixer, communicating valve, two-dimensional liquid chromatography, the second splitter, mass spectrometric apparatus, ultraviolet detection Device, the sampling valve are connected through pipeline with the sample introduction end of the first dimension liquid chromatogram, the first splitter respectively, the connection Valve is connected through pipeline with the mixer, two-dimensional liquid chromatography, the second splitter, mass spectrometric apparatus, UV detector respectively, The mixer is also connected through pipeline with the sample outlet end of first splitter.

Preferably, the sampling valve be two six-way valves, the sampling valve be equipped with the 1st interface, the 2nd interface, interface 3, 4th interface, the 5th interface, the 6th interface, the 4th interface are injection port, and the interface 3 is waste discharge mouth, the 1st interface warp Pipeline is connected with the first dimension liquid chromatogram, and the 6th interface is connected through pipeline with the sample introduction end of the first splitter.

Preferably, the communicating valve be two six-way valves, the communicating valve be equipped with the 1st interface, the 2nd interface, interface 3, 4th interface, the 5th interface, the 6th interface, the 1st interface are connected through pipeline with the sample introduction end of the second splitter, and the described 4th connects Mouth is connected through pipeline with the sample outlet end of the second splitter, and the 2nd interface is connected through pipeline with two-dimensional liquid chromatography, institute It states interface 3 and is connected through pipeline with mass spectrometric apparatus, the 5th interface is waste discharge mouth, and the 5th interface is through pipeline and ultraviolet detection Device is connected, and the 6th interface is connected through pipeline with mixer.

Preferably, the Detection wavelength of the UV detector is 220-240nm.Preferably, the inspection of the UV detector Survey wavelength is 230nm.

Preferably, the mixer is also connected with compensation pump, the compensation pump is connected through pipeline with mixer.

Preferably, the compensation pump is that unitary pumps.

Preferably, the condition of the compensation pump is：Compensating mobile phase is：1-10mmol/L K₂HPO₄；Flow velocity：0.6- 1.0mL/min。

It is highly preferred that the condition of the compensation pump is：Compensating mobile phase is：5mmol/L K₂HPO₄；Flow velocity：0.8mL/ min。

The first dimension liquid chromatogram in the twin columns Liquid Chromatography-Tandem Mass Spectrometry system, sampling valve, the first splitter, mixing Device, communicating valve, two-dimensional liquid chromatography, the second splitter, mass spectrometric apparatus, UV detector, compensation pump will match.Described With referring to above-mentioned each component after being connected to as a whole in use, can effectively run.

Third aspect present invention provides a kind of application method of twin columns Liquid Chromatography-Tandem Mass Spectrometry system, including following step Suddenly：

1) the first dimension liquid chromatogram and sampling valve are opened, the first dimension mobile phase is entered by the 1st interface of sampling valve, while to Sample solution is entered by the 4th interface of sampling valve, through the 5th interface, with the first dimension mobile phase after the 6th interface mixes, through pipe Line enters the first splitter and carries out fraction；

2) the first extra dimension mobile phase, is interfaced to interface 3 through the 2nd by the 1st interface switching of sampling valve and arranges through pipeline Out；

3) communicating valve is opened, the solution without ingredient to be measured after fraction will be carried out by the first splitter in step 1), through pipe Line is entered by the 6th interface of communicating valve, is flowed to the 5th interface, is entered after UV detector is measured through pipeline and be discharged；

4) switching communicating valve is to trapping position, the first splitter will carry out after fraction containing the molten of ingredient to be measured in step 1) Liquid is entered by the 6th interface of communicating valve, and switching enters the second splitter through pipeline and carry out separation and collection through the 1st interface；

5) switching connection valve position, then opens two-dimensional liquid chromatography, while cutting off the 6th interface and the 1st of communicating valve The connection of interface, the second dimension mobile phase are entered by the 2nd interface reverse phase of communicating valve, are switched through the 1st interface, through pipeline by second point It is flushed out from the ingredient to be measured isolated in column；

6) ingredient to be measured flushed out in step 5), switching successively the 4th interface, interface 3 through communicating valve, through pipeline into Enter mass spectrometric apparatus to be measured.

Preferably, in step 4), opening compensation is pumped, and is contained after compensation mobile phase and first splitter progress fraction to be measured The solution of ingredient mixes after pipeline enters mixer, enters back into the 6th interface of communicating valve.

