CN106841461A - The extraction of N nitrosamine and assay method in a kind of product for quitting smoking that SPE is exchanged based on hydrophobic nonionic - Google Patents
The extraction of N nitrosamine and assay method in a kind of product for quitting smoking that SPE is exchanged based on hydrophobic nonionic Download PDFInfo
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Abstract
Extraction and assay method the present invention relates to N nitrosamine in a kind of product for quitting smoking that SPE is exchanged based on hydrophobic nonionic, belong to tobacco chemistry inspection technology field.The extracting method of N nitrosamine includes being extracted the ammonium acetate solution that product for quitting smoking addition concentration is 5~100mmol/L in product for quitting smoking of the present invention based on hydrophobic nonionic exchange SPE, the crude extract for obtaining flows through the HiCapt MCX solid-phase extraction columns after activation, then drip washing is carried out, wash-out, obtains final product.The extracting method of N nitrosamine in product for quitting smoking of the present invention based on hydrophobic nonionic exchange SPE, the removal of impurity and chaff interference can effectively be removed, shorten process time, improve the degree of accuracy and the precision of assay method, the relative recovery of method between 92.2%~108.9%, 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 extraction and survey of N- nitrosamine in a kind of product for quitting smoking that SPE is exchanged based on hydrophobic nonionic
Determine method, belong to tobacco chemistry inspection technology field.
Background technology
The peculiar N- nitrosamine of tobacco is that aminated compounds reacts and generates with nitrosification agent in acid condition, is one
The carcinogen that class is received significant attention, be not only Huffman inventory and food and medicine Surveillance Authority of the U.S. " tobacco product and
Important substance in harmful and potentially harmful substance list in flue gas ", is also international cancer research institution's " smoke-free tobacco product
In 28 kinds of harmful substances " important component in list.Therefore, the accurate analysis peculiar N- content of nitrosamines of tobacco, to ensureing product
Consumption safety is significant.
Sucking product for quitting smoking can obtain the satisfaction of common cigarette, in order to obtain this satisfaction, part product for quitting smoking sample
Tobacco leaf ingredient is with the addition of in product, and then also has the presence of N- nitrosamine.However, there is presently no N- nitrosamine in product for quitting smoking
The relevant report of measure.
Application publication number for CN102012407A patent of invention disclose one grow tobacco and tobacco product in tobacco it is peculiar
The detection method of N- nitrosamine, in this scenario by extract flow through solid phase extraction column, then using formic acid water wash, finally
Eluted with ammonia hydroxide/methanol, obtain prepare liquid.Solid phase extraction filler in the program is that NVP-benzene sulfonic acid is common
Polymers, organic polymer fillers in organic solvent can be swelling, can to a certain extent influence enrichment and the clean-up effect of filler.
In addition, also not optimized to the various parameters for influenceing effect of extracting in the program.And would generally in product for quitting smoking sample
There is the addition of traditional Chinese medicine ingredients, therefore its sample substrate has the technical side in very big difference, therefore the program with tobacco sample matrix
Case may not be suitable for product for quitting smoking sample.
Therefore, the assay method for developing N- nitrosamine in a kind of product for quitting smoking is extremely urgent.
The content of the invention
It is an object of the invention to provide N- nitrosamine in a kind of product for quitting smoking that SPE is exchanged based on hydrophobic nonionic
Extracting method.
It is sub- it is a further object to provide N- in a kind of product for quitting smoking that SPE is exchanged based on hydrophobic nonionic
The assay method of nitramine.
In order to realize the above object the technical solution adopted in the present invention is：
The extracting method of N- nitrosamine in a kind of product for quitting smoking that SPE is exchanged based on hydrophobic nonionic, including following step
Suddenly：
1) ammonium acetate solution that product for quitting smoking addition concentration is 5~100mmol/L is extracted, is obtained crude extract；
2) by step 1) obtained by crude extract flow through the HiCapt MCX solid-phase extraction columns after activation process, in completion
Sample；
3) drip washing is carried out with methanol aqueous solution；
4) eluted with any one in acetone, acetone ammonia water mixture, acetone formic acid mixed liquor, obtained final product N- nitrous
The extract solution of amine；Acetone, the volume ratio of ammoniacal liquor are 97~99 in the acetone ammonia water mixture:3~1；Acetone formic acid mixed liquor
Middle acetone, the volume ratio of formic acid are 97~99:3~1；The mass fraction of the ammoniacal liquor is 35%.
