CN110531006A - Method that is a kind of while analyzing 8 kinds of alkaloids in tobacco leaf - Google Patents
Method that is a kind of while analyzing 8 kinds of alkaloids in tobacco leaf Download PDFInfo
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- CN110531006A CN110531006A CN201910973361.7A CN201910973361A CN110531006A CN 110531006 A CN110531006 A CN 110531006A CN 201910973361 A CN201910973361 A CN 201910973361A CN 110531006 A CN110531006 A CN 110531006A
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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
- G01N30/02—Column chromatography
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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
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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
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Abstract
The invention discloses a kind of methods for analyzing 8 kinds of alkaloids in tobacco leaf simultaneously, and the method includes sample pre-treatments, standard curve quantitative analysis step.The present invention is to make alkaloid separate out from sample substrate by sodium hydroxide solution immersion, alkaloid is transferred to chloroform phase by the way that chloroform is added and is vortexed to shake, by taking chloroform layer to carry out instrument analysis after centrifugation layering, compared with prior art, beneficial effects of the present invention are as follows: with existing gas chromatography ratio, analysis time of the invention is shorter, analysis indexes are more；With existing gas chromatography-mass spectrography and gas-chromatography-tandem mass spectrometry ratio, operation of the present invention is simpler, stability is higher.
Description
Technical field
The invention belongs to technical field of analytical chemistry, and in particular to a kind of side for analyzing 8 kinds of alkaloids in tobacco leaf simultaneously
Method.
Background technique
It is the significant secondary metabolites of tobacco using nicotine as the nicotiana alkaloids of representative.Common alkaloid in tobacco
Including nicotine (nicotine), nornicotine (nornicotine), anabasine (Anabasine), dehydrogenation anabasine (
Anatabine), nicotyrine (Nicotyrine), 2,3'- bipyridyl (2,3'-Dipyridyl), myosmine (
Myosmine), cotinine (Cotinine) etc..Due to the content between the total content and different alkaloids of nicotiana alkaloids
Ratio is directly related with the quality of tobacco leaf and safety, establishes simple, the quick nicotiana alkaloids analysis method of one kind for cigarette
Leaf production and cigarette industry all have a very important significance.
Currently, the analysis method of alkaloid mainly has connected neighbor set, gas chromatography, gas-chromatography-in tobacco leaf
Quadrupole rod mass spectrography and gas-chromatography-Quadrupole Mass Spectrometry.Wherein connected neighbor set is due to carrying out chromatographic isolation,
Alkaloid component can not be measured, also not can avoid the influence of the interfering substance of same absorbent wavelength, gas-chromatography-quadrupole rod mass spectrum
Method and gas-chromatography-Quadrupole Mass Spectrometry have many advantages, such as specific good, high sensitivity, but due to nicotine and other lifes
Alkaloids content difference is huge (such as nicotine is differed with nicotyrine content more than 3 orders of magnitude), and mass detector is difficult
Meet the linear response requirement of all alkaloids simultaneously, in addition, the more other detectors of the stability of mass detector are poor, Maintenance Difficulty
It spends larger.Gas-chromatography-hydrogen flame ionization detector method has the characteristics that high sensitivity, stability are good, operation and maintenance is simple,
But it needs object and matrix that can avoid the appearance of false positive results in the case where chromatography is kept completely separate.Currently, being based on
Though the analysis method of high-content biological alkali has been reported that in gas-chromatography-hydrogen flame ionization detector tobacco, gas phase color there is no
Spectrum while the report for analyzing 8 kinds of alkaloids in tobacco leaf.
Summary of the invention
The purpose of the present invention is to provide a kind of methods for analyzing 8 kinds of alkaloids in tobacco leaf simultaneously.
