CN108519455A - The assay method of alkaloid in a kind of tobacco gene editor material - Google Patents
The assay method of alkaloid in a kind of tobacco gene editor material Download PDFInfo
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- CN108519455A CN108519455A CN201810146536.2A CN201810146536A CN108519455A CN 108519455 A CN108519455 A CN 108519455A CN 201810146536 A CN201810146536 A CN 201810146536A CN 108519455 A CN108519455 A CN 108519455A
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- sample
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- inner sleeve
- alkaloid
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Classifications
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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
-
- 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
Abstract
The invention discloses a kind of assay methods of alkaloid in tobacco gene editor material, use following device：Sample extraction bottle, including：Outer tube (1), inner sleeve (2), catheter (4), sieve plate；Sample extraction frame (5)；Sample collection frame (6)；Turn to take out (7) and lower platen (8).Step is：1. extracting；2. filtering；3. collecting sample filtrate；4. being measured using high performance liquid chromatography.The assay method of the present invention can be used for detecting the content of alkaloid in tobacco gene editor's material, has the advantages that pre-treatment is simple and convenient, quantitative accurate, reproducible, is suitable for the analysis of batch samples.
Description
Technical field
The invention belongs to tobacco chemistry research fields, and in particular to alkaloid contains in a kind of suitable tobacco gene editor material
Measure the method for high-flux analysis of detection.
Background technology
With tobacco business " batch production breeding side of the tobacco gene group plan key special subjects based on genome editing technique
The propulsion of case " screens material by chemical composition analysis, and pass through tobacco leaf using all gene editing materials as object
Compatibility test and cigarette composition verification, determine the strong kind of industrial applicability, become the pass of tobacco business active demand
Key technology content.
Nicotiana alkaloids is the very high a kind of vegetable soda of cigarette category content, including nearly 50 kinds of substances, is not present in about more than 60 not
In tobacco of the same race, the alkaloid in tobacco mainly has nicotine, nornicotine, anabasine and anatabine, and wherein nicotine is tobacco
The middle highest alkaloid of content, typically constitutes from 95% of alkaloid or more.Nicotiana alkaloids is a kind of physiology excitant, has and shrinks
The effect of blood vessel, the nervous centralis that promotes blood circulation, adjusts；Simultaneously nicotiana alkaloids again be the key that influence cigarette quality at
Point, the appropriate nicotiana alkaloids that sucks can refresh oneself emerging to the jealous of smoker's physiological stimulation appropriate, pleasant fragrance and alcohol sum
It puts forth energy, cheer up, dispelling fatigue etc., but the content of alkaloid increases in tobacco, the fragrance quality of extreme influence tobacco leaf, availability
With the physiological strengths of cigarette products, smoke transportation and suck safety.Therefore the Accurate Determining of biological tobacco alkali content is to tobacco
Quality evaluation is of great significance.
Currently, measure the main photometry of method of nicotiana alkaloids, gas chromatography (such as Capillary GC method, GC/MS methods,
GC-MS/MS methods) and high performance liquid chromatography (HPLC methods).Common several photometries can not meet sensitivity and selection simultaneously
Property in terms of requirement, most commonly used at present is chromatography, and compared with GC methods, HPLC methods have it is simple, quickly, sensitivity
The features such as height, favorable reproducibility.Ultra performance liquid chromatography (UPLC) is the new technology that waters companies in 2004 are proposed, principle
It is identical as HPLC, but this technological break-through limitation of traditional chromatography, it is greatly improved point of high performance liquid chromatography
Speed is analysed, into being in the past few years widely applied at home.
With the startup of tobacco business tobacco gene editor's batch production breeding work, tens thousand of will be generated by gene editing
Material.Rapid chemical assay is carried out to the material of these magnanimity, the conventional liquid phase chromatography period is long, it is difficult to meet magnanimity
Material quickly screens the demand of chemical analysis.
Invention content
In order to find easier, quick analysis method, we measure the life in tobacco using ultra-performance liquid chromatography
Alkaloids, sample analysis time and conventional liquid phase chromatography compare big shortening.In order to simplify sample pre-treatments, we are also by independently setting
The sample pretreatment device of meter makes the sample pre-treatments period also greatly shorten.Improved method and current conventional liquid phase chromatography
Method is compared, day sample analysis amount greatly improve.This method be established as gene editing material Chemical Screening provide it is quick, accurate
Really, reliable method for high-flux analysis.