Preferably, the runing time of step 1) and step 2) is 0~7min；The runing time of step 3) and step 4) is 7 ~12.5min；The runing time of step 5) is 12.5~20min；The runing time of step 6) is 20~28min.The step 1) and the runing time of step 2) is that the first splitter removes miscellaneous time.The runing time of the step 3) and step 4) is second point From post separation trap time.The runing time of the step 5) is the second splitter washing time.When the operation of the step 6) Between be the mass spectroscopy time.

Preferably, in step 3), the UV detector is for preventing component damages to be measured.

As described above, providing one kind the invention belongs to the technical field of chemical composition analysis important in cigarette mainstream flue gas Twin columns liquid chromatography tandem mass spectrometry is to the analysis method of nitrosamine burst size in cigarette smoke, by fume sample after extracting Object fraction pH value is adjusted to neutrality by loading using SCX strong cat ion exchange column separation, impurity removal, is carried out heartcut, is evaporated Point enter after trapping column inverted liquid chromatogram separation and MRM scanning and quantitative is carried out by mass spectrum.This method has below beneficial to effect Fruit：

(1) a kind of analysis method provided by the invention, by ion exchange and the two different clastotype strings of reverse phase liquid Connection, it is orthogonal according to two root chromatogram column separation principles, largely remove impurity.Its good separating effect and have good compatibility Property, it can be effectively removed interfering substance in flue gas, matrix interference effect substantially reduces, and 4 kinds of TSNAs is made all to realize baseline point From achieving excellent separating degree, sensitivity.

(2) a kind of analysis method provided by the invention, compared with existing single-column method, the technology of the present invention can be significantly improved The effect of cigarette mainstream flue gas nitrosamine burst size measurement, sample pre-treatments are simple, can loading after solvent extraction.

(3) a kind of analysis method provided by the invention, the on-line automatic operation of instrument, detection efficiency is high, and testing result is accurate Reliably, it adapts to various types cigarette and is especially low TSNAs burst size cigarette smoke measurement.

Detailed description of the invention

Fig. 1 is shown as a kind of overall structure diagram of twin columns Liquid Chromatography-Tandem Mass Spectrometry system of the invention.

In figure,

1, the first dimension liquid chromatogram,

2, sampling valve,

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 splitter,

4, mixer,

5, the second splitter,

6, communicating valve,

61, the 1st interface,

62, the 2nd interface,

63, interface 3,

64, the 4th interface,

65, the 5th interface,

66, the 6th interface,

7, UV detector

8, two-dimensional liquid chromatography,

9, mass spectrometric apparatus,

10, compensation pump.

Fig. 2 is shown as the MRM of traditional one-dimensional liquid chromatography tandem mass spectrometry analysis Virginian-type cigarette flue gas TSNAs burst size Spectrogram 2a, 2b, 2c, 2d, 2e, wherein：

Fig. 2 a is total MRM spectrogram of 4 kinds of TSNAs burst sizes；

Fig. 2 b is the MRM spectrogram of NNN burst size in 4 kinds of TSNAs；

Fig. 2 c is the MRM spectrogram of NAT burst size in 4 kinds of TSNAs；

Fig. 2 d is the MRM spectrogram of NAB burst size in 4 kinds of TSNAs；

Fig. 2 e is the MRM spectrogram of NNK release amount in 4 kinds of TSNAs.

Fig. 3 is shown as twin columns liquid chromatography tandem mass spectrometry analysis Virginian-type cigarette flue gas TSNAs burst size of the invention MRM spectrogram 3a, 3b, 3c, 3d, 3e, wherein：

Fig. 3 a is total MRM spectrogram of 4 kinds of TSNAs burst sizes；

Fig. 3 b is the MRM spectrogram of NNN burst size in 4 kinds of TSNAs；

Fig. 3 c is the MRM spectrogram of NAT burst size in 4 kinds of TSNAs；

Fig. 3 d is the MRM spectrogram of NAB burst size in 4 kinds of TSNAs；

Fig. 3 e is the MRM spectrogram of NNK release amount in 4 kinds of TSNAs.

Specific embodiment

The present invention is further explained combined with specific embodiments below, it should be appreciated that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.

