CN107727784A - One grow tobacco and tobacco product in the chiral analysis method of nicotine and nornicotine - Google Patents
One grow tobacco and tobacco product in the chiral analysis method of nicotine and nornicotine Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
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Abstract
The present invention provide one grow tobacco and tobacco product in the chiral analysis method of nicotine and nornicotine, specifically include following steps：1）Sample pre-treatments：Offal sample is obtained after taking tobacco and tobacco product milling, offal sample is added into NaOH solution and methyl tertiary butyl ether(MTBE), is fully stood after vibration, is added organic solvent diluting after taking supernatant liquor filtering, obtain prepare liquid；2）Sample detection：Prepare liquid is subjected to ultra high efficiency and closes phase chromatographic tandem Mass Spectrometer Method, the chiral relative amount formed of nicotine and nornicotine in prepare liquid is determined using areas of peak normalization method.Provided by the invention one grow tobacco and tobacco product in the chiral analysis method of nicotine and nornicotine, using nicotine and nornicotine as research object, processing is optimized to tobacco and tobacco product, its separating effect is excellent, high sensitivity, it is reproducible, the measure of the chiral composition of nicotine and the chiral composition of nornicotine can be achieved.
Description
Technical field
The invention belongs to tobacco components analysis field, be related to one grow tobacco and tobacco product in nicotine and nornicotine
Chiral analysis method.
Background technology
Nicotine is the alkaloid that content is most abundant in tobacco and flue gas, accounts for the 90%~95% of biological total alkali content, can be made
It is the major impetus of smoker's smoking for human central nervous system.Nicotine contains a chiral centre, two mappings be present
Body：S- (-)-nicotine and R- (+)-nicotine, both content, metabolic mechanism and physiological properties in tobacco are entirely different.Natural tobacco
Nicotine in grass is mainly S- (-)-configuration, but by highly sensitive analytical technology, can be in different types of tobacco material
Detect R- (+)-nicotine accounted within total nicotine content 1%.
The content of nornicotine is usually less than the 5% of total alkaloid, is the catabolite of nicotine, shadow be present to tobacco flavor
Ring, there is also two enantiomers.Research at present to the chiral composition of nornicotine is seldom, analysis shows, different tobaccos and tobacco system
The chiral composition of nornicotine is different in product, needs further to be furtherd investigate.
It is higher to the sensitivity requirement of method due to two Enantiomeric excess very differents of nicotine, and method sensitivity with
S- (-)-nicotine, the separating degree of R- (+)-nicotine are closely related；Simultaneously based on the complex matrix of tobacco, effective despumation
Interference is the guarantee of the method degree of accuracy.Ultra high efficiency closes phase chromatogram and combines supercritical fluid chromatography technology, has conventional ultra high effect concurrently
The advantages of liquid chromatogram, with supercritical fluid CO2For mobile phase main body, separated by the solvability of mobile phase.
Due to supercritical fluid CO2The characteristics of with low viscosity, high diffusivity speed and high quality transfer ability, its in chipal compounds and
Homologue separation field has unique advantage.Ultra high efficiency is closed into phase chromatogram and mass spectrometry, separating degree has concurrently with selectivity, can be real
Existing nicotine while nornicotine chiral isomer with detecting.At present, have no that close phase chromatogram on ultra high efficiency uses with MS
Nicotine and the report of the chiral composition of nornicotine analysis simultaneously in tobacco and tobacco product.
The content of the invention
In view of the above the shortcomings that prior art, it is an object of the invention to provide one grow tobacco and tobacco product in cigarette
The chiral analysis method of alkali and nornicotine, using nicotine and nornicotine as research object, pre-treatment is carried out to tobacco and tobacco product
Technique study and ultra high efficiency close phase chromatogram-tandem mass spectrum condition optimizing, establish nicotine and nornicotine ultra high efficiency close phase chromatogram-
Tandem mass spectrum chiral analysis method.
