CN106970178B - It is a kind of based on hydrophobic nonionic exchange Solid Phase Extraction cigarette mainstream flue gas in the peculiar N- nitrosamine of tobacco extraction and assay method - Google Patents
It is a kind of based on hydrophobic nonionic exchange Solid Phase Extraction cigarette mainstream flue gas in the peculiar N- nitrosamine of tobacco extraction and assay method Download PDFInfo
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Abstract
The present invention relates to a kind of extraction of the peculiar N- nitrosamine of tobacco in cigarette mainstream flue gas exchanging Solid Phase Extraction based on hydrophobic nonionic and assay methods, belong to tobacco chemistry inspection technology field.The extracting method of the peculiar N- nitrosamine of tobacco in cigarette mainstream flue gas of the present invention, by trapping there is the filter disc of mainstream smoke total particulate matter to be placed in ammonium acetate solution, filter, using HiCapt MCX solid-phase extraction columns carry out Solid Phase Extraction to get.The extracting method of the peculiar N- nitrosamine of tobacco in cigarette mainstream flue gas of the present invention, impurity and chaff interferent can be effectively removed, accuracy and the precision of assay method are improved, the relative recovery of method is between 92.2%~108.9%, and in a few days and day to day precision is respectively smaller than 4.6% and 4.2%.
Description
Technical field
The present invention relates to the peculiar N- nitrous of tobacco in a kind of cigarette mainstream flue gas exchanging Solid Phase Extraction based on hydrophobic nonionic
The extraction of amine and assay method belong to tobacco chemistry inspection technology field.
Background technology
The peculiar N- nitrosamine of tobacco is that aminated compounds is reacted with nitrosification agent and generated in acid condition, is one
The carcinogen that class receives significant attention, be not only Huffman inventory and U.S. Food and Drug Administration " tobacco product and
Important substance in harmful and potentially harmful substance list in flue gas " and international cancer research institution's " smoke-free tobacco product
In 28 kinds of harmful substances " important component in list.Therefore, the peculiar N- nitrosamine of tobacco in cigarette mainstream flue gas is accurately analyzed
Content to evaluation tobacco and tobacco product, ensures that tobacco product consumption safety is of great significance.
Cigarette smoke is extremely complex mixture, and the smoke components identified at present are more than more than 5000 to plant, and wherein
The contents level of the peculiar N- nitrosamine of tobacco is generally in ng/ branch ranks.Therefore, the peculiar N- nitrosamine of tobacco in main flume is measured
Burst size when often be faced with following difficult point：Object content is low, there are matrix interferences.Therefore, in the peculiar N- nitrosamine of tobacco
It needs that for the suitable extraction of its property selection, purifying and method for concentration subsequent instrument could be carried out when burst size is measured
It measures.
Currently, in cigarette mainstream flue gas there are two types of the detection methods of the peculiar N- nitrosamine of tobacco：First method is with two
Chloromethanes extracts the peculiar N- nitrosamine of tobacco in granule phase substance, and concentrated extracting solution is simultaneously carried out net using alkali alumina chromatographic column
Change, combined instrument separation detection is analyzed by gas-chromatography-thermal energy.But the above method will carry out concentrated extracting solution, and alkalinity
The filling of alumina chromatographic column and use process are relatively time consuming, cause sample pre-treatments step lengthy and tedious, are unfavorable for the fast of a large amount of samples
Speed analysis.In addition, though thermal energy analyzer is to analyze the exclusive type detector of the peculiar N- nitrosamine of tobacco, there are still pre-treatments for it
The problems such as time is long, efficiency is low.Second method is the peculiar N- nitrosamine of tobacco extracted with ammonium acetate solution in granule phase substance, will
Liquid chromatography-tandem mass spectrometry analysis is carried out after extracting solution filtering.This method pre-treatment is easy to operate, but sample solution carries out instrument
Without passing through purified treatment before device analysis, therefore there are stronger matrix interference phenomenon, increase instrument maintenance frequency.
Application publication number is that the patent of invention of CN102012407A discloses a detection side for growing tobacco peculiar N- nitrosamine
Then method uses formic acid water wash, is finally eluted with ammonia hydroxide/methanol in this scenario by extract flow through solid phase extraction column,
Obtain prepare liquid.Solid phase extraction filler in the program is n-vinyl pyrrolidone-benzene sulfonic acid copolymer, and organic polymer is filled out
Material can be swollen in organic solvent, can influence enrichment and the clean-up effect of filler to a certain extent.In addition, not having in the program yet
There are the various parameters to influencing effect of extracting to optimize.