Illustrate embodiments of the present invention below by way of specific specific example, those skilled in the art can be by this specification Other advantages and efficacy of the present invention can be easily understood for disclosed content.The present invention can also pass through in addition different specific realities The mode of applying is embodied or practiced, the various details in this specification can also based on different viewpoints and application, without departing from Various modifications or alterations are carried out under spirit of the invention.

Reagent and experiment appliance used in the following embodiment are conventional use of reagent and experiment appliance, the city Jun Kecong It buys and obtains on field.Specifically used reagent and instrument are as follows：

1, reagent

Reference cigarette 1R5F, 3R4F are purchased from Kentucky tobacco centre of research and development (Kentucky Tobacco Research and Development Center,Lexington,USA)；Finished cigarettes are commercial goods；NNN, NNK, NAT, NAB, NNN- D4, NNK-d4, NAT-d4 and NAB-d4 (purity >=98%, Canadian Toronto Research chemicals company)；Second Nitrile, methanol (chromatographically pure, German Merk company)；Phosphoric acid, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium acetate (analyze pure, traditional Chinese medicines collection Chemical reagent Co., Ltd of group), ultrapure water (Milli-Q pure water meter, Millpore company of the U.S.).

2, instrument

First dimension liquid chromatogram, two-dimensional liquid chromatography (Agilent Technologies 1260Infinity system, Agilent company)；UV detector (is included in Agilent Technologies 1260Infinity system, Agilent Company)；Mass spectrometric apparatus (the triple level four bars mass spectrographs of API4000, American AB SCIEX company)；SCX chromatographic column (Waters Spherisorb S5SCX column, 4.6*150mm, 5 μm)；Trap trapping column (Thermo scientificAcclaim PolarAdvantage II C18,4.6*50mm, 3 μm)；Sampling valve, communicating valve (two six-way valves, Agilent company of the U.S.)； Compensation pump (unitary pump, Agilent company of the U.S.)；Electronic balance (sensibility reciprocal：0.0001g, Mettler Toledo company of Switzerland)； SW12H Ultrasound Instrument (Sono Swiss company of Switzerland)；Milli-Q pure water meter (Millpore company of the U.S.)；The rotation of RM200A type Formula smoking machine (German Borgwaldt company)；Mixer (80 μ L threeway mixers, Agilent company).

As shown in Figure 1, one of present invention twin columns Liquid Chromatography-Tandem Mass Spectrometry system, includes the first dimension liquid chromatogram (1), sampling valve (2), the first splitter (3), mixer (4), communicating valve (6), two-dimensional liquid chromatography (8), the second splitter (5), mass spectrometric apparatus (9), UV detector (7), the sampling valve (2) respectively through pipeline and it is described first dimension liquid chromatogram (1), The sample introduction end of first splitter (3) is connected, and the communicating valve (6) is respectively through pipeline and the mixer (4), the second dimension liquid phase Chromatography (8), the second splitter (5), mass spectrometric apparatus (9), UV detector (7) are connected, the mixer (4) also through pipeline with The sample outlet end of first splitter (3) is connected.

Embodiment 1

1, sample pre-treatments

20 Cigarettes are chosen, cigarette smoke is trapped using filter disc Trapping ways after lighting, is specifically turned using 20 ducts Disc type smoking machine smoking cigarette traps flue gas according to prescriptive procedure in GB/T 19609-2004, flue gas is made to be adsorbed in cambridge filter On.Cambridge filter is placed in middle addition extraction solution in 100mL conical flask, is placed in supersonic generator and carries out ultrasonic extraction 30-50min, extraction solution are 10-20mL 0.1mol/L NH₄Ac aqueous solution, NH₄Ac aqueous solution is used by weighing ammonium acetate It after water is completely dissolved, is transferred in volumetric flask, water constant volume is added to obtain.Inner mark solution is additionally added in extraction solution, inner mark solution is logical Excessively also known as take NNN-d₄、NNK-d₄、NAT-d₄、NAB-d₄, obtained after methanol constant volume is added.The additional amount of inner mark solution is 1- 2mL 400ng/mL.Then it after 0.22 μm of water phase membrane filtration, moves in chromatography bottle to get testing sample solution.