The object of the present invention is achieved like this, and the method that 8 kinds of alkaloids in tobacco leaf are analyzed while described includes sample
Product pre-treatment, standard curve quantitative analysis step, specifically include:
A, sample pre-treatments:
1) tobacco sample drying and crushing is sieved to obtain material a；
2) precise material a is placed in centrifuge tube, and the sodium hydroxide solution of 8 ~ 12 times of material a mass volume ratio is added, shakes up,
10 ~ 20min is stood, the extract liquor of the containing the internal standard of 5 ~ 8 times of material a mass volume ratio is added and is obtained after filtering after concussion centrifugation
Liquid b before sample；
B, standard curve quantitative analysis:
1) it accurately weighs 8 kinds of alkaloid 100.0mg respectively to be placed in different 100ml volumetric flasks, chloroform constant volume is made into 1mg/ml
Single mark stock solution, the working solution of gradient concentration is diluted to, for drawing quantitation curves；
2) liquid b and standard working solution before sample are analyzed to obtain using gas-chromatography-hydrogen flame ionization detector each
The corresponding chromatographic peak area of concentration gradient sample and its internal standard peak area, by obtained peak area divided by after internal standard peak area and its
Corresponding concentration gradient carries out linear fit, obtains calibration curve fit equation and linearly dependent coefficient, the practical sample that will be obtained
The chromatographic peak area of alkaloid in product is calculated opposite divided by calibrating curve equation is inputted after corresponding internal standard peak area
The material concentration answered calculates content of the alkaloid in tobacco leaf according to following formula:
Wherein:
The content of alkaloid in X- representative sample, unit mg/g；
C- represents the concentration for the tested component that standard curve obtains, unit is μ g/mL.
The present invention is to make alkaloid separate out from sample substrate by sodium hydroxide solution immersion, by the way that chloroform is added
And alkaloid is transferred to chloroform phase by the concussion that is vortexed, by taking chloroform mutually to carry out instrument analysis after centrifugation layering, with prior art phase
Than beneficial effects of the present invention are as follows:
1, with existing gas chromatography ratio, analysis time of the invention is shorter, analysis indexes are more；
2, with existing gas chromatography-mass spectrography and gas-chromatography-tandem mass spectrometry ratio, operation of the present invention is simpler, stability more
It is high.
Detailed description of the invention
Fig. 1 is the chemical structure schematic diagram of alkaloid in the present invention 8；
Fig. 2 is the gas-chromatography separation chromatogram schematic diagram of alkaloid；
Wherein: 1 is quinoline (internal standard)；2 be nicotine；3 be nornicotine；4 be myosmine；5 be nicotyrine；6 be anabasine；7 are
Anatabine；8 be 2,3- bipyridyl；9 be cotinine.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention is further illustrated, but is not subject in any way to the present invention
Limitation, based on present invention teach that it is made it is any transform or replace, all belong to the scope of protection of the present invention.
The method that 8 kinds of alkaloids in tobacco leaf are analyzed while of the present invention includes that sample pre-treatments, standard curve are fixed
Analytical procedure is measured, is specifically included:
A, sample pre-treatments:
1) tobacco sample drying and crushing is sieved to obtain material a；
2) precise material a is placed in centrifuge tube, and the sodium hydroxide solution of 8 ~ 12 times of material a mass volume ratio is added, shakes up,
10 ~ 20min is stood, the extract liquor of the containing the internal standard of 5 ~ 8 times of material a mass volume ratio is added and is obtained after filtering after concussion centrifugation
Liquid b before sample；
B, standard curve quantitative analysis:
1) it accurately weighs 8 kinds of alkaloid 100.0mg respectively to be placed in different 100ml volumetric flasks, chloroform constant volume is made into 1mg/ml
Single mark stock solution, the working solution of gradient concentration is diluted to, for drawing quantitation curves；
2) liquid b and standard working solution before sample are analyzed to obtain using gas-chromatography-hydrogen flame ionization detector each
The corresponding chromatographic peak area of concentration gradient sample and its internal standard peak area, by obtained peak area divided by after internal standard peak area and its
Corresponding concentration gradient carries out linear fit, obtains calibration curve fit equation and linearly dependent coefficient, the practical sample that will be obtained
The chromatographic peak area of alkaloid in product is calculated opposite divided by calibrating curve equation is inputted after corresponding internal standard peak area
The material concentration answered calculates content of the alkaloid in tobacco leaf according to following formula:
Wherein:
The content of alkaloid in X- representative sample, unit mg/g；
C- represents the concentration for the tested component that standard curve obtains, unit is μ g/mL.