Therefore, develop that a kind of high sensitivity, matrix removal effect is good and environmental pollution is small, quickly detects cigarette main flue
The method of the peculiar N- nitrosamine of tobacco is extremely urgent in gas.
Invention content
The purpose of the present invention is to provide tobaccos in a kind of cigarette mainstream flue gas exchanging Solid Phase Extraction based on hydrophobic nonionic
The extracting method of peculiar N- nitrosamine, can effectively remove impurity and chaff interferent.
The present invention second be designed to provide it is a kind of based on hydrophobic nonionic exchange Solid Phase Extraction cigarette mainstream flue gas in
The assay method of the peculiar N- nitrosamine of tobacco.
To achieve the above object, the technical scheme is that：
It is a kind of based on hydrophobic nonionic exchange Solid Phase Extraction cigarette mainstream flue gas in the peculiar N- nitrosamine of tobacco extraction side
Method includes the following steps：
1) by trapping have the filter disc of mainstream smoke total particulate matter be added the ammonium acetate solution of a concentration of 5~100mmol/L into
Row extraction, filtering obtain crude extract；
2) crude extract obtained by step 1) is flowed through into the HiCapt MCX solid-phase extraction columns after activation, completes loading；
3) it is eluted with methanol aqueous solution；
4) it is eluted to get N- nitrous with any one in acetone, acetone ammonia water mixture, acetone formic acid mixed liquor
The extracting solution of amine；Acetone in the acetone ammonia water mixture, ammonium hydroxide volume ratio be 97~99:3~1；Acetone formic acid mixed liquor
Middle acetone, formic acid volume ratio be 97~99:3~1；The mass fraction of the ammonium hydroxide is 35%.
The peculiar N- nitrosamine of tobacco includes NNN, NNK, NAT and NAB in the cigarette mainstream flue gas.
The HiCapt MCX solid-phase extraction columns are hydrophobic/ion exchange mixed-mode retention mechanism solid-phase extraction column.
The HiCapt MCX solid-phase extraction columns are purchased from Weitaike Technology (Wuhan) Co., Ltd..
It is extracted as having the filter disc of mainstream smoke total particulate matter to carry out ultrasonic extraction trapping with ammonium acetate solution in step 1).
280~700W of ultrasonic power of the ultrasonic extraction, 20~40kHz of frequency, time are no less than 10min.The ultrasonic extraction time is excellent
It is selected as 30min.
Step 1) is filtered into be filtered using water phase filter membrane.The water phase filter membrane preferred, polyethers sulfone film.The polyether sulfone
The poly (ether sulfone) film that preferably a diameter of 0.22 μm or 0.45 μm of film.
The dosage that ammonium acetate solution used in the mainstream smoke total particulate matter of 5 cigarette is often extracted in step 1) is no less than
10mL.Preferably：Often extract dosage 15mL~25mL of ammonium acetate solution used in the mainstream smoke total particulate matter of 5 cigarette.
Amount of filler is in step 2) HiCapt MCX solid-phase extraction columns：It will often correspond to and use when 6mL crude extract loadings
50~1000mg HiCapt MCX solid phase extraction fillers.Further preferably 500mg HiCapt MCX solid phase extraction fillers.
In step 2) activation for be successively 5% with acetone, methanol volume fraction methanol aqueous solution, water clean Solid Phase Extraction
Column.The acetone, methanol aqueous solution, water volume ratio be 1:2:2.
The crude extract adjusts pH to 3.0~11.0 before loading.Further, it is preferable to be pH to 4.0~6.0.
PH adjusting agent used in the tune pH is acetic acid or ammonium hydroxide.When selecting acetic acid to adjust pH, preferred mass score is
10% acetic acid solution.
The volume fraction of methanol is 5%~20% in methanol aqueous solution described in step 3).Methanol in methanol aqueous solution
Volume fraction preferably 8%~12%.In step 3) when the dosage selection of methanol aqueous solution, often by 6mL crude extract loadings, elution
When methanol aqueous solution dosage be >=0.5mL.Preferably 1mL.
In step 3) leacheate is drained under the suction function of vacuum pump after elution.
Acetone and ammonium hydroxide volume ratio are preferably 99 in step 4):1.The volume ratio of acetone and formic acid is preferably 99:1.
Elution, the dosage of eluent are in step 4)：Often by 6mL crude extract loadings, elution, the elution used after dry
Liquid product >=0.5mL.Preferably 1~2mL.