In order to achieve the above objects and other related objects, the present invention provide one grow tobacco and tobacco product in nicotine and drop cigarette
The chiral analysis method of alkali, specifically includes following steps：
1) sample pre-treatments：Offal sample is obtained after taking tobacco and tobacco product milling, it is molten that offal sample is added into NaOH
Stood after liquid and methyl tertiary butyl ether(MTBE), fully vibration, take supernatant liquor to add organic solvent diluting after filtering, obtain prepare liquid；
2) sample detection：The prepare liquid that step 1) is prepared carries out ultra high efficiency and closes phase chromatogram-tandem mass spectrum detection, using peak
The relative amount of the chiral composition of nicotine and nornicotine in area normalization method measure prepare liquid.
Preferably, the nicotine includes S- (-)-nicotine and R- (+)-nicotine.S- (-)-nicotine and R- (+)-cigarette
Alkali is chiral isomer.The nicotine is racemic nicotine.
Preferably, the nornicotine includes S- (-)-nornicotine and R- (+)-nornicotine.S- (-)-nornicotine with
R- (+)-nornicotine is chiral isomer.The nornicotine is racemic nornicotine.
Preferably, in step 1), the tobacco and tobacco product appointing in tobacco leaf, offal, thin section for tobacco or pipe tobacco
Meaning is a kind of.
It is highly preferred that the one kind of the tobacco leaf in burley tobaccos tobacco leaf, Turkish tobaccos tobacco leaf or cured tobacco leaf.
It is highly preferred that the pipe tobacco is cigarette shreds.
Preferably, in step 1), the NaOH solution is weight percentage as the 5-15%NaOH aqueous solution.It is highly preferred that
The NaOH solution is weight percentage as the 10%NaOH aqueous solution.
Preferably, in step 1), the offal sample adds the ratio between volume (mL) that quality (g) adds with NaOH solution and is
1:1-5.It is highly preferred that it is 1 that the offal sample, which adds the ratio between volume (mL) that quality (g) adds with NaOH solution,:2.
Preferably, in step 1), the offal sample add volume (mL) that quality (g) adds with methyl tertiary butyl ether(MTBE) it
Than for 1:2-8.It is highly preferred that the ratio between volume (mL) that the offal sample addition quality (g) adds with methyl tertiary butyl ether(MTBE) is
1:4。
Preferably, in step 1), the duration of oscillation is 1-10min.It is highly preferred that the duration of oscillation is 5min.
Preferably, in step 1), the static conditions are：Dwell temperature is room temperature；Time of repose is 6-18h.More preferably
Ground, the static conditions are：Dwell temperature is 20-25 DEG C；Time of repose is 10-14h.
Preferably, in step 1), the organic solvent is methyl tertiary butyl ether(MTBE).
Preferably, in step 1), the ratio between volume that the supernatant liquor adds with organic solvent is 1:1-100.
Preferably, in step 1), the filter type is membrane filtration mode.It is highly preferred that the aperture of the filter membrane is
0.22μm。
Preferably, in step 2), the testing conditions that the ultra high efficiency closes phase chromatogram are：Series connection chiral column：Analytical column 1：Change
Property polysaccharide stationary phase chiral chromatographic column；Analytical column 2：Positive Chiral liquid chromatography post；Mobile phase：Supercritical CO2Fluid-containing different
The methanol solution of propylamine, wherein, A phases are supercritical CO2Fluid, it containing percent by volume is 0.02-0.08% isopropylamines that B phases, which are,
Methanol solution；Flow rate of mobile phase：1.0-1.2mL/min；ISM compensates flow path phase：It is 0.05-0.15% containing percent by volume
The methanol solution of formic acid；ISM compensates flow path phase flow velocity：0.2-0.4mL/min；Analysis time：32min；Gradient elution.
It is highly preferred that the testing conditions that the ultra high efficiency closes phase chromatogram are：Series connection chiral column：Analytical column 1：Trefoil
CEL1 posts, column length 150mm, internal diameter 3.0mm, 2.5 μm of stationary phase particle diameter；Analytical column 2：Chiralcel OD-H liquid-phase chromatographic columns,
Column length 250mm, internal diameter 4.6mm, 5 μm of stationary phase particle diameter；Mobile phase：Supercritical CO2Fluid-the methanol solution containing isopropylamine, its
In, A phases are supercritical CO2Fluid, B phases are containing the methanol solution that percent by volume is 0.05% isopropylamine；Flow rate of mobile phase：
1.1mL/min；ISM compensates flow path phase：Containing the methanol solution that percent by volume is 0.1% formic acid；ISM compensates flow path
Phase flow velocity：0.3mL/min；Analysis time：32min；Gradient elution.