8 kinds of alkaloids are nicotine, nornicotine, nicotyrine, cotinine, anabasine, dehydrogenation anabasine, 2,3- connection
Pyridine and myosmine.
Step A 1) described in drying be drying.
The temperature of the drying is 40 DEG C.
Step A 1) described in pulverize and sieve be that crushed 40 meshes.
Step A 2) described in sodium hydroxide solution mass concentration be 10%.
The extract liquor of the containing the internal standard is to take 5ml internal standard stock solution to 500ml volumetric flask, and chloroform constant volume is added, prepares
At the extract liquor of 10 μ g/mL quinoline.
The internal standard stock solution is to weigh 100.0mg quinoline to be made into 1mg/ml with chloroform constant volume in 100ml volumetric flask
Internal standard stock solution.
Step B 2) described in gas-chromatography-hydrogen flame ionization detector analysis condition are as follows: chromatographic column, DB-5MS(30 m
× 0. 25 mm × 0. 25 μm)；Sample volume, 1 μ L；Split ratio, 40:1；Injector temperature, 250 DEG C.Temperature program
Condition: 100 DEG C of beginnings, 20 DEG C/min rise to 180 DEG C, and 6 DEG C/min rises to 250 DEG C, keeps 4 min.Detector
(FID) temperature, 250 DEG C；Carrier gas (nitrogen) flow velocity, 1.0 mL/min；Fuel gas (hydrogen) flow velocity, 35 mL/min, auxiliary
350 mL/min of gas (air)；40 mL/min of make-up gas flow.
The method that 8 kinds of alkaloids in tobacco leaf are analyzed while of the present invention, concrete operations are as follows:
1, instrument and reagent
Gas-chromatography-hydrogen flame ionization detector (GC-FID, Thermo Fisher company, the U.S.), Millipore ultrapure water
Machine (Millipore company, the U.S.), 5804 supercentrifuge of Eppendorf (German Eppendorf company), Talboys
Digital display multitube vortex mixer (Troemner company, the U.S.).
2, solution is prepared
Internal standard stock solution: 100.0 mg quinoline are weighed in 100 mL volumetric flasks and are made into 1 mg/mL internal standard deposit with chloroform constant volume
Liquid, -20 DEG C are kept in dark place.
Standard solution is prepared: being accurately weighed 8 kinds of 100.0 mg of alkaloid respectively and is placed in 100 different mL volumetric flasks, chlorine
Imitative constant volume is made into the single mark stock solution of 1 mg/mL, and -20 DEG C are kept in dark place.According to sample actual conditions, it is diluted to gradient
The working solution of concentration, for drawing quantitation curves.
Extract liquor is prepared: taking 5 mL internal standard stock solutions to 500 mL volumetric flasks, chloroform constant volume is added, is made into 10 μ g/mL
The extract liquor of (quinoline), -20 DEG C are kept in dark place.
3, sample treatment
40 DEG C of tobacco sample drying, it is to be measured to crushed 40 meshes.1.00 g cigarette samples are accurately weighed in 50 mL centrifuge tubes,
The NaOH solution of 10 mL 10% is added, shakes up, stands 15 min, the extract liquor of 7 mL containing the internal standards is added, be vortexed concussion (frequency
2500 Hz of rate) 1 min, be centrifuged (6 000 r/min) 5 min after, take 2 mL lower layer chloroform solutions to cross equipped with the anhydrous sulphur of 2 g
After the filter (0.44 μm) of sour sodium, upper gas chromatographic analysis.