It is a kind of based on hydrophobic nonionic exchange Solid Phase Extraction cigarette mainstream flue gas in the peculiar N- nitrosamine of tobacco measurement side
Method includes the following steps：
1. the preparation of extracting solution
1.1 by the trapping containing Isotopic Internal Standard have the filter disc of mainstream smoke total particulate matter be added it is a concentration of 5~
100mmol/L ammonium acetate solutions extract, and obtain crude extract；
1.2 flow through step 1.1 gained crude extract the HiCapt MCX solid-phase extraction columns after activation, complete loading；
1.3 are eluted with methanol aqueous solution, are dried in vacuo later；
1.4 are eluted with any one in acetone, acetone ammonia water mixture, acetone formic acid mixed liquor to get the Asias N-
The extracting solution of nitramine；Acetone in the acetone ammonia water mixture, ammonium hydroxide volume ratio be 97~99:3~1；Acetone formic acid mixes
Acetone in liquid, formic acid volume ratio be 97~99:3~1；
2. the measurement of the peculiar N- nitrosamine of tobacco in cigarette mainstream flue gas
Step 1.4 gained extracting solution is dried up, is carried out with the redissolution liquid that ammonium formate solution, formic acid acetonitrile solution are formulated
It redissolves, carries out liquid chromatography tandom mass spectrometry determination later.
Isotopic Internal Standard includes NNN-d4, NNK-d4, NAT-d4, NAB-d4 in step 1.1.
The dosage that ammonium acetate solution used in the mainstream smoke total particulate matter of 5 cigarette is often extracted in step 1.1 is no less than
10mL.Preferably：Often extract dosage 15mL~25mL of ammonium acetate solution used in the mainstream smoke total particulate matter of 5 cigarette.
The addition of Isotopic Internal Standard is on step 1.1 filter disc：Often corresponded in various isotopes using 1mL ammonium acetate solutions
Target addition is 10ng.
It is molten for 0.1% formic acid acetonitrile by the ammonium formate solution and formic acid volume fraction of 0.01mol/L that liquid is redissolved in step 2
Liquid is according to 1~2:1~2 volume ratio is formulated.It is described redissolve liquid dosage be：It is final to survey often by 6mL crude extract loadings
It is needed before fixed using the volume >=0.1mL for redissolving liquid.Further preferably 0.2mL.
Acetone and ammonium hydroxide volume ratio are preferably 99 in step 1.4:1.The volume ratio of acetone and formic acid is preferably 99:1.
Elution, the dosage of eluent are in step 4)：Often by 6mL crude extract loadings, elution, the elution used after dry
Liquid product >=0.5mL.Preferably 1~2mL.
The peculiar N- nitrosamine of tobacco in a kind of cigarette mainstream flue gas exchanging Solid Phase Extraction based on hydrophobic nonionic of the present invention
In assay method, involved in the preparation process of extracting solution to design parameter and extraction conditions and cigarette mainstream flue gas in tobacco it is special
Have identical in the extracting method of N- nitrosamine.
Liquid chromatography tandom mass spectrometry determination includes the preparation of the mixed standard solution of series concentration in above-mentioned steps 2, is prepared
Step is：
The Isotopic Internal Standard (NNN-d4, NNK-d4, NAT-d4 and NAB-d4) for growing tobacco peculiar N- nitrosamine with four is molten
Matter, methanol are solvent, prepare inner mark solution；Peculiar N- nitrosamine (NNN, NNK, NAT and NAB) is grown tobacco as solute with four, is added
Enter the inner mark solution of fixed volume, methanol is solvent, prepares the mixed standard solution of series concentration.
The making step of the assay method standard curve of the peculiar N- nitrosamine of tobacco of the present invention is：
The Isotopic Internal Standard (NNN-d4, NNK-d4, NAT-d4 and NAB-d4) for growing tobacco peculiar N- nitrosamine with four is molten
Matter, methanol are solvent, prepare inner mark solution；Peculiar N- nitrosamine (NNN, NNK, NAT and NAB) is grown tobacco as solute with four, is added
Enter the inner mark solution of fixed volume, methanol is solvent, prepares the mixed standard solution of series concentration, by the mixing mark of series concentration
Quasi- solution carries out liquid chromatography-tandem mass spectrometry analysis, with target analytes peak area and corresponding isotopic peak area ratio to mesh
Mark analytical concentration does regression analysis to get the standard curve of each target analytes.