It is highly preferred that the gradient elution program is specially：
0-0.5min, A phase：B phase volume ratios are 97：3-97：3；
0.5-8.0min, A phase：B phase volume ratios are 97：3-92：8；
8.0-12.0min A phases：B phase volume ratios are 92：8-92：8；
12.0-30.0min, A phase：B phase volume ratios are 92：8-90：10；
30.0-32.0min, A phase：B phase volume ratios are 90：10-97：3.
It is highly preferred that the ultra high efficiency, which closes phase chromatogram, also includes following testing conditions：Column temperature：35-45℃；Sample introduction room temperature
Degree：8-12℃；Sample size：1-10μL；Back pressure：1550-1850psi.
It is further preferred that the ultra high efficiency, which closes phase chromatogram, also includes following testing conditions：Column temperature：40℃；Sample Room
Temperature：10℃；Sample size：2μL；Back pressure：1600psi.
Preferably, in step 2), the mass spectrographic testing conditions are：Ion gun：ESI sources；Scan mode：Cation is swept
Retouch；Ion source temperature：300℃；Electron spray voltage：5000V；Atomization gas pressure：40psi；Detection mode：Multiple-reaction monitoring
(MRM), the numerical value of MRM parameters is shown in Table 1.
The Mass Spectrometry Conditions of table 1 (MRM parameters)
Preferably, in step 2), the areas of peak normalization method refers to：Phase chromatogram-tandem mass spectrum measure is closed through ultra high efficiency
Afterwards, the chromatographic peak area of the chiral constituent of nicotine and nornicotine is obtained respectively, calculates the chiral constituent sum of nicotine,
Total chromatographic peak area of nicotine is obtained, calculates the chiral constituent sum of nornicotine, obtains total chromatographic peak area of nornicotine.
Then, by the chromatographic peak area of the chiral constituent of nicotine respectively divided by nicotine total chromatographic peak area, obtain the hand of nicotine
The chromatographic peak area of property constituent accounts for the phase of the percentage, the as chiral constituent of nicotine of total chromatographic peak area of nicotine
To content.By the chromatographic peak area difference of the chiral constituent of nornicotine divided by total chromatographic peak area of nornicotine, dropped
The chromatographic peak area of the chiral constituent of nicotine accounts for the chirality of the percentage, as nornicotine of total chromatographic peak area of nornicotine
The relative amount of constituent.Specifically, the chiral composition of nicotine accounts for total nicotine percentage with R- (+) nicotine and represented, that is, passes through
After ultra high efficiency closes phase chromatogram-tandem mass spectrum measure, the chromatographic peak area of two chiral isomers of nicotine is respectively obtained, with R- (+)
The chromatographic peak area of nicotine divided by total chromatographic peak area of nicotine are the relative amount that can obtain the chiral composition of nicotine.Nornicotine
Chirality composition accounts for total nornicotine percentage with R- (+) nornicotine and represented, i.e., after ultra high efficiency closes phase chromatogram-tandem mass spectrum measure,
The chromatographic peak area of two chiral isomers of nornicotine is respectively obtained, with the chromatographic peak area divided by nornicotine of R- (+) nornicotine
Total chromatographic peak area be the relative amount that can obtain the chiral composition of nornicotine.
As described above, provided by the invention one grow tobacco and tobacco product in the chiral analysis method of nicotine and nornicotine,
Using nicotine and nornicotine as research object, carry out pre-treating method research to tobacco and tobacco product and ultra high efficiency close phase chromatogram-
Tandem mass spectrum condition optimizing, establish the analysis method of chiral isomer composition in nicotine and nornicotine.Analysis side in the present invention
Method can realize the analysis of nicotine chiral isomer and nornicotine chiral isomer simultaneously, both separating degrees in actual sample
Up to more than 2.5.In the analysis method of the present invention, close phase chromatogram and mass spectrometry by ultra high efficiency effectively can disturb in despumation,
Realize the Accurate Determining of the chiral composition of nicotine in sample, nornicotine.Analysis method is easy to be quick in the present invention, high sensitivity, weight
Renaturation is good, can fully meet nicotine and nornicotine chirality composition analysis needs in tobacco and tobacco product.