2, the preparation of standard solution

The standard items for pipetting NAB, NAT, NNN, NNK that 10ml concentration is 0.1mg/ml respectively, are placed in 100mL volumetric flask In, methanol constant volume is added, is made into hybrid standard stock solution.The concentration of NAB, NAT, NNN, NNK in hybrid standard stock solution It is 10 μ g/ml.Meanwhile d4-NNN, d4-NAT, d4-NAB, d4-NNK internal standard compound of certain volume are pipetted respectively, methanol is added Constant volume is made into inner mark solution, and the concentration of d4-NNN, d4-NAT, d4-NAB, d4-NNK are 400ng/ml in inner mark solution.

Different volumes hybrid standard stock solution is accurately pipetted respectively, is placed in multiple 100mL volumetric flasks, then accurate respectively The inner mark solution that certain volume is added is formulated as a series of mixed standard solutions with methanol constant volume.The serial hybrid standard of preparation Solution concentration is 0-100ng/ml, and wherein internal standard compound concentration is 10ng/ml.

3, the measurement of sample size

Testing sample solution in the standard solution and 1 of 2 preparations is subjected to twin columns Liquid Chromatography-Tandem Mass Spectrometry system respectively respectively Hybrid standard stock solution and testing sample solution are passed through the first separation under the first dimension liquid chromatogram effect by system detection After column carries out fraction, under two-dimensional liquid chromatography effect, separated by second splitter, then by the mass spectrometric apparatus Be measured, compare retention time, scanning of the mass spectrum carry out it is qualitative, determine 4 kinds of nitrous amine components in prepare liquid；Internal standard is used simultaneously Calibration curve method carries out MRM and quantifies, and obtains the content of 4 kinds of nitrous amine components in prepare liquid.

Specifically, Internal standard curve method is first to distinguish the mixed standard solution of various concentrations a series of in above-mentioned 2 Carry out the detection of twin columns liquid chromatography mass combined system, obtain respectively 4 kinds of nitrous amine components/corresponding internal standard compound second level select from The linear relationship of sub- peak area ratio and 4 kinds of nitrous amine components/corresponding internal standard compound mass concentration ratio draws corresponding standard work Curve selects ion peak areas ratio as ordinate (Y-axis), 4 kinds of nitrous using the second level of 4 kinds of nitrous amine components and corresponding internal standard compound The mass concentration ratio of amine component and corresponding internal standard compound is abscissa (X-axis), calculates separately to obtain 4 kinds of nitrosamine ingredient standard work The regression equation of curve.Prepare liquid in above-mentioned 1 is subjected to the detection of twin columns liquid chromatography mass combined system again, by the to be measured of acquisition 4 kinds of nitrous amine components/corresponding internal standard compound second level selects ion peak areas ratio in liquid, substitutes into corresponding 4 kinds of nitrous amine component mark The regression equation of quasi- working curve, and according to the known quality concentration that corresponding internal standard compound is added, 4 kinds of Asias in prepare liquid are calculated The mass concentration of nitramine ingredient.

Wherein, the condition of the first dimension liquid chromatogram is：First dimension liquid chromatogram：Positive liquid chromatography pump；Column temperature： 25-35℃；Flow velocity：0.5-1.5mL/min；Sample volume：5-15μl；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 elution.

As shown in table 1, the specific procedure of the 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 splitter is strong cation exchange chromatography column (SCX).

Under the first dimension liquid chromatogram effect, during the first splitter be adjusted to the pH value of object fraction after fraction Property, using heartcut method, obtain the fraction of 4 kinds of nitrous amine components.The chromatography for the object fraction that first splitter is isolated The cutting at peak is complete, under two-dimensional liquid chromatography effect, is transferred to the trapping of the second splitter and retains, other non-targeted object fractions Not into the second splitter.

The condition of the two-dimensional liquid chromatography is：Second dimension liquid chromatography pump：Reversed-phase liquid chromatography pumps (RPLC)；Column Temperature：25-35℃；Flow velocity：0.2-0.3mL/min；Sample volume：5-15μl；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 the 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.

Second splitter is Trap trapping column.

The condition of the mass spectrometric apparatus is：

In addition, twin columns liquid chromatography mass combined system of the invention is in specific measurement, as shown in Figure 1, described first It ties up and is equipped with sampling valve between liquid chromatogram and the first splitter.

As shown in Figure 1, the sampling valve is two six-way valves, the sampling valve connects equipped with the 1st interface, the 2nd interface, the 3rd Mouth, the 4th interface, the 5th interface, the 6th interface, the 4th interface are injection port, and the interface 3 is waste discharge mouth, and the described 1st connects Mouth is connected through pipeline with the first dimension liquid chromatogram, and the 6th interface is connected through pipeline with the first splitter.