The condition of liquid chromatography-tandem mass spectrometry analysis is：Chromatographic column：Poroshell 120 EC-C18(3.0mm×
100mm, 2.7 μm)；Column temperature：40℃；Sample size：5μL；Mobile phase is -0.1% formic acid acetonitrile of 0.01mol/L ammonium formates (A)
(B)；Flow velocity：0.4mL/min；Eluent gradient program：T=0min, 10%B, t=2.0min, 40%B, t=4.0min,
60%B, t=6.0min, 90%B, t=9.0min, 90%B, t=9.5min, 10%B, t=15.0min, 10%B；Mass spectrum item
Part：Electron spray voltage 5000V；Atomization gas (Gas1) pressure 50psi；Assisted atomization gas (Gas2) pressure 50psi；Gas curtain gas
(Gas1) pressure 35psi；450 DEG C of ion source temperature；Import voltage 8V；Exit potential 10V；Remove cluster voltage 40V；Residence time
40ms；Monitoring pattern：Multiple-reaction monitoring.
The quota ion pair of NNN, NNK, NAT and NAB are followed successively by 178.1 in the multiple-reaction monitoring>148.2,208.1>
122.1,190.1>160.1 and 192.1>162.2, corresponding collision voltage is followed successively by 15,16,15 and 17eV；NNN,NNK,NAT
It is followed successively by 178.1 with the qualitative ion pair of NAB>120.1,208.1>106.1,190.1>106.1 and 192.1>133.1, accordingly
Collision voltage be followed successively by 15,16,15 and 17eV；The quota ion pair of NNN-d4, NNK-d4, NAT-d4 and NAB-d4 are followed successively by
182.1>152.2,212.1>126.1,194.1>164.1 and 196.1>166.2, corresponding collision voltage is followed successively by 15,16,15
And 17eV.
The beneficial effects of the invention are as follows：
The present invention uses tobacco in Solid Phase Extraction-Liquid Chromatography-Tandem Mass Spectrometry qualitative and quantitative analysis cigarette mainstream flue gas
Peculiar N- nitrosamine, sample pre-treatments are easy to operate, quick, and the removal efficiency of impurity and chaff interferent is high, processing time and instrument
Analyze short advantage.There is the peculiar N- nitrosamine of tobacco itself hydrophobic performance to have along with pyridine ring is all contained in its structure
Ion-exchange capacity, carrying out extraction to sample extracting solution using HiCapt MCX solid-phase extraction columns has good clean-up effect,
Impurity and chaff interferent can be effectively removed, improve accuracy and the precision of assay method, the relative recovery of method between
Between 92.2%~108.9%, in a few days and day to day precision is respectively smaller than 4.6% and 4.2%.
Description of the drawings
Fig. 1 is the chromatogram of mixed standard solution 3；
Fig. 2 is the measurement result figure of hundreds of cigarette mainstream flue gas；
Fig. 3 is the optimum results figure of packing quality in HiCapt MCX solid-phase extraction columns；
Fig. 4 is the optimum results figure of load solution pH value；
Fig. 5 is the optimum results figure of methanol content in leacheate；
Fig. 6 is the optimum results figure of eluent type；
Fig. 7 is the optimum results figure of effluent volume.
Specific implementation mode
Embodiments of the present invention are described further below in conjunction with the accompanying drawings.
Embodiment 1
The peculiar N- nitrosamine of tobacco in a kind of cigarette mainstream flue gas exchanging Solid Phase Extraction based on hydrophobic nonionic of the present invention
Extracting method includes the following steps：
1) according to GB/T 19609-2004,4387 ISO:2000 regulation aspirates 5 cigarette with linear type smoking machine,
There is the filter disc of mainstream smoke total particulate matter to be placed in container trapping, 15mL, 5mmol/L ammonium acetate solution is added, is placed in ultrasound
Wave producer (is purchased from Kunshan Ultrasonic Instruments Co., Ltd., model：KQ-700DB ultrasonic extraction 30min on), ultrasonic power
280W, frequency 40kHz, extracting solution cross 0.22 μm of water phase filter membrane to get sample crude extract, and adjusting sample crude extract pH value is
4, as load solution；
2) 500mg HiCapt MCX solid phase extraction fillers are weighed in the Solid Phase Extraction void column pipe with sieve plate, are constantly struck
Beating keeps filler filling uniform, and upper end cover upper sieve plate compresses, for use.Before loading, 1.0mL acetone, 2.0mL methanol volumes point are used successively
Methanol aqueous solution, 2mL water cleaning activated solid extraction pillar of the number for 5%；Then under the effect of gravity by 6mL load solutions from
So solid phase extraction column is flowed through to complete loading process；
3) methanol aqueous solution for being 10% with 1mL methanol volume fractions after end of the sample is eluted, and waits for that leacheate is complete
After flowing through extraction column, leacheate is drained under the suction function of vacuum pump；
4) the 2mL acetone concentrated ammonia liquor of 1% volume fraction (contain) elution target analytes are used, collect to get.