Above-mentioned product for quitting smoking is the filler for lighting type Ling " for giving up smoking.
N- nitrosamine includes NNN, NNK, NAT and NAB in above-mentioned product for quitting smoking.
Above-mentioned steps 1) in extract operation be：By the ammonium acetate solution that product for quitting smoking addition concentration is 5~100mmol/L
In, carry out ultrasonically treated, insoluble matter is then peeled off, obtain crude extract.
The concentration of above-mentioned ammonium acetate solution is preferably 10mmol/L~80mmol/L.
The above-mentioned consumption per ammonium acetate solution used by 1g product for quitting smoking is 10~100mL.
It is above-mentioned ultrasonically treated, 280~700W of ultrasonic power, 20~40kHz of frequency, time >=10min.Ultrasonic time is preferred
It is 10~60min.Ultrasonic time is more preferably 30min.
Above-mentioned separation insoluble matter is by the way of filtering or centrifugation.
Above-mentioned filtering uses water phase filter membrane.
The aperture of above-mentioned water phase filter membrane is 0.20~0.50 μm.Preferably 0.22 μm or 0.45 μm.
Above-mentioned water phase filter membrane is poly (ether sulfone) film.
Above-mentioned centrifugation, be will be ultrasonically treated after mixed liquor stand, be then centrifuged, finally take supernatant liquor.
Above-mentioned time of repose is 3~10min.Preferably 5min.
Above-mentioned 5000~10000rpm of centrifugal rotational speed, the time is 3~10min.Centrifugation time is preferably 5min.
Above-mentioned HiCapt MCX solid-phase extraction columns are hydrophobic/ion exchange mixed-mode retention mechanism solid-phase extraction column.
Above-mentioned HiCapt MCX solid-phase extraction columns are purchased from Weitaike Technology (Wuhan) Co., Ltd..
Above-mentioned HiCapt MCX solid-phase extraction columns have hydrophobic and strong ion exchange.
The consumption of the filler in above-mentioned HiCapt MCX solid-phase extraction columns used is：6mL crude extracts are often completed into loading
When, accordingly use the HiCapt MCX solid phase extraction fillers of 50~1000mg.Preferably, 6mL crude extracts correspondence uses 500mg
HiCapt MCX solid phase extraction fillers.
Above-mentioned crude extract adjusts pH to 3.0~11 before loading.Preferably adjust pH to 4~6.
The conditioning agent of above-mentioned regulation pH value is for acetic acid is molten or ammoniacal liquor.When pH is adjusted from acetic acid, preferred mass fraction is
10% acetic acid solution.
Above-mentioned activation process is operated：Successively HiCapt MCX solid-phase extraction columns are flowed through with acetone, methanol aqueous solution, water.
Above-mentioned activation is to flow through HiCapt MCX with acetone, the methanol aqueous solution that methyl alcohol volume fraction is 5%, water successively to consolidate
Phase extraction column.The acetone, methanol aqueous solution, the volume ratio of water are 1:2:2.
Step 3) in methanol aqueous solution the volume fraction of methyl alcohol be 5~20%.
Step 3) in drain leacheate under the suction function of vavuum pump after drip washing.
Step 4) acetone is preferably 99 with the volume ratio of ammoniacal liquor in acetone ammonia water mixture:1.
Step 4) acetone is preferably 99 with the volume ratio of formic acid in acetone formic acid mixed liquor:1.
Above-mentioned wash-out, the consumption of eluent is：Often by 6mL crude extract loadings, drip washing, the eluting liquid used after drying
Product >=0.5ml.Preferably 1~2ml.More preferably 1ml.