Brief description of the drawings
Fig. 1 is shown as racemic nicotine in the present invention (i.e. S- (-)-nicotine and R- (+)-nicotine respectively accounts for 50%) standard sample
With the ultra high efficiency of racemic nornicotine (i.e. S- (-)-nornicotine and R- (+)-nornicotine respectively accounts for 50%) standard sample close phase chromatogram-
Mass spectrometry total ion current figure.
Fig. 2 is shown as in the present invention the super of nicotine chiral isomer and nornicotine chiral isomer in flue-cured tobacco offal prepare liquid
Efficiently close phase chromatograph-mass spectrometer coupling total ion current figure.
Embodiment
The present invention is expanded on further with reference to specific embodiment, it should be appreciated that these embodiments are merely to illustrate the present invention
Rather than limit the scope of the invention.
Illustrate embodiments of the present invention below by way of specific instantiation, those skilled in the art can be by this specification
Disclosed content understands other advantages and effect of the present invention easily.The present invention can also pass through specific realities different in addition
The mode of applying is embodied or practiced, the various details in this specification can also be based on different viewpoints with application, without departing from
Various modifications or alterations are carried out under the spirit of the present invention.
The reagent and instrument that following examples use are as follows：
1st, reagent
Burley tobaccos tobacco leaf, Turkish tobaccos tobacco leaf, cured tobacco leaf, cigarette rags (commercially available)；Racemic nicotine standard product (S-
(-)-nicotine and R- (+)-nicotine respectively account for 50wt%, Sigma Co., USA)；Racemic nornicotine standard items (S- (-)-nornicotine
50wt%, Sigma Co., USA are respectively accounted for R- (+)-nornicotine)；Methanol, methyl tertiary butyl ether(MTBE), isopropylamine (chromatographically pure,
Amethyst Chemicals companies)；Sodium hydroxide (analyzes pure, Chemical Reagent Co., Ltd., Sinopharm Group)；Supercritical CO2Stream
Body (ultra high efficiency is closed phase chromatogram and prepared).
2nd, instrument
ACQUITY UPC2Type ultra high efficiency closes phase chromatogram (Waters, US)；The triple quadrupole rods tandem mass spectrometries of 6410B
(Agilent companies of the U.S.).
Embodiment 1
1st, sample pre-treatments
Take cured tobacco leaf to obtain offal sample after being milled, take 1g offal samples to be placed in 15mL test tubes, add 10%NaOH
Aqueous solution 2mL and methyl tertiary butyl ether(MTBE) 4mL, screws spiral cover, and after fully vibrating 5min, 20-25 DEG C of room temperature stands 10-14h and stayed overnight.
Take supernatant liquor to add methyl tertiary butyl ether(MTBE) after 0.22 μm of membrane filtration to be diluted, obtain prepare liquid.Supernatant liquor and first
The ratio between volume that base tertbutyl ether adds is 1:10.Each sample parallel determination is three times.
2nd, determine
The prepare liquid prepared in above-mentioned steps 1 is subjected to ultra high efficiency and closes phase chromatogram-tandem mass spectrum detection, is returned using peak area
One changes the relative amount of the chiral composition of nicotine and nornicotine in method measure prepare liquid.Wherein, the chiral composition of nicotine in prepare liquid
Composition be：S- (-)-nicotine, R- (+)-nicotine, nornicotine chiral isomer composition：S- (-)-nornicotine, R- (+)-drop cigarette
Alkali.Specific testing result is shown in Fig. 1,2.From Fig. 1,2, method is to cigarette in tobacco and tobacco product such as cured tobacco leaf in the present invention
The separating effect of alkali chiral isomer and nornicotine chiral isomer is very good.
During specific measure, the chiral composition of nicotine accounts for total nicotine percentage with R- (+) nicotine and represented, i.e., closes phase through ultra high efficiency
After chromatogram-tandem mass spectrum measure, the chromatographic peak area of two chiral isomers of nicotine is respectively obtained, with the chromatogram of R- (+) nicotine
Total chromatographic peak area of peak area divided by nicotine is the relative amount that can obtain the chiral composition of nicotine.The chiral composition of nornicotine with
R- (+) nornicotine accounts for total nornicotine percentage and represented, i.e., after ultra high efficiency closes phase chromatogram-tandem mass spectrum measure, respectively obtains drop
The chromatographic peak area of two chiral isomers of nicotine, with the chromatographic peak area of R- (+) nornicotine divided by total chromatographic peak of nornicotine
Area is the relative amount that can obtain the chiral composition of nornicotine.