The peculiar N- nitrosamine of tobacco in a kind of cigarette mainstream flue gas exchanging Solid Phase Extraction based on hydrophobic nonionic of the present invention
Assay method includes the following steps：
1. the extraction of the peculiar N- nitrosamine of tobacco in cigarette mainstream flue gas
1.1 according to GB/T 19609-2004,4387 ISO:2000 regulation aspirates 5 cigarette with linear type smoking machine,
Isotopic Internal Standard addition is had in trapping on the filter disc of mainstream smoke total particulate matter, is placed in container, 15mL, 5mmol/L is added
Ammonium acetate solution is placed in supersonic generator and (is purchased from Kunshan Ultrasonic Instruments Co., Ltd., model：KQ-700DB ultrasound carries on)
30min, ultrasonic power 280W, frequency 40kHz, extracting solution is taken to cross 0.22 μm of water phase filter membrane to get sample crude extract, adjust thick
Extracting liquid pH value is 4, as load solution；
1.2 accurately weigh 500mg HiCapt MCX solid phase extraction fillers in the Solid Phase Extraction void column pipe with sieve plate,
Constantly beaing keeps filler filling uniform, and upper end cover upper sieve plate compresses, for use.Before loading, 1.0mL acetone, 2.0mL methanol are used successively
Methanol aqueous solution, 2mL water activated solid extraction pillar of the volume fraction for 5%；Then under the effect of gravity by 6mL load solutions
Naturally solid phase extraction column is flowed through to complete loading process；
The methanol aqueous solution for being 10% with 1mL methanol volume fractions after 1.3 ends of the sample is eluted, and waits for that leacheate is complete
After flowing through extraction column, leacheate is drained under the suction function of vacuum pump；
1.4 with 2mL acetone (containing 1% volume fraction ammonium hydroxide) elution target analytes, collect to get；
2. the measurement of the peculiar N- nitrosamine of tobacco in cigarette mainstream flue gas
By step 1.4 gained extracting solution, nitrogen dries up at 35 DEG C, then redissolves liquid with 0.2mL and redissolved to get to be measured
Liquid.The formic acid acetonitrile solution that liquid is 0.1% by the ammonium formate solution and formic acid volume fraction of 0.01mol/L is redissolved according to 1:1
Volume ratio is formulated.
The preparation of mixed standard solution：With four grow tobacco peculiar N- nitrosamine Isotopic Internal Standard (NNN-d4, NNK-d4,
NAT-d4 and NAB-d4) it is solute, methanol is solvent, prepares inner mark solution；With four grow tobacco peculiar N- nitrosamine (NNN, NNK,
NAT and NAB) it is solute, the inner mark solution of fixed volume is added, methanol is solvent, prepares the mixed standard solution of series concentration,
It is specifically shown in the following table 1.Wherein interior target concentration is 10ng/mL.Mixed standard solution is subjected to liquid chromatography-tandem mass spectrometry point
Analysis；
The concentration table of 1 mixed standard solution of table
The making of standard curve：Mixed standard solution is taken to carry out liquid chromatography-tandem mass spectrometry analysis, analysis condition is：Color
Compose column：120 EC-C18 of Poroshell (3.0mm × 100mm, 2.7 μm)；Column temperature：40℃；Sample size：5μL；Mobile phase is
- 0.1% formic acid acetonitrile (B) of 0.01mol/L ammonium formates (A)；Flow velocity：0.4mL/min；Eluent gradient program：T=0min,
10%B, t=2.0min, 40%B, t=4.0min, 60%B, t=6.0min, 90%B, t=9.0min, 90%B, t=
9.5min, 10%B, t=15.0min, 10%B；Mass Spectrometry Conditions：Electron spray voltage 5000V；Atomization gas (Gas1) pressure 50psi；
Assisted atomization gas (Gas2) pressure 50psi；Gas curtain gas (Gas1) pressure 35psi；450 DEG C of ion source temperature；Import voltage 8V；Go out
Mouth voltage 10V；Remove cluster voltage 40V；Residence time 40ms；Monitoring pattern：Multiple-reaction monitoring.