The assay method of the peculiar N- nitrosamine of tobacco, bag in a kind of product for quitting smoking that SPE is exchanged based on hydrophobic nonionic
Include following steps：
The preparation of 1.N- nitrosamine extract solutions
1.1 sequentially add Isotopic Internal Standard and ammonium acetate solution that concentration is 5~100mmol/L is carried out in product for quitting smoking
Extract, obtain crude extract；
The crude extract of step 1.1 gained is flowed through the HiCapt MCX solid-phase extraction columns after activation process by 1.2, is completed
Loading；
1.3 carry out drip washing with methanol aqueous solution；
1.4 are eluted with any one in acetone, acetone ammonia water mixture, acetone formic acid mixed liquor, obtain final product N- sub-
The extract solution of nitramine；
The measure of 2.N- nitrosamine
By the extract solution drying of gained in step 1.4, the redissolution liquid being formulated with ammonium formate solution, formic acid acetonitrile solution
Redissolved, liquid chromatography tandom mass spectrometry determination is carried out afterwards.
Above-mentioned Isotopic Internal Standard includes NNN-d4, NNK-d4, NAT-d4, NAB-d4.
The above-mentioned consumption per ammonium acetate solution used by 1g product for quitting smoking is 10~100mL.
The addition of above-mentioned Isotopic Internal Standard is：1mL ammonium acetate solutions correspondence is often added to add every kind of Isotopic Internal Standard
Quality is 10ng.
Gained crude extract pH before loading is adjusted to 3.0~11.0 in step 1.1.
Acetone is preferably 99 with ammoniacal liquor volume ratio in step 1.4:1.Acetone is preferably 99 with the volume ratio of formic acid:1.
Above-mentioned wash-out, the consumption of eluent is：Often by 6mL crude extract loadings, drip washing, the eluting liquid used after drying
Product >=0.5mL.Preferably 1~2mL.More preferably 1mL.
Above-mentioned redissolution solution is molten with the formic acid acetonitrile that formic acid volume fraction is 0.1% by the ammonium formate solution of 0.01mol/L
Liquid is according to 1~2:1~2 volume ratio is formulated.
The consumption of above-mentioned redissolution solution is：Per loading 6mL crude extracts, follow-up redissolution liquor capacity >=0.1mL used.It is excellent
Elect 0.1~1mL as.More preferably 0.2mL.
In product for quitting smoking of the present invention in the assay method of N- nitrosamine, the specific ginseng being related in the preparation process of extract solution
Number and extraction conditions are identical with the extracting method of N- nitrosamine in product for quitting smoking.
Liquid chromatography tandom mass spectrometry determination includes the preparation of the mixed standard solution of series concentration in above-mentioned steps 2, prepares
Step is：
It is molten with four Isotopic Internal Standards (NNN-d4, NNK-d4, NAT-d4 and NAB-d4) for growing tobacco peculiar N- nitrosamine
Matter, methyl alcohol is solvent, prepares inner mark solution；Peculiar N- nitrosamine (NNN, NNK, NAT and NAB) is grown tobacco for solute with four, plus
Enter the internal standard of fixed volume, methyl alcohol is solvent, prepare the mixed standard solution of series concentration.
The making step of the assay method standard curve of N- nitrosamine is in product for quitting smoking of the present invention：
It is molten with four Isotopic Internal Standards (NNN-d4, NNK-d4, NAT-d4 and NAB-d4) for growing tobacco peculiar N- nitrosamine
Matter, methyl alcohol is solvent, prepares inner mark solution；Peculiar N- nitrosamine (NNN, NNK, NAT and NAB) is grown tobacco for solute with four, plus
Enter the internal standard of fixed volume, methyl alcohol is solvent, prepare the mixed standard solution of series concentration, the hybrid standard of series concentration is molten
Liquid carries out liquid chromatography-tandem mass spectrometry analysis, with target analytes peak area and corresponding isotopic peak area ratio to target point
Analysis concentration does regression analysis, obtains final product the standard curve of each target analytes.
The condition of above-mentioned liquid chromatography-tandem mass spectrometry analysis is：Chromatographic column：Poroshell 120EC-C18；Column temperature：40
℃；Sample size：5μL；Mobile phase A：0.01mol/L ammonium formate solutions, Mobile phase B：0.1% formic acid acetonitrile solution；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 pressure 50psi；Assisted atomization atmospheric pressure 50psi；Gas curtain atmospheric pressure 35psi；Ion source temperature 450
℃；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 is followed successively by 178.1 in above-mentioned 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
Qualitative ion pair with NAB is followed successively by 178.1>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 is 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.
Above-mentioned chromatographic column：Poroshell 120EC-C18 3.0mm × 100mm, 2.7 μm.