Wherein, the testing conditions of the ultra high efficiency conjunction phase chromatogram are：Series connection chiral column：Analytical column 1：Trefoil CEL1
Post, column length 150mm, internal diameter 3.0mm, 2.5 μm of stationary phase particle diameter；Analytical column 2：Chiralcel OD-H liquid-phase chromatographic columns, column length
250mm, internal diameter 4.6mm, 5 μm of stationary phase particle diameter；Mobile phase：Supercritical CO2Fluid-the methanol solution containing isopropylamine, wherein, A
It is mutually supercritical CO2Fluid, B phases are containing the methanol solution that percent by volume is 0.05% isopropylamine；Flow rate of mobile phase：1.1mL/
min；ISM compensates flow path phase：Containing the methanol solution that percent by volume is 0.1% formic acid；ISM compensation flow path mutually flows
Speed：0.3mL/min；Analysis time：32min；Gradient elution.
The gradient elution program be specially：
0-0.5min, A phase：B phase volume ratios are 97：3-97：3；
0.5-8.0min, A phase：B phase volume ratios are 97：3-92：8；
8.0-12.0min A phases：B phase volume ratios are 92：8-92：8；
12.0-30.0min, A phase：B phase volume ratios are 92：8-90：10；
30.0-32.0min, A phase：B phase volume ratios are 90：10-97：3.
The ultra high efficiency closes phase chromatogram and also includes following testing conditions：Column temperature：40℃；Sample introduction room temperature：10℃；Sample introduction
Amount：2μL；Back pressure：1600psi.
The mass spectrographic testing conditions are：Ion gun：ESI sources；Scan mode：Cation scans；Ion source temperature：300
℃；Electron spray voltage：5000V；Atomization gas pressure：40psi；Detection mode：Multiple-reaction monitoring (MRM), the numerical value of MRM parameters are shown in
Table 1.
Embodiment 2
1st, sample pre-treatments
Tobacco and tobacco product is taken to obtain offal sample after being milled, tobacco and tobacco product are selected from tobacco leaf, offal, cigarette with thin
Any one in piece or pipe tobacco, wherein, the one kind of tobacco leaf in burley tobaccos tobacco leaf, Turkish tobaccos tobacco leaf or cured tobacco leaf, cigarette
Silk is cigarette shreds.Take 1g offal samples to be placed in 15mL test tubes, add 5-15%NaOH aqueous solution 1-5mL and methyl tertbutyl
Ether 2-8mL, screws spiral cover, after fully vibrating 1-10min, is stored at room temperature 6-18h and stays overnight.Supernatant liquor is taken through 0.22 μm of filter membrane mistake
Methyl tertiary butyl ether(MTBE) is added after filter to be diluted, and obtains prepare liquid.The ratio between volume that supernatant liquor adds with methyl tertiary butyl ether(MTBE)
For 1:1-100.Each sample parallel determination is three times.
2nd, determine
The prepare liquid prepared in above-mentioned steps 1 is subjected to ultra high efficiency and closes phase chromatogram-tandem mass spectrum detection, is returned using peak area
One changes the relative amount of the chiral composition of nicotine and nornicotine in method measure prepare liquid.Wherein, nicotine chiral photo-isomerisation in prepare liquid
Body composition：S- (-)-nicotine, R- (+)-nicotine, nornicotine chiral isomer composition：S- (-)-nornicotine, R- (+)-nornicotine,
In the present invention method to the separating effect of nicotine chiral isomer and nornicotine chiral isomer in tobacco and tobacco product very
It is good.