The quota ion pair of NNN, NNK, NAT and NAB are followed successively by 178.1 in the multiple-reaction monitoring>148.2,208.1>
122.1,190.1>160.1 and 192.1>162.2, corresponding collision voltage is followed successively by 15,16,15 and 17eV；NNN,NNK,NAT
It is followed successively by 178.1 with the qualitative ion pair of NAB>120.1,208.1>106.1,190.1>106.1 and 192.1>133.1, accordingly
Collision voltage be followed successively by 15,16,15 and 17eV；The quota ion pair of NNN-d4, NNK-d4, NAT-d4 and NAB-d4 are followed successively by
182.1>152.2,212.1>126.1,194.1>164.1 and 196.1>166.2, corresponding collision voltage is followed successively by 15,16,15
And 17eV.The end of the program makees target analysis concentration with target analytes peak area and corresponding isotopic peak area ratio
To get the standard curve of each target analytes, parameter see the table below 2 for regression analysis, and the chromatogram of mixed standard solution 3 is shown in Fig. 1.
The corresponding standard curve of 2 each target analytes of table, quantitative limit and detection limit
Note：Detection limit corresponding concentration when with quantitative limit being signal-to-noise ratio (S/N) 3 and 10.
The measurement of the peculiar N- nitrosamine of tobacco in sample：Above-mentioned solution to be measured is taken to carry out liquid chromatography-tandem mass spectrometry analysis,
Analysis condition carries out the analysis condition of liquid chromatography-tandem mass spectrometry analysis, each substance peak face that will be measured with mixed standard solution
Product substitutes into following formula 1, calculates the content of target analytes in sample.
Formula 1：
In formula, m is the content of target analytes in sample, ng/g；X is the peak area of target analytes and Isotopic Internal Standard
The ratio between；A is the slope of standard curve, and b is the intercept of standard curve；V is the volume of sample extracting solution, mL；N is sample quality,
g。
Embodiment 2
By assay method described in embodiment 1, in addition hundreds of commercial cigarettes are chosen, the wherein peculiar N- nitrous of tobacco is measured
The content of amine, the results are shown in Figure 2.
In other embodiments of the present invention, in HiCapt MCX solid-phase extraction columns filler dosage can also be 50mg,
100mg, 200mg, 300mg or 1000mg, the other the same as in Example 1.By packing quality in Fig. 3 HiCapt MCX solid-phase extraction columns
Optimum results figure it is found that the quality of filler within the scope of 50-1000mg, as quality is continuously improved, the peak face of target analytes
Product is continuously improved, and when reaching 500mg, continues growing packing quality, the peak area of target analytes improves unobvious, comprehensive
Consider the effect of extracting of all target analytes, the quality of preferred filler is 500mg.
In other embodiments of the present invention, the pH value of load solution can also be 3.4,6,10.5 or 11, other with implementation
Example 1.As shown in the optimum results figure of Fig. 4 load solution pH value, when pH is 4.0, all analytes have preferable extraction
Effect；When pH value is higher than 6.0 or less than 4.0, the extraction efficiency of target analytes is declined；Due in solid-phase extraction column
Filler hydrophobic and cationic exchange interaction, pH major part targets in 4.0-6.0 are mainly leaned on to the extractions of target analytes
Analyte molecule exists with protonated form, and the cation exchange group in filler exists in the form of anion, at this time filler
Hydrophobic effect and cationic exchange interaction power between target analytes is stronger, therefore has best effect of extracting.
In other embodiments of the present invention, the volume fraction of methanol can also be in methanol aqueous solution in step 3) and 1.3
5%, 8%, 12%, 15% or 20%, the other the same as in Example 1.As shown in the optimum results figure of methanol content in Fig. 5 leacheates,
When methanol volume fraction is 10% in methanol aqueous solution, target analytes have preferable extraction efficiency, continue growing methanol
Content reduces the hydrophobic interaction between target analytes and filler, is unfavorable for the raising of extraction efficiency.Therefore, methanol
The volume fraction of methanol is preferably 10% in aqueous solution.