Beneficial effects of the present invention：
Employed in the extracting method of N- nitrosamine in product for quitting smoking of the present invention based on hydrophobic nonionic exchange SPE
HiCapt MCX solid-phase extraction columns, HiCapt MCX solid phase extraction fillers have good clean-up effect, energy to sample extracting solution
The removal of impurity and chaff interference are effectively removed, the degree of accuracy and the precision of assay method is improved.
The assay method of N- nitrosamine in product for quitting smoking of the present invention based on hydrophobic nonionic exchange SPE, by using
HiCapt MCX solid-phase extraction columns are extracted to target analytes, and hydrophobic and cation exchange is carried out to target analytes, are reached
It is then sub- with N- in Liquid Chromatography-Tandem Mass Spectrometry qualitative and quantitative analysis product for quitting smoking to the purification to target analytes
Nitramine, assay method of the invention has sample pre-treatments simple to operate, quick, and the removal efficiency of impurity and chaff interference is high, place
Reason time short advantage.
Brief description of the drawings
Fig. 1 is the chromatogram of mixed standard solution 3；
Fig. 2 is the measurement result figure of product for quitting smoking；
Fig. 3 is the optimum results figure of crude extract pH value；
Fig. 4 is the optimum results figure of eluent species；
Fig. 5 is the optimum results figure of effluent volume；
Fig. 6 is the optimum results figure of methanol content in leacheate；
Fig. 7 is the optimum results figure of packing quality in HiCapt MCX solid-phase extraction columns.
Specific embodiment
Following embodiments are described in further detail to the present invention.
Embodiment 1
In the present embodiment in product for quitting smoking N- nitrosamine extracting method, comprise the following steps：
1) filler (being accurate to 0.1mg) that 1.0g lights type Ling " for giving up smoking is weighed, is inserted in 30mL tool plug centrifuge tubes, accurately
The ammonium acetate solution of 30mL concentration 10mmol/L is added, supersonic generator is placed in (purchased from the limited public affairs of city of Kunshan's ultrasonic instrument
Department, model：KQ-700DB ultrasonically treated, ultrasonic extraction 30min, ultrasonic power 700W, frequency 40kHz are carried out on), 5min is stood
Afterwards, it is placed in and 5min is centrifuged on centrifuge, centrifugal rotational speed 10000rpm takes supernatant liquor, obtains final product crude extract；Crude extract is upper
PH value before sample is adjusted to 4, used as load solution；
2) 500mg HiCapt MCX solid phase extraction fillers accurately are weighed in the SPE void column pipe with sieve plate, no
Disconnected beaing makes filler fill uniformly, and upper end cover upper sieve plate is compressed, stand-by；Before loading, successively with 1.0mL acetone, 2.0mL methyl alcohol bodies
Fraction is 5% methanol aqueous solution, 2mL water cleaning activated solid extraction pillar；Afterwards under gravity by 6mL steps 1)
The load solution of middle gained flows through solid phase extraction column to complete loading process naturally；
3) drip washing is carried out with the methanol aqueous solution that 1mL methyl alcohol volume fractions are 10% after end of the sample, treats that leacheate is complete
After flowing through extraction column, leacheate is drained under the suction function of vavuum pump；
4) with 2mL acetone (concentrated ammonia liquor containing 1% volume fraction) wash-out target analytes, collect, obtain final product.