During specific measure, the areas of peak normalization method refers to：After ultra high efficiency closes phase chromatogram-tandem mass spectrum measure, point
Not Huo get nicotine and nornicotine chiral constituent chromatographic peak area, calculate the chiral constituent sum of nicotine, obtain
Total chromatographic peak area of nicotine, the chiral constituent sum of nornicotine is calculated, obtain total chromatographic peak area of nornicotine.So
Afterwards, by the chromatographic peak area of the chiral constituent of nicotine respectively divided by nicotine total chromatographic peak area, obtain the chirality of nicotine
The chromatographic peak area of constituent accounts for the percentage of total chromatographic peak area of nicotine, and as the chiral constituent of nicotine is relative
Content.By the chromatographic peak area difference of the chiral constituent of nornicotine divided by total chromatographic peak area of nornicotine, drop cigarette is obtained
The chromatographic peak area of the chiral constituent of alkali accounts for chiral group of the percentage of total chromatographic peak area of nornicotine, as nornicotine
Into the relative amount of composition.
Wherein, the testing conditions of the ultra high efficiency conjunction phase chromatogram are：Series connection chiral column：Analytical column 1：Modified polysaccharides are fixed
Phase chiral chromatographic column；Analytical column 2：Positive Chiral liquid chromatography post；Mobile phase：Supercritical CO2Fluid-methanol containing isopropylamine is molten
Liquid, wherein, A phases are supercritical CO2Fluid, B phases are containing the methanol solution that percent by volume is 0.02-0.08% isopropylamines；Stream
Dynamic phase flow velocity：1.0-1.2mL/min；ISM compensates flow path phase：Containing the methanol that percent by volume is 0.05-0.15% formic acid
Solution；ISM compensates flow path phase flow velocity：0.2-0.4mL/min；Analysis time：32min；Gradient elution.
The gradient elution program be specially：
0-0.5min, A phase：B phase volume ratios are 97：3-97：3；
0.5-8.0min, A phase：B phase volume ratios are 97：3-92：8；
8.0-12.0min A phases：B phase volume ratios are 92：8-92：8；
12.0-30.0min, A phase：B phase volume ratios are 92：8-90：10；
30.0-32.0min, A phase：B phase volume ratios are 90：10-97：3.
The ultra high efficiency closes phase chromatogram and also includes following testing conditions：Column temperature：35-45℃；Sample introduction room temperature：8-12
℃；Sample size：1-10μL；Back pressure：1550-1850psi.
The mass spectrographic testing conditions are：Ion gun：ESI sources；Scan mode：Cation scans；Ion source temperature：300
℃；Electron spray voltage：5000V；Atomization gas pressure：40psi；Detection mode：Multiple-reaction monitoring (MRM), the numerical value of MRM parameters are shown in
Table 1.
Embodiment 3
Racemic nicotine standard sample and racemic nornicotine standard sample is taken to carry out pre-treatment by the step 1 of embodiment 1,
It is measured again by the condition of the step 3 of embodiment 1, using 3 times of signal to noise ratio as test limit, obtains the test limit of method, specific knot
Fruit is as shown in table 2.Wherein, racemic nicotine standard product are the hybrid standard product of S- (-)-nicotine and R- (+)-nicotine, S- (-)-
The mass ratio of nicotine and R- (+)-nicotine is 1:1.Nornicotine standard items are the mixed of S- (-)-nornicotine and R- (+)-nornicotine
The mass ratio of standardization product, S- (-)-nornicotine and R- (+)-nornicotine is 1:1.As shown in Table 2, due to two enantiomers it
Between there is good separating degree, the test limit of its method is less than 0.064 μ g/mL, has higher method sensitivity.
The methodological study of table 2：Test limit
| Object | Test limit (μ g/mL) |
| S- (-)-nicotine | 0.028 |
| R- (+)-nicotine | 0.031 |
| S- (-)-nornicotine | 0.064 |
| R- (+)-nornicotine | 0.061 |
Embodiment 4
Racemic nicotine standard sample and racemic nornicotine standard sample is taken to carry out pre-treatment by the step 1 of embodiment 1,
It is measured again by the condition of the step 3 of embodiment 1, respectively repeatedly Parallel testing 5 times (n=5), the results are shown in Table 3.Wherein, outside
Racemization nicotine standard product are the matter of the hybrid standard product of S- (-)-nicotine and R- (+)-nicotine, S- (-)-nicotine and R- (+)-nicotine
The ratio between amount is 1:1.Nornicotine standard items are the hybrid standard product of S- (-)-nornicotine and R- (+)-nornicotine, S- (-)-nornicotine
Mass ratio with R- (+)-nornicotine is 1:1.