In other embodiments of the present invention, the eluent in step 4) and 1.4 can also be containing 3% volume fraction ammonium hydroxide
Acetone mixture, acetone mixture or acetone containing 1% or 3% volume fraction formic acid, the other the same as in Example 1.Such as Fig. 6 eluents
Shown in the optimum results figure of type and Fig. 7 eluent dosages, a certain proportion of ammonium hydroxide is added in acetone, can improve target point
The elution efficiency of object is analysed, this is because the cation that can be destroyed under alkaline condition between filler and target analytes exchanges and makees
With.Other than NAB, remaining target analytes can completely be eluted by 1mL eluents liquid, in order to ensure preferably to elute effect
The volume of rate, eluent is preferably 1~2mL.
In other embodiments of the present invention, in cigarette mainstream flue gas in the assay method step 2 of the peculiar N- nitrosamine of tobacco
Redissolving liquid can also be by the formic acid acetonitrile solution that the ammonium formate solution and formic acid volume fraction of 0.01mol/L are 0.1% according to 1:2
Or 2:1 volume ratio is formulated, the other the same as in Example 1.
Test example
1) precision and mark-on reclaims test
To investigate the reproducibility of said determination method, in the trapping of embodiment 1 has a filter disc of mainstream smoke total particulate matter
A certain amount of mixed standard solution is added, is configured to sample (the corresponding value point of basic, normal, high concentration of basic, normal, high three kinds of concentration
It Wei not be 2,10 and 40 times of each target analytes range of linearity minimum).Under the analysis condition of optimization, target analytes with it is dry
Disturb object separation well, chaff interferent does not influence quantifying for target analytes.
It is measured with 5 samples prepared in one day, calculates the in a few days relative standard deviation under various concentration；With continuous
The sample prepared is measured within 3 days, calculates the relative standard deviation in the daytime under various concentration；By the ratio between the peak area of analysis gained
It substitutes into standard curve, the content of target analytes in solution to be measured is calculated, and obtained relatively compared with actual interpolation amount
As a result the rate of recovery see the table below 3.
3 precision of table and mark-on reclaims test result
From table 3 it can be seen that target analytes under various concentration in a few days and day to day precision is respectively smaller than 4.6% He
4.2%, illustrate that this method has preferable reproducibility.Meanwhile under various concentration target analytes relative recovery between
Between 92.2%~108.9%.
In order to further verify the peculiar N- nitrosamine of tobacco of the present invention assay method accuracy, using this method and existing
Commercial cigarettes are analyzed in row standard method (YQ/T 17-2012), and the results are shown in Table 4.
The result (ng/ branch) that table 4 analyzes commercial cigarettes using this method and standard method
The result shows that the measurement result consistency that two methods obtain is good, illustrate that the accuracy of this method is good, Ke Yiman
Sufficient routine analysis requirement.
Claims (9)
1. the extracting method of the peculiar N- nitrosamine of tobacco in a kind of cigarette mainstream flue gas exchanging Solid Phase Extraction based on hydrophobic nonionic,
It is characterized by comprising the following steps：
The ammonium acetate solution that trapping has the filter disc of mainstream smoke total particulate matter that a concentration of 5 ~ 100 mmol/L is added is extracted,
Filtering, obtains crude extract；
By step 1）Gained crude extract flows through the HiCapt MCX solid-phase extraction columns after activation, completes loading；
It is eluted with methanol aqueous solution；
Carrying to get N- nitrosamine is eluted with any one in acetone, acetone ammonia water mixture, acetone formic acid mixed liquor
Take liquid；Acetone in the acetone ammonia water mixture, ammonium hydroxide volume ratio be 97 ~ 99:3~1；Acetone, first in acetone formic acid mixed liquor
The volume ratio of acid is 97 ~ 99:3~1；The mass fraction of the ammonium hydroxide is 35%.
2. the peculiar N- of tobacco in a kind of cigarette mainstream flue gas exchanging Solid Phase Extraction based on hydrophobic nonionic as described in claim 1
The extracting method of nitrosamine, which is characterized in that the peculiar N- nitrosamine of tobacco includes NNN, NNK, NAT in the cigarette mainstream flue gas
And NAB.
3. the peculiar N- of tobacco in a kind of cigarette mainstream flue gas exchanging Solid Phase Extraction based on hydrophobic nonionic as described in claim 1
The extracting method of nitrosamine, which is characterized in that the crude extract adjusts pH to 3.0 ~ 11.0 before loading.
4. the peculiar N- of tobacco in a kind of cigarette mainstream flue gas exchanging Solid Phase Extraction based on hydrophobic nonionic as claimed in claim 3
The extracting method of nitrosamine, which is characterized in that it is acetic acid or ammonium hydroxide to adjust pH adjusting agent used in pH.