The assay method of N- nitrosamine, comprises the following steps in the product for quitting smoking of the present embodiment：
1. in product for quitting smoking N- nitrosamine extraction
1.1 weigh the filler (being accurate to 0.1mg) that 1.0g lights type Ling " for giving up smoking, insert in 30mL tool plug centrifuge tubes, plus
After entering Isotopic Internal Standard, the ammonium acetate solution of accurate addition 30mL concentration 10mmol/L is placed in supersonic generator (purchased from elder brother
Ultrasonic instrument Co., Ltd of mountain city, model：KQ-700DB ultrasonically treated, ultrasonic extraction 30min, ultrasonic power 700W are carried out on),
Frequency 40kHz, after standing 5min, is placed in and 5min is centrifuged on centrifuge, and centrifugal rotational speed 10000rpm takes supernatant liquor, obtains final product thick
Extract solution；PH value of the crude extract before loading is adjusted to 4, used as load solution；
1.2 it is accurate weigh 500mg HiCapt MCX solid phase extraction fillers in the SPE void column pipe with sieve plate,
Constantly beaing makes filler fill uniformly, and upper end cover upper sieve plate is compressed, stand-by.Before loading, successively with 1.0mL acetone, 2.0mL methyl alcohol
Volume fraction is 5% methanol aqueous solution, 2mL water cleaning activated solid extraction pillar.Afterwards under gravity by step 1.1
The 6mL load solutions of middle gained flow through solid phase extraction column to complete loading process naturally；
Drip washing is carried out with the methanol aqueous solution that 1mL methyl alcohol volume fractions are 10% after 1.3 ends of the sample, treats that leacheate is complete
After flowing through extraction column, leacheate is drained under the suction function of vavuum pump；
1.4, with 2mL acetone (containing 1% volume fraction ammoniacal liquor) wash-out target analytes, collect, and obtain final product；
2. in product for quitting smoking N- nitrosamine measure
By gained extract solution nitrogen drying at 35 DEG C in step 1.4, then redissolved with 0.2mL redissolution solution, must be treated
Survey liquid.Redissolve solution by 0.01mol/L ammonium formate solution and formic acid acetonitrile mixed solution that formic acid volume fraction is 0.1% by
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, methyl alcohol is solvent, prepares inner mark solution；With four grow tobacco peculiar N- nitrosamine (NNN, NNK,
NAT and NAB) it is solute, the internal standard of fixed volume is added, methyl alcohol is solvent, prepares the mixed standard solution of series concentration, specifically
See the table below 1.Wherein interior target concentration is 10ng/mL.Mixed standard solution is carried out into liquid chromatography-tandem mass spectrometry analysis.
The concentration table of the mixed standard solution of table 1
The making of standard curve：Taking mixed standard solution carries out liquid chromatography-tandem mass spectrometry analysis, and analysis condition is：Color
Spectrum post：Poroshell 120EC-C18 (3.0mm × 100mm, 2.7 μm)；Column temperature：40℃；Sample size：5μL；Mobile phase is
The formic acid acetonitrile (B) of 0.01mol/L ammonium formates (A) -0.1%；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 is 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
Qualitative ion pair with NAB is followed successively by 178.1>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 is 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.Program end of run, with target analytes peak area and corresponding isotopic peak area ratio to target analyte concentration
Make regression analysis, obtain final product the standard curve of each target analytes, parameter see the table below 2, and the chromatogram of mixed standard solution 3 is shown in Fig. 1.
The corresponding standard curve of each target analytes of table 2, quantitative limit and test limit
Note：Corresponding concentration when test limit and quantitative limit are signal to noise ratios (S/N) 3 and 10
The measure of N- nitrosamine in sample：Taking above-mentioned solution to be measured carries out liquid chromatography-tandem mass spectrometry analysis, analysis condition
Ibid, each material peak area that will be measured is substituted 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
Assay method as described in embodiment 1, chooses several product for quitting smoking in addition, determines the content of wherein N- nitrosamine,
Result (is only listed and detects all 4 kinds of samples of N- nitrosamine) as shown in Figure 2.
In other embodiment in the present invention, the pH of crude extract can be adjusted to 3.4-11.0, in the further present invention
Other embodiment in the pH of crude extract can select to be 3.4,6,10.5 or 11, the other the same as in Example 1.Due to SPE
Extraction of the filler to target analytes is mainly by the effect of hydrophobic and cation exchange in post, pH most of mesh in 4.0-6.0
Mark analyte molecule exists with protonated form, and the cation exchange group in filler exists in anion form, now fills out
Hydrophobic effect and cation exchange active force between material and target analytes is stronger, therefore with optimal effect of extracting.Such as
Shown in Fig. 3, in the pH value range (3.4-11.0) investigated, when pH is 4.0, all analytes have preferably extraction
Effect.