As shown in Figure 1, communicating valve is equipped between first splitter and two-dimensional liquid chromatography, the communicating valve and the Mixer is equipped between one splitter, the mixer is connected with first splitter, communicating valve respectively.

As shown in Figure 1, the communicating valve is two six-way valves, the communicating valve connects equipped with the 1st interface, the 2nd interface, the 3rd Mouthful, the 4th interface, the 5th interface, the 6th interface, the 1st interface is connected through pipeline with the sample introduction end of the second splitter, described the 4 interfaces are connected through pipeline with the sample outlet end of the second splitter, and the 2nd interface is connected through pipeline with two-dimensional liquid chromatography Logical, the interface 3 is connected through pipeline with mass spectrometric apparatus, and the 5th interface is waste discharge mouth, and the 6th interface is through pipeline and mixes Clutch is connected.

As shown in Figure 1, the 5th interface is also connected with UV detector, the UV detector is through pipeline and the 5th interface It is connected.The Detection wavelength of the UV detector is 220-240nm.

As shown in Figure 1, the mixer is also connected with compensation pump, the compensation pump is connected through pipeline with mixer.Institute Compensation pump is stated for adjusting the pH value of One-Dimensional flows phase and diluting acetonitrile concentration in One-Dimensional flows phase.The compensation pump is unitary Pump.It is described compensation pump condition be：Mobile phase is：1-10mmol/L K₂HPO₄；Flow velocity：0.6-1.0mL/min.

As shown in Figure 1, when being measured using twin columns liquid chromatography mass combined system, open the first dimension liquid chromatogram and Sampling valve, first dimension mobile phase by sampling valve the 1st interface enter, while testing sample solution by sampling valve the 4th interface into Enter, enters the first splitter with the first dimension mobile phase after the 6th interface mixes through the 5th interface through pipeline and carry out fraction；It is extra The first dimension mobile phase, be interfaced to interface 3 by the 1st interface switching of sampling valve through the 2nd and be discharged through pipeline；Above-mentioned runing time For 0~7min.Communicating valve is opened, the solution of ingredient to be measured will be free of after the first splitter carries out fraction, through pipeline by being connected to 6th interface of valve enters, and flows to the 5th interface, enters after UV detector is measured through pipeline and be discharged；Switching communicating valve is to catching Collect position, the solution containing ingredient to be measured after the first splitter carries out fraction is entered by the 6th interface of communicating valve, switching warp 1st interface enters the second splitter through pipeline and carries out separation and collection；Above-mentioned runing time is 7~12.5min.Switch communicating valve Then two-dimensional liquid chromatography is opened in position, while cutting off the connection of the 6th interface and the 1st interface of communicating valve, two-dimensional flow Mutually entered by the 2nd interface reverse phase of communicating valve, switching through the 1st interface, through pipeline by isolated in the second splitter it is to be measured at Divide and flushes out；Above-mentioned runing time is 12.5~20min.The ingredient to be measured that will be flushed out again switches the 4th successively through communicating valve Interface, interface 3 enter mass spectrometric apparatus through pipeline and are measured；Above-mentioned runing time is 20~28min.Benefit can also be opened Pump is repaid, compensation mobile phase and first splitter carry out the solution containing ingredient to be measured after fraction, after pipeline enters mixer Mixing, enters back into the 6th interface of communicating valve.

Embodiment 2

1, sample pre-treatments

20 Cigarettes are chosen, cigarette smoke is trapped using filter disc Trapping ways after lighting, is specifically turned using 20 ducts Disc type smoking machine smoking cigarette traps flue gas according to prescriptive procedure in GB/T 19609-2004, flue gas is made to be adsorbed in cambridge filter On.Cambridge filter is placed in 100mL conical flask, extraction solution is added, is placed in supersonic generator and carry out ultrasonic extraction 40min, extraction solution are 20ml 0.1mol/L NH₄Ac aqueous solution, 0.1mol/L NH₄Ac aqueous solution is by weighing 3.85g Ammonium acetate after being completely dissolved with water, is transferred in 500ml volumetric flask, water constant volume is added to obtain.It is molten that internal standard is additionally added in extraction solution Liquid, inner mark solution by weighing NNN-d respectively₄、NNK-d₄、NAT-d₄、NAB-d₄, obtained after methanol constant volume is added.Inner mark solution Additional amount be 1mL 400ng/mL.Then it after 0.22 μm of water phase membrane filtration, moves in chromatography bottle to get to test sample Product solution.