5. the peculiar N- of tobacco in a kind of cigarette mainstream flue gas exchanging Solid Phase Extraction based on hydrophobic nonionic as described in claim 1
The extracting method of nitrosamine, which is characterized in that step 3）Described in methanol aqueous solution the volume fraction of methanol be 5% ~ 20%.
6. the assay method of the peculiar N- nitrosamine of tobacco in a kind of cigarette mainstream flue gas exchanging Solid Phase Extraction based on hydrophobic nonionic,
It is characterized by comprising the following steps：
（1）The preparation of extracting solution
1）There is the filter disc of mainstream smoke total particulate matter that a concentration of 5 ~ 100mmol/L acetic acid is added the trapping containing Isotopic Internal Standard
Ammonium salt solution extracts, and obtains crude extract；
2）By step 1）Gained crude extract flows through the HiCapt MCX solid-phase extraction columns after activation, completes loading；
3）It is eluted with methanol aqueous solution, the volume fraction of the methanol aqueous solution is 5% ~ 20%；
4）It is eluted to get N- nitrosamine with any one in acetone, acetone ammonia water mixture, acetone formic acid mixed liquor
Extracting solution；Acetone in the acetone ammonia water mixture, ammonium hydroxide volume ratio be 97 ~ 99:3~1；Acetone in acetone formic acid mixed liquor,
The volume ratio of formic acid is 97 ~ 99:3~1；
（2）The measurement of the peculiar N- nitrosamine of tobacco in cigarette mainstream flue gas
By step 4）Gained extracting solution dries up, and the redissolution liquid being formulated with ammonium formate solution, formic acid acetonitrile solution is redissolved,
Carry out liquid chromatography tandom mass spectrometry determination later, the chromatographic condition when liquid chromatography tandom mass spectrometry determination is：Chromatographic column：
Poroshell 120 EC-C18；Column temperature：40℃；Sample size：5μL；Mobile phase is 0.01mol/L ammonium formate A-0.1% formic acid second
Nitrile B；Flow velocity：0.4 mL/min；Eluent gradient program：T=0 min, 10% B, t=2.0 min, 40% B, t=4.0min, 60%
B, t=6.0min, 90% B, t=9.0 min, 90% B, t=9.5min, 10% B, t=15.0min, 10% B；Mass Spectrometry Conditions：EFI
Mist voltage 5000V；50 psi of atomization gas pressure；Assisted atomization atmospheric pressure 50psi；35 psi of gas curtain atmospheric pressure；Ion source temperature
450℃；Import voltage 8V；Exit potential 10V；Remove cluster voltage 40V；40 ms of residence time；Monitoring pattern：Multiple-reaction monitoring.
7. the peculiar N- of tobacco in a kind of cigarette mainstream flue gas exchanging Solid Phase Extraction based on hydrophobic nonionic as claimed in claim 6
The assay method of nitrosamine, which is characterized in that step 1）Middle Isotopic Internal Standard includes NNN-d4, NNK-d4, NAT-d4, NAB-
d4。
8. the peculiar N- of tobacco in a kind of cigarette mainstream flue gas exchanging Solid Phase Extraction based on hydrophobic nonionic as claimed in claim 6
The assay method of nitrosamine, which is characterized in that step（2）It is middle to redissolve ammonium formate solution and formic acid volume of the liquid by 0.01mol/L
The formic acid acetonitrile solution that score is 0.1% is according to 1 ~ 2:1 ~ 2 volume ratio is formulated.
9. the peculiar N- of tobacco in a kind of cigarette mainstream flue gas exchanging Solid Phase Extraction based on hydrophobic nonionic as claimed in claim 6
The assay method of nitrosamine, which is characterized in that the quota ion pair of NNN, NNK, NAT and NAB is successively in the multiple-reaction monitoring
It is 178.1>148.2,208.1>122.1,190.1>160.1 and 192.1>162.2, corresponding collision voltage is followed successively by 15,16,
15 and 17eV；The qualitative ion pair of NNN, NNK, NAT and NAB are followed successively by 178.1>120.1,208.1>106.1,190.1>
106.1 and 192.1>133.1, corresponding collision voltage is followed successively by 15,16,15 and 17eV；NNN-d4, NNK-d4, NAT-d4 and
The quota ion pair of NAB-d4 is followed successively by 182.1>152.2,212.1>126.1,194.1>164.1 and 196.1>166.2, accordingly
Collision voltage be followed successively by 15,16,15 and 17eV.
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