In other embodiment in the present invention, step 4) and 1.4 in eluent can select acetone, acetone ammonia water mixture,
Any one in acetone formic acid mixed liquor.Further, can also be the acetone mixture containing 3% volume fraction ammoniacal liquor, contain
The acetone mixture or acetone of 1% or 3% volume fraction formic acid, the other the same as in Example 1.As Fig. 4 eluents species, Fig. 5 are eluted
Shown in liquid product optimum results figure, a certain proportion of ammoniacal liquor is added in acetone, it is possible to increase the desorption efficiency of target analytes,
Because the cation exchange effect that can be destroyed between filler and target analytes under alkalescence condition.
In other embodiment in the present invention, step 3) and 1.3 methanol aqueous solutions in the volume fraction of methyl alcohol can also be
5%th, 8%, 10%, 12%, 15% or 20%, the other the same as in Example 1.Such as the optimum results figure of methanol content in Fig. 6 leacheates
Shown, when methanol content is 10%, target analytes have preferable extraction efficiency.
In other embodiment in the present invention, the packing quality in HiCapt MCX SPEs can be in 50-1000mg
In the range of.Further, the consumption of the filler in HiCapt MCX SPEs can also for 50mg, 100mg, 200mg,
300mg or 1000mg, the other the same as in Example 1.As shown in fig. 7, the quality of filler is in the range of 50-1000mg, with quality not
Disconnected to improve, the peak area of target analytes is significantly increased, and when packing quality reaches 500mg, continues to increase packing quality, mesh
The peak area for marking analyte is improved not substantially, considers the effect of extracting of all target analytes, and preferred filler quality is
500mg。
In other embodiments of the present invention, redissolving liquid in the assay method step 2 of N- nitrosamine in Ling " for giving up smoking can also be by
The ammonium formate solution of 0.01mol/L and formic acid acetonitrile solution that formic acid volume fraction is 0.1% are according to 1:2 or 2:1 volume ratio
It is formulated, the other the same as in Example 1.
In other embodiments of the present invention, the concentration of ammonium acetate solution can also be 5mmol/L, 15mmol/L, 50mmol/
L, 80mmol/L or 100mmol/L, the other the same as in Example 1.
In others embodiment of the invention, ultrasonic extraction, the condition of vortex oscillation can be adjusted suitably.
Test example
Precision and mark-on reclaims are tested
To investigate the anti-interference of said determination method, added in the filler for lighting type product for quitting smoking of embodiment 1
A certain amount of mixed standard solution, (the corresponding value of basic, normal, high concentration is respectively to be configured to the sample of basic, normal, high three kinds of concentration
2,10 and 40 times of each target analytes range of linearity minimum).Under the analysis condition of optimization, target analytes and chaff interference
Separate well, chaff interference does not influence quantifying for target analytes.
It is measured with 5 samples prepared in a day, calculates the in a few days relative standard deviation under various concentrations；With continuous
The sample prepared for 3 days is measured, and calculates the relative standard deviation in the daytime under various concentrations；By the ratio between peak area obtained by analysis
Substitute into standard curve, be calculated the content of target analytes in solution to be measured, and obtain relative compared with actual interpolation amount
The rate of recovery, as a result see the table below 3.From table 3 it can be seen that target analytes under various concentrations in a few days and day to day precision difference
Less than 4.6% and 4.2%, illustrate that the method has preferable reappearance.Meanwhile, the relative of target analytes is returned under various concentrations
Yield is between 92.2%~108.9%.
The precision of table 3 and mark-on reclaims test result
Claims (10)
1. it is a kind of based on hydrophobic nonionic exchange SPE product for quitting smoking in N- nitrosamine extracting method, it is characterised in that：
Comprise the following steps：
1) ammonium acetate solution that product for quitting smoking addition concentration is 5~100mmol/L is extracted, is obtained crude extract；
2) by step 1) obtained by crude extract flow through the HiCapt MCX solid-phase extraction columns after activation process, complete loading；
3) drip washing is carried out with methanol aqueous solution；
4) eluted with any one in acetone, acetone ammonia water mixture, acetone formic acid mixed liquor, obtained final product N- nitrosamine
Extract solution；Acetone, the volume ratio of ammoniacal liquor are 97~99 in the acetone ammonia water mixture:3~1；Third in acetone formic acid mixed liquor
Ketone, the volume ratio of formic acid are 97~99:3~1；The mass fraction of the ammoniacal liquor is 35%.