4, instrumental conditions
Chromatographic column, the mm × 0. 25 μm of DB-5MS(30 m × 0. 25)；Sample volume, 1 μ L；Split ratio, 40:1；Sample introduction
Mouth temperature, 250 DEG C.Temperature program condition: 100 DEG C of beginnings, 20 DEG C/min rise to 180 DEG C, and 6 DEG C/min rises to 250
DEG C, keep 4 min.Detector (FID) temperature, 250 DEG C；Carrier gas (nitrogen) flow velocity, 1.0 mL/min；Fuel gas (hydrogen) stream
Speed, 35 mL/min assist gas (air) 350 mL/min；40 mL/min of make-up gas flow.Alkaloid in 8 in tobacco sample
And interior target chromatogram such as Fig. 1.
5, standard curve is quantitative
Accurately weigh 8 kinds of 100.0 mg of alkaloid respectively and be placed in 100 different mL volumetric flasks, chloroform constant volume, be made into 1 mg/
Single mark stock solution of mL, -20 DEG C are kept in dark place.According to sample actual conditions, it is diluted to the working solution of gradient concentration, is used
In drafting quantitation curves.By the sample made according to " sample pre-treatments " and " instrumental conditions " part the method into
Row analysis, obtains the corresponding chromatographic mass spectrometry peak area of each concentration gradient sample and its internal standard peak area.The peak area that will be obtained
Linear fit is carried out divided by concentration gradient corresponding with its after internal standard peak area, obtains calibration curve fit equation and linear correlation
Coefficient.The chromatographic peak area of alkaloid in obtained actual sample is bent divided by input calibration after corresponding internal standard peak area
Corresponding material concentration (ug/mL) is calculated in line equation.According to formula, once formula calculates alkaloid containing in tobacco leaf
Amount:
In formula:XThe content of alkaloid in representative sample, unit are milligrams per kilogram (mg/g)；C represents the quilt that standard curve obtains
The concentration of component is surveyed, unit is micrograms per millilitre (μ g/mL).
Embodiment 1
--- the content analysis of alkaloid in " cinnabar cigarette " and normal cured tobacco leaf
Experimental material: the tobacco leaf of " the cinnabar cigarette " and common flue-cured tobacco that are determined after just roasting by external appearance characteristic
Experimental method: accurately weighing 1.00 g water-removing sample in 50 mL centrifuge tubes, the NaOH solution of 10 mL 10% be added,
It shakes up, stands 15 min, the extract liquor of 7 mL containing the internal standards is added, be vortexed concussion (2500 Hz of frequency) 1 min, centrifugation (6 000
R/min) after 5 min, after taking 2 mL lower layer chloroform solutions to cross the filter (0.44 μm) equipped with 2 g anhydrous sodium sulfates, upper gas
Analysis of hplc.
Instrumental conditions: chromatographic column, the mm × 0. 25 μm of DB-5MS(30 m × 0. 25)；Sample volume, 1 μ
L；Split ratio, 40:1；Injector temperature, 250 DEG C.Temperature program condition: 100 DEG C of beginnings, 20 DEG C/min rise to 180 DEG C,
6 DEG C/min rises to 250 DEG C, keeps 4 min.Detector (FID) temperature, 250 DEG C；Carrier gas (nitrogen) flow velocity, 1.0 mL/
min；Fuel gas (hydrogen) flow velocity, 35 mL/min assist gas (air) 350 mL/min；40 mL/min of make-up gas flow.