2, the preparation of standard solution

Different volumes hybrid standard stock solution is accurately pipetted respectively, is placed in multiple 100mL volumetric flasks, then accurate respectively The inner mark solution that certain volume is added is formulated as a series of mixed standard solutions with methanol constant volume.The serial hybrid standard of preparation Solution concentration is 0.25,0.5,1.0,2.5,25,50 and 100ng/mL, and wherein internal standard compound concentration is 10ng/ml.

3, the measurement of sample size

Testing sample solution in the standard solution and 1 of 2 preparations is subjected to twin columns liquid chromatography mass combination system respectively respectively Hybrid standard stock solution and testing sample solution are passed through the first separation under the first dimension liquid chromatogram effect by system detection After column carries out fraction, under two-dimensional liquid chromatography effect, separated by second splitter, then by the mass spectrometric apparatus Be measured, compare retention time, scanning of the mass spectrum carry out it is qualitative, determine 4 kinds of nitrous amine components in prepare liquid；Internal standard is used simultaneously Calibration curve method carries out MRM and quantifies, and obtains the content of 4 kinds of nitrous amine components in prepare liquid.

Wherein, the condition of the first dimension liquid chromatogram is：First dimension liquid chromatogram：Agilent Technologies 1260Infinity positive liquid chromatogram；Column temperature：30℃；Flow velocity：1mL/min；Sample volume：10μl；Mobile phase A phase：10mmol/ L KH₂PO₄+5mmol/L H₃PO₄+ 85%H₂O+15%CH₃CN (pH=2.8)；Mobile phase B phase：10mmol/L KH₂PO₄+ 5mmol/L H₃PO₄+ 0.5mol/L KCl+85%H₂O+15%CH₃CN (pH=2.8)；Gradient elution.

As shown in table 1, the specific procedure of the 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 splitter is strong cation exchange chromatography column (SCX), and the strong cation exchange chromatography column (SCX) is Waters Spherisorb S5SCX column (4.6*150mm, 5 μm).

The condition of the two-dimensional liquid chromatography is：Two-dimensional liquid chromatography：Agilent Technologies 1260Infinity reversed-phase liquid chromatography (RPLC)；Column temperature：30℃；Flow velocity：0.25mL/min；Sample volume：10μl；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 the 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.

Second splitter is Trap trapping column, and the Trap trapping column is Thermo scientificAcclaim II C18 column of PolarAdvantage (4.6*50mm, 3 μm).

The condition of the mass spectrometric apparatus is：

As shown in Figure 1, the 5th interface is also connected with UV detector, the UV detector is through pipeline and the 5th interface It is connected.The Detection wavelength of the UV detector is 230nm.

Embodiment 3

As shown in 2 in above-described embodiment 2, hybrid standard stock solution is accurately pipetted respectively, using methanol constant volume, is prepared dense Degree is a series of mixed standard solutions of 0.25,0.5,1.0,2.5,25,50 and 100ng/mL, and wherein internal standard compound concentration is 10ng/ml。

A series of mixed standard solution of above-mentioned prepared various concentrations is subjected to twin columns liquid chromatography mass connection respectively With system detection, select ion peak areas ratio as ordinate (Y-axis) using the second level of 4 kinds of nitrous amine components and corresponding internal standard compound, 4 The mass concentration ratio of kind nitrous amine component and corresponding internal standard compound is abscissa (X-axis), carries out regression analysis, obtains regression equation Linear relationship is good, within the scope of the linear concentration of 0.25~100.00ng/mL, the phase of the regression equation of 4 kinds of nitrous amine components It closes coefficients R and is all larger than 0.999.

It to the object response signal in mixed standard solution, is repeated sample introduction 10 times, is carried out double using minimum concentration standard specimen Column liquid chromatographic mass spectrometry network analysis, using 3 times of signal-to-noise ratio as the detection limit (LOD) of method, 10 times of signal-to-noise ratio are quantitative limit (LOQ), it show that the method detection of NNK is limited to 0.77ng/cig after being scaled sample size, is quantitatively limited to 2.56ng/cig；NAB Method detection be limited to 0.16ng/cig, be quantitatively limited to 0.55ng/cig；The method detection of NAT is limited to 0.57ng/cig, quantitative It is limited to 1.89ng/cig；The method detection of NNN is limited to 0.46ng/cig, is quantitatively limited to 1.52ng/cig；It is with higher sensitive Degree.