The purpose of the present invention is to provide the high throughputs that alkaloid in a kind of suitable tobacco gene editor material detects
Analysis method.
The purpose of the present invention is what is be achieved through the following technical solutions：
The assay method of alkaloid, uses following device in a kind of tobacco gene editor material：
Sample extraction bottle, including：Outer tube 1, sealed bottom and to be flat, oral area has a sealing ring 11；Inner sleeve
2, outer diameter is adapted with the internal diameter of the outer tube 1, length be more than the outer tube 1 length；Catheter 4, is located at
In the inner sleeve 2, lower end has an extension mouth and the lower end inner wall of the inner sleeve 2 to be sealedly and fixedly connected, and described interior
2 upper port of casing is fixedly connected to form compression face 21, and upper part is stretched out outside the inner sleeve 2, and back bending is downward；Sieve plate 3, position
In in the lower ending opening of the inner sleeve 2, for one or more；Sample extraction frame 5 inside has several sample extraction bottle putting holes
51, for placing the sample extraction bottle；Sample collection frame 6 inside has several sample bottle putting holes 61 and several waste collections
Bottle putting hole 62, for placing sample bottle 610 and waste collection bottle 620；Turn to take out 7, is located at 6 central part of sample collection frame；
The sample collection frame 6 and the sample extraction frame 5 can surround the pumping 7 that turns and rotate respectively；Lower platen 8, surface is extended with
Several push-down heads 81；
This approach includes the following steps：
1. accurately weighing the tobacco powder sample of 0.25g in outer tube, the extractant of 25mL, parallel processing 100 is added
A sample；It is inserted into inner sleeve and is placed on the sample extraction frame and carry out ultrasonic extraction 35min；
2. being fixed using shaft, the nozzle of the catheter is made to be directed at waste collection bottle 620, push inner sleeve by
Pressure surface 21 simultaneously makes extraction solution pass through after filtering sieve plate 3 filters to enter in catheter, and the initial filtrate 2-3mL that passes out enters
In waste collection bottle 620；
3. centered on shaft, sample extraction frame is rotated, the downward back bending nozzle of the catheter is made to be directed at the sample
610 bottlenecks of bottle continue the compression face 21 for pushing inner sleeve, 1-1.5mL sample filtrates are transferred in sample bottle 610, are waited for
The sample filtrate of analysis；
4. being measured using efficient liquid phase chromatographic analysis, quantified using peak area method, contrast sample tobacco gene
Edit material in alkaloid peak area and standard solution working curve peak area method, you can determine tobacco gene editor
The content of alkaloid in material.
Preferably, 1. used extractant is 80v/v% ethanol solutions to step, and it includes the sodium hydroxide of 0.5wt%.
Preferably, the aperture of the step 2. filtering sieve plate (3) is 0.45 μm.
Preferably, the efficient liquid phase chromatographic analysis condition of step 4. is as follows：Chromatographic column：ACQUITYUPLC BEH C18
(2.1 × 50 μm, 1.7 μm) column；Flow velocity：0.6mL/min；Column temperature：45℃；Mobile phase：The acetonitrile solution of 10v/v%；Sample size：
5.0μL；Detection wavelength：259nm.
Preferably, the also ammonium acetate containing 0.1wt% in the mobile phase.
Preferably, the alkaloid in detection tobacco is：Cotinine, nornicotine, myosmine, anabasine and nicotine are examined
It surveys, wherein detector is diode array detector.
Wherein standard working curve is plotted as：Within the scope of 0.2-200 μ g/mL with acetontrile cotinine, nornicotine,
The standard solution of at least five concentration of myosmine, anabasine standard items uses acetontrile nicotine within the scope of 8-8000 μ g/mL
The standard solution of at least five concentration of standard items recycles high performance liquid chromatograph molten to the standard of above-mentioned all concentration respectively
The content of five kinds of alkaloid standard items in liquid is detected, and according to the peak area and corresponding concentration difference of five kinds of standard items
It draws the standard working curve of five kinds of standard items and its regression equation is calculated.