Standard curve is quantitative: accurately weighing 8 kinds of 100.0 mg of alkaloid respectively and is placed in 100 different mL volumetric flasks, chlorine
Imitative constant volume is made into the single mark stock solution of 1 mg/mL, and -20 DEG C are kept in dark place.According to sample actual conditions, it is diluted to gradient
The working solution of concentration, for drawing quantitation curves.By the sample made is according to " sample pre-treatments " and " instrument analyzes item
Part " part the method is analyzed, and the corresponding chromatographic mass spectrometry peak area of each concentration gradient sample and its internal standard peak face are obtained
Product.Obtained peak area is subjected to linear fit divided by concentration gradient corresponding with its after internal standard peak area, obtains calibration curve
Fit equation and linearly dependent coefficient.By the chromatographic peak area of the alkaloid in obtained actual sample divided by corresponding internal standard
Calibrating curve equation is inputted after peak area, and corresponding material concentration (ug/mL) is calculated.According to formula, once formula calculates life
Content of the alkaloids in tobacco leaf:
In formula:XThe content of alkaloid in representative sample, unit are every gram of milligram (mg/g)；It is tested to represent that standard curve obtains by c
The concentration of component, unit are micrograms per millilitre (μ g/mL)；
The alkaloid such as table 1 of different parts different colours cinnabar cigarette and common flue-cured tobacco is calculated.As can be seen from the table,
Cinnabar cigarette significantly rises relative to normal flue-cured tobacco alkaloid total amount.It is embodied in nornicotine, myosmine, anabasine, new tobacco
Alkali equal size then significantly increases, and nicotine, nicotyrine, 2,3'- bipyridyl, cotinine equal size significantly reduce.
The content distribution situation of alkaloid in 1 cinnabar cigarette of table and normal flue-cured tobacco.
Embodiment 2
Method comparison:
Claims (9)
1. a kind of method for analyzing 8 kinds of alkaloids in tobacco leaf simultaneously, it is characterised in that analyze 8 kinds in tobacco leaf while described
The method of alkaloid includes sample pre-treatments, standard curve quantitative analysis step, is specifically included:
A, sample pre-treatments:
1) tobacco sample drying and crushing is sieved to obtain material a；
2) precise material a is placed in centrifuge tube, and the sodium hydroxide solution of 8 ~ 12 times of material a mass volume ratio is added, shakes up,
10 ~ 20min is stood, the extract liquor of the containing the internal standard of 5 ~ 8 times of material a mass volume ratio is added and is obtained after filtering after concussion centrifugation
Liquid b before sample；
B, standard curve quantitative analysis:
1) it accurately weighs 8 kinds of alkaloid 100.0mg respectively to be placed in different 100ml volumetric flasks, chloroform constant volume is made into 1mg/ml
Single mark stock solution, the working solution of gradient concentration is diluted to, for drawing quantitation curves；
2) liquid b and standard working solution before sample are analyzed to obtain using gas-chromatography-hydrogen flame ionization detector each
The corresponding chromatographic peak area of concentration gradient sample and its internal standard peak area, by obtained peak area divided by after internal standard peak area and its
Corresponding concentration gradient carries out linear fit, obtains calibration curve fit equation and linearly dependent coefficient, the practical sample that will be obtained
The chromatographic peak area of alkaloid in product is calculated opposite divided by calibrating curve equation is inputted after corresponding internal standard peak area
The material concentration answered calculates content of the alkaloid in tobacco leaf according to following formula:
Wherein:
The content of alkaloid in X- representative sample, unit mg/g；
C- represents the concentration for the tested component that standard curve obtains, unit is μ g/mL.
2. method that is according to claim 1 while analyzing 8 kinds of alkaloids in tobacco leaf, it is characterised in that described 8 kinds
Alkaloid is nicotine, nornicotine, nicotyrine, cotinine, anabasine, dehydrogenation anabasine, 2,3- bipyridyl and myosmine.
3. method that is according to claim 1 while analyzing 8 kinds of alkaloids in tobacco leaf, it is characterised in that step A 1) in
The drying is drying.
4. method that is according to claim 1 or 2 while analyzing 8 kinds of alkaloids in tobacco leaf, it is characterised in that described
Dry temperature is 40 DEG C.
5. method that is according to claim 1 while analyzing 8 kinds of alkaloids in tobacco leaf, it is characterised in that step A 1) in
Described pulverizing and sieving is to crushed 40 meshes.
6. method that is according to claim 1 while analyzing 8 kinds of alkaloids in tobacco leaf, it is characterised in that step A 2) in
The mass concentration of the sodium hydroxide solution is 10%.