2. the extraction side of N- nitrosamine in the product for quitting smoking of SPE is exchanged based on hydrophobic nonionic as claimed in claim 1
Method, it is characterised in that the peculiar N- nitrosamine of tobacco includes NNN, NNK, NAT and NAB in the product for quitting smoking.
3. it is according to claim 1 based on hydrophobic nonionic exchange SPE product for quitting smoking in N- nitrosamine extraction side
Method, it is characterised in that：Step 2) described in activation process be：HiCapt MCX are flowed through with acetone, methanol aqueous solution, water successively to consolidate
Phase extraction column.
4. it is according to claim 1 based on hydrophobic nonionic exchange SPE product for quitting smoking in N- nitrosamine extraction side
Method, it is characterised in that：Step 3) volume fraction of methyl alcohol is 5~20% in methanol aqueous solution.
5. it is according to claim 1 based on hydrophobic nonionic exchange SPE product for quitting smoking in N- nitrosamine extraction side
Method, it is characterised in that：Step 1) in gained crude extract pH to 3.0~11.0 is adjusted before loading.
6. it is a kind of based on hydrophobic nonionic exchange SPE product for quitting smoking in N- nitrosamine assay method, it is characterised in that：
Comprise the following steps：
The preparation of 1.N- nitrosamine extract solutions
1.1 sequentially add Isotopic Internal Standard and ammonium acetate solution that concentration is 5~100mmol/L is carried in product for quitting smoking
Take, obtain crude extract；
Step 1.1 gained crude extract is flowed through the HiCapt MCX solid-phase extraction columns after activation process by 1.2, completes loading；
1.3 carry out drip washing with methanol aqueous solution；
1.4 are eluted with any one in acetone, acetone ammonia water mixture, acetone formic acid mixed liquor, obtain final product N- nitrosamine
Extract solution；
2. in product for quitting smoking N- nitrosamine measure
The extract solution drying of gained in step 1.4, the redissolution liquid being formulated with ammonium formate solution, formic acid acetonitrile solution are carried out
Redissolve, liquid chromatography tandom mass spectrometry determination is carried out afterwards.
7. it is according to claim 6 based on hydrophobic nonionic exchange SPE product for quitting smoking in N- nitrosamine measure side
Method, it is characterised in that：Ammonium formate solution and formic acid that formic acid volume fraction be 0.1% of the liquid by 0.01mol/L are redissolved in step 2
Acetonitrile solution is according to 1~2:1~2 volume ratio is formulated.
8. it is according to claim 6 based on hydrophobic nonionic exchange SPE product for quitting smoking in N- nitrosamine measure side
Method, it is characterised in that：Isotopic Internal Standard includes NNN-d4, NNK-d4, NAT-d4, NAB-d4 in step 1.1.
9. it is according to claim 6 based on hydrophobic nonionic exchange SPE product for quitting smoking in N- nitrosamine measure side
Method, it is characterised in that：The condition of liquid chromatography-tandem mass spectrometry analysis is：Chromatographic column：Poroshell120EC-C18；Post
Temperature：40℃；Sample size：5μL；Mobile phase A is 0.01mol/L ammonium formate solutions, and Mobile phase B is 0.1% formic acid acetonitrile solution；Stream
Speed：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 pressure 50psi；Assisted atomization atmospheric pressure 50psi；Gas curtain atmospheric pressure 35psi；Ion source temperature 450
℃；Import voltage 8V；Exit potential 10V；Remove cluster voltage 40V；Residence time 40ms；Monitoring pattern：Multiple-reaction monitoring.
10. it is according to claim 9 based on hydrophobic nonionic exchange SPE product for quitting smoking in N- nitrosamine measure
Method, it is characterised in that：The quota ion pair of NNN, NNK, NAT and NAB is 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；The qualitative ion pair of NNN, NNK, NAT and NAB is followed successively by 178.1>120.1、208.1>106.1、190.1>106.1 Hes
192.1>133.1, corresponding collision voltage is followed successively by 15,16,15 and 17eV；NNN-d4, NNK-d4, NAT-d4 and NAB-d4's
Quota ion pair is followed successively by 182.1>152.2、212.1>126.1、194.1>164.1 and 196.1>166.2, corresponding collision electricity
Pressure is followed successively by 15,16,15 and 17eV.
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Application publication date: 20170613 |