The sample extraction bottle that the method for the present invention uses integrates extraction, filtering, transfer, and sample extracts in outer tube 1
After taking；Inner sleeve 2 is firmly pushed at compression face 21, extract liquor enters after being filtered by sieve plate 3 in catheter 4, and
The downward mouth of back bending from catheter 4 exits into sample bottle, obtains sample filtrate and carries out chromatography.This process sample
Extraction, filtering, transfer are integrated, and avoid experimental error caused by operation.
1 oral area of outer tube dress fixes inner sleeve 2 there are one sealing ring 11 when for extracting, with prevent extract liquor from splashing out with
And it is buffered when sample filtering.
In the present invention, 4 diameter of catheter is preferably 1-2mm.
It is as follows using multiple sample extraction bottle operating process of apparatus of the present invention：Several sample extraction bottles (such as eight) are put
In the extraction flask putting hole 51 for entering sample extraction frame 5, it is respectively put into sample and carries out extracting operation；Start the part solution collected
(such as 2ml) is abandoned, therefore is started first respectively the waste collection bottle 620 on the downward back bending of catheter 4 alignment sample collection frame 6
Oral area, lower platen 8 is covered, push-down head 81 is made to be respectively aligned to compression face 21, the liquid in waste collection bottle 620 reaches requirement
Afterwards, rotation sample extraction frame 5 or sample collection frame 6 make the downward back bending of catheter 4 be directed at the oral area of sample bottle 610, continue to
Lower platen 8 is pushed, the fluid sample in eight sample extraction bottles enters respective drain after being filtered respectively by respective sieve plate 3
It in pipe 4, enters back into sample bottle 610, continues to push the bottom that the lower platen 8 makes inner sleeve 2 be depressed into outer tube 1, then institute
There is the liquid in sample extraction bottle to be transferred to respectively in sample bottle 610 by respective catheter 4, completes sample transfer behaviour
Make.Sample in sample bottle 610 can be respectively used to analyze.
The assay method of the present invention：
1. standard curve and detection limit：It is measured using the series standard solution of 5 kinds of alkaloids of selected chromatographic condition pair,
Using each component peak area as ordinate (Y), corresponding each component mass concentration is that abscissa (X) draws standard curve, obtains the side of recurrence
Journey and related coefficient, detection limit take continuous 10 sample introductions of minimum concentration standard solution, calculate corresponding as signal-to-noise ratio S/N=3
Standard detection limit
Table 1：Regression equation, related coefficient and the detection limit of 5 kinds of alkaloids
Detection limit is being ng grades of levels, accuracy height.
2. repeatability and recovery of standard addition
Sample introduction is analyzed measures by selected sample pre-treatments condition processing, and by selected chromatographic condition for sample, a copy of it
On the basis of, another be added known quantity alkaloid standard specimen (set be similar to actual sample content 0.5,1.0,2.0 times three add
Dosage), the rate of recovery is calculated by the amount of measuring divided by Standard entertion amount that standard is added.The rate of recovery of each alkaloid is obtained in 88.6-
Between 102%, the illustration method rate of recovery is very high.
Tobacco sample parallel determination under the same conditions 7 times (same to batch processeds), and calculate the phase of 7 parallel determinations
To standard deviation, the RSD that can obtain cotinine is 2.6%, the RSD of nornicotine is 2.4%, the RSD of myosmine is 1.8%, new cigarette
Alkali RSD is 3.2%, and nicotine RSD is 1.1%, and illustration method precision is good.Tobacco sample is measured in different time (as often
It is measured 1 time, 7 times totally), the relative standard deviation of 7 measurement results is calculated, the RSD that can obtain cotinine is 2.9%, nornicotine
RSD be 2.6%, the RSD of myosmine is 2.2%, anabasine RSD is 3.7%, nicotine RSD be 1.4%；Illustrate when different
Interior measurement, method repeatability are still good.