7. method that is according to claim 1 while analyzing 8 kinds of alkaloids in tobacco leaf, it is characterised in that described containing is interior
Target extract liquor is to take 5ml internal standard stock solution to 500ml volumetric flask, and chloroform constant volume is added, is configured to the extraction of 10 μ g/mL quinoline
Liquid.
8. method that is according to claim 7 while analyzing 8 kinds of alkaloids in tobacco leaf, it is characterised in that the internal standard
Stock solution is to weigh 100.0mg quinoline to be made into 1mg/ml internal standard stock solution with chloroform constant volume in 100ml volumetric flask.
9. method that is according to claim 1 while analyzing 8 kinds of alkaloids in tobacco leaf, it is characterised in that step B 2) in
The gas-chromatography-hydrogen flame ionization detector analysis condition are as follows: chromatographic column, the mm of DB-5MS(30 m × 0. 25 ×
0. 25 μm)；Sample volume, 1 μ L；Split ratio, 40:1；Injector temperature, 250 DEG C, temperature program condition: 100 DEG C of beginnings,
20 DEG C/min rises to 180 DEG C, and 6 DEG C/min rises to 250 DEG C, keeps 4 min；Detector (FID) temperature, 250 DEG C；It carries
Gas (nitrogen) flow velocity, 1.0 mL/min；Fuel gas (hydrogen) flow velocity, 35 mL/min assist gas (air) 350 mL/min；
40 mL/min of make-up gas flow.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113917045A (en) * | 2021-11-17 | 2022-01-11 | 云南省烟草农业科学研究院 | Method for quantitatively analyzing 11 kinds of amide alkaloids in tobacco leaves by gas chromatography-hydrogen flame detector |
| CN113917044A (en) * | 2021-11-16 | 2022-01-11 | 云南省烟草农业科学研究院 | Method for quantitatively analyzing 11 kinds of amide alkaloids in tobacco leaves by gas chromatography-tandem mass spectrometry |
| CN114034796A (en) * | 2021-11-16 | 2022-02-11 | 云南省烟草农业科学研究院 | Method for quantitatively analyzing 11 kinds of amide alkaloids in tobacco leaves by gas chromatography-quadrupole mass spectrometry |
| CN114460209A (en) * | 2022-01-28 | 2022-05-10 | 云南中烟工业有限责任公司 | Method for measuring contents of seven alkaloids in fresh tobacco leaves |
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| CN113917044A (en) * | 2021-11-16 | 2022-01-11 | 云南省烟草农业科学研究院 | Method for quantitatively analyzing 11 kinds of amide alkaloids in tobacco leaves by gas chromatography-tandem mass spectrometry |
| CN114034796A (en) * | 2021-11-16 | 2022-02-11 | 云南省烟草农业科学研究院 | Method for quantitatively analyzing 11 kinds of amide alkaloids in tobacco leaves by gas chromatography-quadrupole mass spectrometry |
| CN113917044B (en) * | 2021-11-16 | 2023-10-20 | 云南省烟草农业科学研究院 | Method for quantitatively preparing 11 kinds of amide alkaloids in tobacco leaves by gas chromatography-tandem mass spectrometry |
| CN113917045A (en) * | 2021-11-17 | 2022-01-11 | 云南省烟草农业科学研究院 | Method for quantitatively analyzing 11 kinds of amide alkaloids in tobacco leaves by gas chromatography-hydrogen flame detector |
| CN113917045B (en) * | 2021-11-17 | 2023-06-30 | 云南省烟草农业科学研究院 | Method for quantitatively analyzing 11 kinds of amide alkaloids in tobacco leaves by gas chromatography-hydrogen flame detector |
| CN114460209A (en) * | 2022-01-28 | 2022-05-10 | 云南中烟工业有限责任公司 | Method for measuring contents of seven alkaloids in fresh tobacco leaves |
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Application publication date: 20191203 |