As can be seen from Table 3, the total nicotine percentage that accounts for of R- (+)-nicotine is 50.0% in racemic nicotine standard sample,
It is consistent with the actual accounting of R- (+)-nicotine in racemic nicotine standard sample, and R- in racemic nicotine standard sample
The relative standard deviation (RSD) of (+)-nicotine is 1.26%.And R- (+)-nornicotine accounts in racemic nornicotine standard sample
Total nornicotine percentage is 50.0%, is consistent with the actual accounting of R- (+)-nornicotine in racemic nornicotine standard sample,
And the relative standard deviation (RSD) of R- (+)-nornicotine is 0.98% in racemic nornicotine standard sample.It can be seen that the present invention
The precision of middle assay method is preferable, and repeatability is good.
The methodological study of table 3：Precision (n=5)
| Sequence number | Sample | R- (+)-nicotine (%)* | R- (+)-nornicotine (%)* | RSD (%) |
| 1 | Racemic nicotine standard sample | 50.0 | / | 1.26 |
| 2 | Racemic nornicotine standard sample | / | 50.0 | 0.98 |
*The average quality percentage accounting (relative to total nicotine content) of R- (+)-nicotine,*The average matter of R- (+)-nornicotine
Measure percentage accounting (relative to total nornicotine content)
Embodiment 5
Nicotine and the chiral composition of nornicotine in different types of tobacco and tobacco product sample are determined using this method, repeated
Determine 3 times (n=3), concrete outcome is shown in Table 4.As shown in Table 4, in different types of tobacco and tobacco product sample, R- (+)-cigarette
Alkali is 0.235-0.426% with respect to the average quality percentage accounting of total nicotine content, and the relatively total nornicotine of R- (+)-nornicotine contains
The average quality percentage accounting of amount is 38.2-66.0%.And according to existing nicotine and the analysis statement of nornicotine chiral isomer
Mode, the percentage that total nicotine is mainly accounted for R- (+)-nicotine are formed to represent that nicotine is chiral, and R- (+)-nicotine is removed in total nicotine
It is S- (-)-nicotine in addition, the percentage that total nornicotine is mainly accounted for R- (+)-nornicotine forms to represent that nornicotine is chiral,
Always it is S- (-)-nornicotine in addition to R- (+)-nornicotine in nornicotine.So as to show to be directed to different types of tobacco and tobacco
Product sample, this method can effectively determine the content of nicotine and nornicotine chiral isomer composition in its actual sample, method
It is easy to operate, applicability is good, as a result accurately, reliably.
Nicotine and nornicotine chiral component analysis result (n=3) in the tobacco of table 4 and tobacco product
aThe percentage accounting of R- (+)-nicotine (relative to total nicotine content).
bThe percentage accounting of R- (+)-nornicotine (relative to total nornicotine content).
So the present invention effectively overcomes various shortcoming of the prior art and has high industrial utilization.
The above-described embodiments merely illustrate the principles and effects of the present invention, not for the limitation present invention.It is any ripe
Know the personage of this technology all can carry out modifications and changes under the spirit and scope without prejudice to the present invention to above-described embodiment.Cause
This, those of ordinary skill in the art is complete without departing from disclosed spirit and institute under technological thought such as
Into all equivalent modifications or change, should by the present invention claim be covered.
Claims (11)
1. one grow tobacco and tobacco product in the chiral analysis method of nicotine and nornicotine, specifically include following steps：
1) sample pre-treatments：Take tobacco and tobacco product to obtain offal sample after being milled, by offal sample add NaOH solution and
Stood after methyl tertiary butyl ether(MTBE), fully vibration, take supernatant liquor to add organic solvent diluting after filtering, obtain prepare liquid；
2) sample detection：The prepare liquid that step 1) is prepared carries out ultra high efficiency and closes phase chromatogram-tandem mass spectrum detection, using peak area
The relative amount of the chiral composition of nicotine and nornicotine in normalization method measure prepare liquid.
2. according to claim 1 one grow tobacco and tobacco product in the chiral analysis method of nicotine and nornicotine, it is special
Sign is that the nicotine includes S- (-)-nicotine and R- (+)-nicotine, and the nornicotine includes S- (-)-nornicotine and R-
(+)-nornicotine.