Beneficial effects of the present invention：
(1) device of the present invention integrate extraction, filters and transfer, is not needed in entire sample pretreatment process
Sample transfer can obtain filtered sample to be analysed, and pre-treatment operates to obtain prodigious simplification, may increase introducing and miss
The link of difference also can be reduced accordingly, and the precision of analysis result is improved.In addition, sample pre-treatments operate according to a conventional method
When, in syringe filters filter process, tobacco fine powder can block filtering head, and the rate of filtration is slower；And the processing side of the present invention
Method is slightly placed after the completion of ultrasonic extraction, and under tobacco sample powder is just sunk to, sieve plate when filtering in inner sleeve is only and upper layer
The stillness of night contacts, and can effectively avoid the sieve plate in filter process from blocking in this way, and faster, filter operation is easier real the rate of filtration
It is existing.
(2) detection method of the invention has the time short：Measuring the alkaloid in tobacco using the present invention only needs 5min left
It is right.
(3) detection method of the invention have the advantages that operation accurately, high sensitivity and reproducible.
Description of the drawings
Fig. 1 is the outer tube schematic diagram of the present invention；
Fig. 2 is the inner sleeve schematic diagram of the present invention；
Inner sleeve is inserted into housing spout part schematic diagram when Fig. 3 is the extraction mixing of the present invention；
Inner sleeve is inserted into outer tube schematic internal view when Fig. 4 is the extract liquor filtering of the present invention, transfer；
Fig. 5 is the sample extraction frame and sample collection frame schematic diagram of the present invention；
Fig. 6 is that the sample extraction bottle of the present invention is placed on sample extraction frame and sample collection bottle is placed on sample receipts
Collect the schematic diagram of sample transfer step when on frame；
Fig. 7 is the transparent lower platen schematic diagram of the present invention；
Fig. 8 is standard solution chromatogram；
Fig. 9 is 1 sample solution chromatogram of embodiment.
Description of the drawings：1- outer tubes；2- inner sleeves；21- compression faces；3- sieve plates；4- catheters；5- sample extraction framves；51-
Sample extraction bottle putting hole；6- sample collection framves；61- sample bottle putting holes；62- waste collection bottle putting holes；610- samples are received
Collect bottle；620- waste collection bottles；7- shafts；8- lower platens；81- push-down heads；82- handles；11- sealing rings；12- samples.
Specific implementation mode
The present invention is further described by following specific examples, but does not limit the present invention.
Embodiment 1：Alkaloid content determination in tobacco sample A
1, instrument and reagent
Cotinine, nornicotine, myosmine, nicotine, anabasine standard items (purity >=98%) (Sigma Co., USA)；Second
Alcohol (analysis is pure), sodium hydroxide (analysis is pure), ammonium acetate (top pure grade) are purchased from Guangzhou Shantou Xi Long chemical reagent works；Acetonitrile (chromatography
It is pure), it is purchased from Merck companies of Germany.
WatersAcquity type ultra performance liquid chromatography systems, equipped with vacuum degassing machine, binary gradient pump, automatic sampling
Device, thermocolumn case, diode array detector and Empower-2 chromatographic work stations (Waters, US)；AE240 electronics point
Analyse balance (accurate 0.0001g, Mettler company)；Electric heating constant temperature ultrasound bath pot (Changzhou Nuo Ji Instrument Ltd.)；
Milli-Q50 ultra-pure waters instrument (Millipore companies of the U.S.).
2, standard solution is prepared
Within the scope of 0.2-200 μ g/mL at least with acetontrile cotinine, nornicotine, myosmine, anabasine standard items
The standard solution of five concentration, with the mark of at least five concentration of acetontrile nicotine standard product within the scope of 8-8000 μ g/mL
Quasi- solution
3, the processing of sample
Using the device of the invention, Fig. 1-7 is seen.
Sample pretreatment process：
1. tobacco sample crushes, 40 mesh sieve is crossed, sample 0.25g is accurately weighed in outer tube, is existed with the liquid-transfering gun of 25mL
The 80v/v% ethyl alcohol (sodium hydroxide for including 0.5wt%) of 25mL is added in casing equipped with sample, then in extraction flask
Oral area of the casing clamp in outer tube.100 samples of parallel processing；The rack for test tube of carry sample bottle is put into constant-temperature ultrasonic after installing
Ultrasonic extraction 35min in water-bath.