Embodiment 4

It takes identical Virginian-type cigarette to investigate the rate of recovery, in a few days repeated, repeated in the daytime of method, that is, chooses known nitrous The mixed standard solution of known concentration is added in amine concentration sample, then carries out in above-described embodiment 21 pre-treatment, and carries out double Column liquid chromatographic mass spectrometry network analysis, calculates its rate of recovery.Simultaneously to same testing sample solution respectively in 1 day, day Between carry out be measured in parallel 5 times (n=5), obtain the precision determination data of 4 kinds of nitrous amine components, the results are shown in Table 4.

As can be seen from Table 4, for the rate of recovery of object method between 93.5~106.2%, in a few days repeated is opposite Standard deviation (RSD) between 2.7~4.9%, relative standard deviation (RSD) repeated in the daytime between 3.8~5.9%, Illustrate the rate of recovery of the method for the present invention high, accuracy and reproducible, can satisfy quantitative needs.

The rate of recovery and precision of 44 kinds of nitrous amine components of table

Embodiment 5

It is compared using twin columns liquid chromatography tandem mass spectrometry with the method for traditional one-dimensional Liquid Chromatography-Tandem Mass Spectrometry, point It Xuan Qu not 11mg Virginian-type cigarette.First group of method for using traditional one-dimensional Liquid Chromatography-Tandem Mass Spectrometry, reference standard YC/ T184-2004, concrete outcome are shown in Fig. 2.Second group uses in above-described embodiment 21 pre-treatment, and carries out twin columns liquid chromatogram matter Combined system analysis is composed, concrete outcome is shown in Fig. 3.

By to shown in 3a~3e in 2a~2e in Fig. 2 and Fig. 3, it is found that use twin columns Liquid Chromatography-Tandem Mass Spectrometry Method makes four kinds of substance separating degrees, is obviously improved in detection sensitivity, and NNN, NAT and NNK are substantially complete in bidimensional liquid phase separation Full removal impurity interference, NAB and Interference Peaks realize baseline separation.

Embodiment 6

The burst size of TSNAs in 3R4F cigarette mainstream flue gas is measured using the analysis method of the invention in such as embodiment 2, Its NNN, NNK, NAT, NAB content is respectively 106,91,113 and 13.8ng/cig, 19 realities with the tissue of CORESTA in 2011 It tests the common experimental data average value in room to compare, mean value RSD is tested using the measurement result and CORESTA of this method jointly Within 5.5%, it is accurate and reliable to show that twin columns liquid chromatography tandem mass spectrometry method surveys cigarette smoke TSNAs burst size.

In addition, the mixed type and Virginian-type cigarette of different box mark tar are chosen, using point of the invention in such as embodiment 2 Analysis method is measured, and the results are shown in Table 5.As shown in Table 5, blended type cigarette NNN burst size range 59.0~347ng/cig, NAT Burst size range 126~201ng/cig, NAB burst size 16.5~25.1ng/cig of range, NNK release amount range 59.0~ 81.5ng/cig.14.6~29.5ng/ of Virginian-type cigarette NNN burst size range 4.6~8.7ng/cig, NAT burst size range Cig, NAB burst size 1.2~2.8ng/cig of range, 4.4~5.4ng/cig of NNK release amount range.Sample is measured with conventional method The contents level of product is consistent, and this method has preferable accuracy.Wherein, the content (ng/cig) of sample is by point of the invention It is obtained after sample concentration (ng/mL) × 20mL/20cig of analysis method measurement.

5 part commercial cigarettes TSNAs burst size of table

In conclusion present invention demonstrates that, cigarette mainstream flue gas nitrosamine is analyzed with twin columns liquid chromatography tandem mass spectrometry Burst size sheet has preferable effect, as the result is shown：This method high sensitivity, reproducible, accuracy is high, standard cigarettes 3R4F quantitative result and CORESTA test mean value relative standard deviation within 5.5% jointly.Therefore, online twin columns liquid phase color Spectrum tandem mass spectrometry is accurate and reliable, detection limit is low, adapts to various types cigarette and is especially low TSNAs burst size cigarette smoke survey It is fixed.So the present invention effectively overcomes various shortcoming in the prior art and has high industrial utilization value.