3. according to claim 1 one grow tobacco and tobacco product in the chiral analysis method of nicotine and nornicotine, it is special
Sign is, in step 1), any one of the tobacco and tobacco product in tobacco leaf, offal, thin section for tobacco or pipe tobacco.
4. according to claim 1 one grow tobacco and tobacco product in the chiral analysis method of nicotine and nornicotine, it is special
Sign is, in step 1), the NaOH solution is weight percentage as the 5-15%NaOH aqueous solution；The offal sample adds matter
It is 1 to measure the ratio between volume mL that g adds with NaOH solution:1-5.
5. according to claim 1 one grow tobacco and tobacco product in the chiral analysis method of nicotine and nornicotine, it is special
Sign is, in step 1), it is 1 that the offal sample, which adds the ratio between volume mL that quality g adds with methyl tertiary butyl ether(MTBE),:2-8.
6. according to claim 1 one grow tobacco and tobacco product in the chiral analysis method of nicotine and nornicotine, it is special
Sign is, in step 1), the duration of oscillation is 1-10min；The static conditions are：Dwell temperature is room temperature；Time of repose
For 6-18h.
7. according to claim 1 one grow tobacco and tobacco product in the chiral analysis method of nicotine and nornicotine, it is special
Sign is, in step 1), the organic solvent is methyl tertiary butyl ether(MTBE)；The volume that the supernatant liquor and organic solvent add it
Than for 1:1-100.
8. according to claim 1 one grow tobacco and tobacco product in the chiral analysis method of nicotine and nornicotine, it is special
Sign is, in step 2), the testing conditions that the ultra high efficiency closes phase chromatogram are：Series connection chiral column：Analytical column 1：Modified polysaccharides
Stationary phase chiral chromatographic column；Analytical column 2：Positive Chiral liquid chromatography post；Mobile phase：Supercritical CO2Fluid-the first containing isopropylamine
Alcoholic solution, wherein, A phases are supercritical CO2Fluid, B phases are molten containing the methanol that percent by volume is 0.02-0.08% isopropylamines
Liquid；Flow rate of mobile phase：1.0-1.2mL/min；ISM compensates flow path phase：It is 0.05-0.15% formic acid containing percent by volume
Methanol solution；ISM compensates flow path phase flow velocity：0.2-0.4mL/min；Analysis time：32min；Gradient elution.
9. according to claim 8 one grow tobacco and tobacco product in the chiral analysis method of nicotine and nornicotine, it is special
Sign is that the gradient elution program is specially：0-0.5min, A phase：B phase volume ratios are 97：3-97：3；0.5-8.0min,
A phases：B phase volume ratios are 97：3-92：8；8.0-12.0min A phases：B phase volume ratios are 92：8-92：8；12.0-30.0min A
Phase：B phase volume ratios are 92：8-90：10；30.0-32.0min, A phase：B phase volume ratios are 90：10-97：3.
10. according to claim 8 one grow tobacco and tobacco product in the chiral analysis method of nicotine and nornicotine, it is special
Sign is that the ultra high efficiency closes phase chromatogram and also includes following testing conditions：Column temperature：35-45℃；Sample introduction room temperature：8-12℃；
Sample size：1-10μL；Back pressure：1550-1850psi.
11. according to claim 1 one grow tobacco and tobacco product in the chiral analysis method of nicotine and nornicotine, it is special
Sign is, in step 2), the mass spectrographic testing conditions are：Ion gun：ESI sources；Scan mode：Cation scans；Ion gun
Temperature：300℃；Electron spray voltage：5000V；Atomization gas pressure：40psi；Detection mode：Multiple-reaction monitoring MRM.
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| CN109619660A (en) * | 2019-02-18 | 2019-04-16 | 湖北和诺生物工程股份有限公司 | A method of extracting R- nicotine from tobacco waste |
| CN109651335A (en) * | 2019-02-18 | 2019-04-19 | 湖北和诺生物工程股份有限公司 | A kind of racemization agent and preparation method thereof improving R- nicotine racemization rate |
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| NL2032578A (en) * | 2022-07-22 | 2022-09-28 | Yunnan Tobacco Quality Supervision And Testing Station | Method for Determining Purity of Nicotine by Use of GCMS-GC Method |
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