2. being fixed using shaft, so that the nozzle of the catheter is directed at waste collection bottle, push the compression of inner sleeve
Face simultaneously enters in catheter after so that extraction solution is passed through filtering sieve plate filtering, and passes out into waste collection bottle, initially
Filtrate 2-3mL enters in waste collection bottle；Centered on shaft, sample extraction frame is rotated, the downward back bending of the catheter is made
Nozzle is directed at the sample bottle bottleneck, continues the compression face for pushing inner sleeve, 1-1.5mL sample filtrates are transferred to sample bottle
In, obtain sample filtrate to be analyzed；
4, chromatographic condition
Chromatographic column：ACQUITY UPLC BEH C18 (2.1 × 50 μm, 1.7 μm) column；Flow velocity：0.6mL/min；Column temperature：45
℃；Mobile phase：10% v/v acetonitriles (including 0.1wt% ammonium acetates)；Type of elution：Isocratic elution；Sample size：5.0μL；Inspection
Survey wavelength：259nm.
Using qualitative, the quantified by external standard method with standard specimen comparison retention time.
5, assay method
Regression analysis is carried out with the chromatographic peak area of object and its respective concentration, obtains standard curve.To what is prepared
Sample A is measured, and measures the chromatographic peak area of detection object, substitutes into standard curve, respectively obtain cotinine in sample,
5 kinds of alkaloids such as nornicotine, myosmine, anabasine, nicotine, are as a result shown in Fig. 9 and table 2.
The content of 5 kinds of alkaloids in table 2, tobacco sample A
| Compound | Content (mg/g) |
| Cotinine | 0.328 |
| Nornicotine | 0.964 |
| Myosmine | 0.322 |
| Anabasine | 0.924 |
| Nicotine | 19.2 |
Embodiment 2：Alkaloid content determination in tobacco sample B
Specific steps are as described in Example 1, select tobacco sample B, measure 5 kinds of alkaloids and be shown in Table 3.
The content of 5 kinds of alkaloids in table 3, tobacco sample B
| Compound | Content (mg/g) |
| Cotinine | 0.486 |
| Nornicotine | 1.350 |
| Myosmine | 0.489 |
| Anabasine | 1.520 |
| Nicotine | 24.8 |
Embodiment 3：Alkaloid content determination in tobacco sample C
Specific steps are as described in Example 1, select tobacco sample C, measure 5 kinds of alkaloids and be shown in Table 4.Table 4, tobacco sample
The content of 5 kinds of alkaloids in product C
| Compound | Content (mg/g) |
| Cotinine | 0.412 |
| Nornicotine | 1.020 |
| Myosmine | 0.457 |
| Anabasine | 0.984 |
| Nicotine | 21.2 |
Claims (5)
1. the assay method of alkaloid in a kind of tobacco gene editor material, which is characterized in that use following device：
Sample extraction bottle, including：Outer tube (1), sealed bottom and to be flat, oral area has a sealing ring (11)；Inner sleeve
(2), outer diameter is adapted with the internal diameter of the outer tube (1), length be more than the outer tube (1) length；Catheter
(4), it is located in the inner sleeve (2), lower end has the lower end inner wall of an extension mouth and the inner sleeve (2) to seal fixed connect
It connecing, compression face (21) is fixedly connected to form with the inner sleeve (2) upper port, upper part stretches out inner sleeve (2) outside,
And back bending is downward；Sieve plate (3) is located in the lower ending opening of the inner sleeve (2), for one or more；Sample extraction frame (5),
There are several sample extraction bottle putting holes (51) in it, for placing the sample extraction bottle；Sample collection frame (6), if inside having
Dry-eye disease bottle putting hole (61) and several waste collection bottle putting holes (62), for placing sample bottle (610) and waste collection bottle
(620)；Turn to take out (7), is located at sample collection frame (6) central part；The sample collection frame (6) and the sample extraction frame
(5) pumping (7) that turns can be surrounded respectively to rotate；Lower platen (8), surface are extended with several push-down heads (81)；
This approach includes the following steps：
1. accurately weighing the tobacco powder sample of 0.25g in outer tube, the extractant of 25mL, 100 samples of parallel processing are added
Product；It is inserted into inner sleeve and is placed on the sample extraction frame (5) and carry out ultrasonic extraction 35min；
2. being fixed using shaft, so that the nozzle of the catheter is directed at waste collection bottle (620), push the compression of inner sleeve
Face (21) simultaneously makes extraction solution pass through after filtering sieve plate (3) filters to enter in catheter, the initial filtrate 2-3mL that passes out into
Enter in waste collection bottle (620)；
3. centered on shaft, sample extraction frame is rotated, the downward back bending nozzle of the catheter is made to be directed at the sample bottle
(610) bottleneck continues the compression face (21) for pushing inner sleeve, 1-1.5mL sample filtrates is transferred in sample bottle (610), cold
But sample filtrate to be analyzed is obtained to room temperature；
4. being measured using efficient liquid phase chromatographic analysis, quantified using peak area method, contrast sample tobacco gene editor
In material alkaloid peak area and standard solution working curve peak area method, you can determine tobacco gene editor's material
The content of middle alkaloid.