The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as At all equivalent modifications or change, should be covered by the claims of the present invention.

Claims

1. a kind of twin columns liquid chromatography tandem mass spectrometry is to the analysis method of nitrosamine burst size in cigarette smoke, to cigarette smoke Sample pre-treatments are carried out, qualitative, quantitative point is carried out to sample to be tested obtained by pre-treatment using twin columns Liquid Chromatography-Tandem Mass Spectrometry system Analysis, the twin columns Liquid Chromatography-Tandem Mass Spectrometry system include the first dimension liquid chromatogram, two-dimensional liquid chromatography, mass spectrometric apparatus, The first dimension liquid chromatogram includes the first splitter, and the two-dimensional liquid chromatography includes the second splitter；

Nitrosamine includes N- nitrosonornicotine in the cigarette smoke, (3- compares pyridine to 4- (methyl nitroso amino)-l- Base) -1- butanone, N- nitroso anabasine and N- nitrosoanatabine；

The sample pre-treatments include the following steps：

2) film is crossed after filter disc being carried out ultrasonic extraction to get sample to be tested；

It is described that qualitative and quantitative analysis is carried out to sample to be tested obtained by pre-treatment using twin columns Liquid Chromatography-Tandem Mass Spectrometry system, including Following steps：

B) sample qualitative detection：By the step A) standard sample prepared and after sample pre-treatments, sample to be tested carries out twin columns respectively Liquid Chromatography-Tandem Mass Spectrometry system detection, under the first dimension liquid chromatogram effect, after carrying out fraction by the first splitter, the Under two-dimensional liquid chromatography effect, is separated by second splitter, then be measured by the mass spectrometric apparatus, compare reservation Time, scanning of the mass spectrum progress are qualitative, determine 4 kinds of nitrous amine components in sample to be tested；

C) sample amounts detect：By the step A) standard sample prepared and after sample pre-treatments, sample to be tested carries out twin columns respectively Liquid Chromatography-Tandem Mass Spectrometry system detection, under the first dimension liquid chromatogram effect, after carrying out fraction by the first splitter, the Under two-dimensional liquid chromatography effect, separated by second splitter, then be measured by the mass spectrometric apparatus, using internal standard Calibration curve method carries out MRM and quantifies, and obtains the content of 4 kinds of nitrous amine components in sample to be tested；

Step B) or C) in, first splitter be strong cation exchange chromatography column；The condition of the first dimension liquid chromatogram For：First dimension liquid chromatogram：Positive liquid chromatogram；Column temperature：25-35℃；Flow velocity：0.5-1.5mL/min；Sample volume：5-15μl； First dimension mobile phase A phase：5-15mmol/L KH₂PO₄+1-10mmol/L H₃PO₄+ 80-90%H₂O+10-20%CH₃CN, pH= 2-4；First dimension 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 elution；

Step B) or C) in, second splitter be Trap trapping column, the Trap trapping column be C18 column；Second dimension The condition of liquid chromatogram is：Two-dimensional liquid chromatography：Reversed-phase liquid chromatography；Column temperature：25-35℃；Flow velocity：0.2-0.3mL/min； Sample volume：5-15μl；Second dimension mobile phase A phase：1-10mmol/L NH₄Ac+92-98%H₂O+2-8%CH₃CN；Two-dimensional flow Dynamic phase B phase：1-10mmol/L NH₄Ac+92-98%CH₃CN+2-8%H₂O；Gradient elution；

Step B) or C) in, the condition of the mass spectrometric apparatus is：

Mass spectrum：6460 Triple Quad MS triple quadrupole bar mass spectrum of Agilent Technologies；Ionization mode：EFI Mist ion source；Scanning mode：Multiple-reaction monitoring MRM；Spray voltage is：5000V；Atomization gas pressure is：482.6kPa；Gas curtain gas Pressure is：206.7kPa；Assisted atomization atmospheric pressure is：482.6kPa；Ion source temperature is：500℃；Clean sample introduction needle program： needle wash 30s；The switching of mass spectrum flow path：It is scanned since 10min, 28min terminates；

The specific procedure of gradient elution of the first dimension liquid chromatogram 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 specific procedure of the gradient elution of the two-dimensional liquid chromatography 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.