2. according to the method described in claim 1, it is characterized in that, step 1. used extractant is 80v/v% ethanol solutions,
And it includes the sodium hydroxide of 0.5wt%.
3. according to the method described in claim 1, it is characterized in that, the aperture of the step 2. filtering sieve plate (3) is 0.45 μ
m。
4. according to the method described in claim 1, it is characterized in that, step 4. the efficient liquid phase chromatographic analysis condition is such as
Under：Chromatographic column：ACQUITY UPLC BEH C18 (2.1 × 50 μm, 1.7 μm) column；Flow velocity：0.6mL/min；Column temperature：45℃；Stream
Dynamic phase：The acetonitrile solution of 10v/v%；Sample size：5.0μL；Detection wavelength：259nm.
5. according to the method described in claim 4, it is characterized in that, the also ammonium acetate containing 0.1wt% in the mobile phase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201810146536.2A CN108519455A (en) | 2018-02-12 | 2018-02-12 | The assay method of alkaloid in a kind of tobacco gene editor material |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114894936A (en) * | 2022-05-27 | 2022-08-12 | 云南中烟工业有限责任公司 | Liquid chromatography detection method for six alkaloids in tobacco and tobacco products |
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| CN102004132A (en) * | 2010-10-20 | 2011-04-06 | 中国烟草总公司郑州烟草研究院 | Method for measuring alkaloid in tobacco and tobacco product |
| CN103257196A (en) * | 2013-05-10 | 2013-08-21 | 云南省烟草农业科学研究院 | Method for detecting alkaloids and nitrosamines in tobaccos simultaneously |
| CN204447405U (en) * | 2015-02-12 | 2015-07-08 | 中国水产科学研究院 | A kind of discoid rotatable solid-phase extraction device |
| CN105784455A (en) * | 2016-05-16 | 2016-07-20 | 张起香 | Extraction structure and liquid pretreatment device |
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2018
- 2018-02-12 CN CN201810146536.2A patent/CN108519455A/en not_active Withdrawn
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102004132A (en) * | 2010-10-20 | 2011-04-06 | 中国烟草总公司郑州烟草研究院 | Method for measuring alkaloid in tobacco and tobacco product |
| CN103257196A (en) * | 2013-05-10 | 2013-08-21 | 云南省烟草农业科学研究院 | Method for detecting alkaloids and nitrosamines in tobaccos simultaneously |
| CN204447405U (en) * | 2015-02-12 | 2015-07-08 | 中国水产科学研究院 | A kind of discoid rotatable solid-phase extraction device |
| CN105784455A (en) * | 2016-05-16 | 2016-07-20 | 张起香 | Extraction structure and liquid pretreatment device |
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| 刘正聪等: "超高效液相色谱法测定卷烟烟丝中的生物碱", 《烟草化学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114894936A (en) * | 2022-05-27 | 2022-08-12 | 云南中烟工业有限责任公司 | Liquid chromatography detection method for six alkaloids in tobacco and tobacco products |
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Application publication date: 20180911 |