CN104101675A - Method used for separating and identifying flavonoid matters in tobacco by adopting liquid chromatography-mass spectrography - Google Patents
Method used for separating and identifying flavonoid matters in tobacco by adopting liquid chromatography-mass spectrography Download PDFInfo
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Abstract
The invention discloses a method used for separating and identifying flavonoid matters in tobacco by adopting liquid chromatography-mass spectrography. The method comprises the following steps: a sample is ground and dried, an extractant is added according to the standard that 0.1 ml of the extractant is added into 1 mg of the sample for extraction and centrifugation, a supernatant liquor is collected, water and chloroform are added according to the standard that 0.5 ml of the water and 1 ml of the chloroform are added into 1 ml of the supernatant liquor for centrifugation, and the supernatant liquor is collected for analysis; 153+ and 151- are adopted as characteristic ions for scanning, parent ions are extracted for second level mass spectrum scanning, and an obtained spectrogram is compared with a spectral library to determine a chemical structure and verified through a reference substance; the molecules and ions of the identified matters and myricetin, dihydromyricetin, catechinic acid/epicatechin, daidzein, glycitein and glycyrrhizin/isoliquiritigenin are adopted as characteristic ions for scanning, the molecules and ions of the searched glycosylation flavonoid matters are used as the parent ions for the second level mass spectrum scanning, an obtained spectrogram is compared with the spectral library to determine a chemical structure and verified through the reference substance. According to the invention, the method is suitable for the analyzing and detecting micro even trace flavonoid matters.
Description
Technical field
The invention belongs to plant extracts and separate authenticate technology field, be specifically related to a kind of liquid chromatograph mass spectrography and flavonoids material in tobacco separated to the method for identifying.
Background technology
Flavonoids material, claims again flavone compound, refers to that basic parent nucleus is 2-phenyl chromone compounds, makes a general reference two phenyl ring with phenolic hydroxyl group by the interconnective a series of compounds of central thricarbon atom.Flavonoids material is the important secondary metabolites of plant, and it all plays an important role to plant itself and the mankind.Aspect plant stress-resistance, flavonoids material all shows significant effect at the aspect such as pest-resistant, antiviral, antibacterial of plant.Aspect the impact of human health, existing a large amount of report shows that flavonoids material has the effect of the aspect such as anticancer, anti-ageing and antiviral.Therefore, the flavonoids material in separation and plant identification has great importance to the mechanism of action of plant and human health for illustrating flavonoids material.Although people separate and have identified part flavonoids material from plant, still have the flavonoids material of a large amount of the unknowns to wait for that we identify.Set up a kind of simple, quick, highly sensitive isolation and identification method, have great importance for the micro-even discovery of trace flavonoids material in plant.
In plant, the classical separating and extracting process of flavonoids material comprises solvent extraction, column chromatography, paper chromatography etc.Structural Identification, based on 3 special ring structures of flavonoids material, adopts the methods such as ultraviolet spectrum, mass spectrum, nuclear magnetic resonance to identify alone or in combination.Along with the development of chromatogram and mass-spectrometric technique, liquid chromatography-ultraviolet spectrum-mass spectrometric hyphenated technique becomes the main method of flavonoids structure of matter qualification.This method utilizes liquid chromatography to classify to flavonoids material conventionally, utilizes feature uv absorption region class flavonoid substance and other compound, finally utilizes mass spectrum to carry out Structural Identification to the flavonoids material of finding.But, because UV-detector sensitivity is limited, can only be used for the analyzing and testing of the flavonoids material that content is higher, and a lot of trace even trace flavonoids material cannot be found by UV-detector.Therefore, develop a kind of high sensitivity and flavonoids is had to specific isolation and identification method and will there is very important realistic meaning.
Summary of the invention
The object of the present invention is to provide a kind of liquid chromatograph mass spectrography flavonoids material in tobacco to be separated to the method for qualification.
The object of the present invention is achieved like this, and described liquid chromatograph mass spectrography separates the method for qualification to flavonoids material in tobacco, comprises sample preparation, instrumental analysis and separate qualification operation, specifically comprises:
A, sample preparation: the tobacco plant tissue of collection is ground, freeze drying, take dry sample, add extraction agent by the consumption of 0.1ml/mg sample, ultrasonic extraction, centrifugal, collect supernatant liquor, add water by the consumption of 0.5ml/ml supernatant liquor, add chloroform to carry out vortex by the consumption of 1ml/ml supernatant liquor and mix, centrifugal, then get supernatant liquor as stratographic analysis sample.
B, instrumental analysis: liquid-phase chromatographic analysis condition: m) chromatographic column of Waters BEH C18(15cm × 2.1mm × 1.7 μ; Mobile phase A is water, and Mobile phase B is acetonitrile, and two-phase is all added 0.1% formic acid and the ammonium acetate of 0.2 mmol/L; Eluent gradient elution program is as follows: 0 ~ 1min, 10%B; 1 ~ 9min, 10%B-90%B; 9 ~ 11min, 90%B-100%B; 11 ~ 11.1min, 100%B-10%B; 11.1 ~ 13min, 10%B; 30 DEG C of column temperatures, sample size 2 μ l, flow velocity 0.25 mL/min.
Mass spectrophotometry condition: ion gun is electric spray ion source; In the time doing parent ion scanning, negative ions scans simultaneously, and result is verified mutually; Electron spray voltage ± 4000 V; Curtain atmospheric pressure 40 psi; 700 DEG C of ion source temperatures; Assisted gas 1,60 psi; Assisted gas 2,50 psi; Remove bunch voltage-100 V.
C, Structural Identification: the Structural Identification of free state flavonoids material: with 153
+, 151
-for characteristic ion carries out parent ion full scan, scanning voltage 30v, sweep limit 250 ~ 320, searches the free state flavonoids not replaced by glycosyl; Scanning result under negative ion mode is carried out to the ion extraction, under positive ion mode, under corresponding retention time, also can find the corresponding hydrogen ion that adds, choose these ions and carry out second order ms scanning as parent ion, scanning voltage 30v, by the second order ms obtaining and the contrast of spectrum storehouse, determine the chemical constitution of flavonoids material, then carry out retention time and second order ms checking with n-compound.
The Structural Identification of glycosylation flavonoids material: according to the Structural Identification result of free state flavonoids material, the molion of having identified material taking these carries out parent ion scanning as characteristic ion, the molion of myricetin, dibydro myricetrin, catechin/epicatechin, Daidzein, Glycitein and glycyrrhizin/isoliquiritigenin is also carried out to parent ion scanning as characteristic ion simultaneously, scanning voltage 30v, sweep limit 250 ~ 1500; In scanning result, search possible glycosylation flavonoids material, the molion of choosing these glycosylation flavonoids carries out second order ms scanning as parent ion, scanning voltage 30v, by the second order ms obtaining and the contrast of spectrum storehouse, determine the chemical constitution of flavonoids material, then carry out retention time and second order ms checking with n-compound.
Isolation and identification method of the present invention is highly sensitive, accuracy good, be specially adapted to the even analyzing and testing of trace flavonoids material of trace, and sample preparation is simple and quick, has effectively improved detection efficiency, has higher application value.
Brief description of the drawings
Fig. 1 is the basic structure of main flavonoids material parent nucleus in tobacco;
Fig. 2 is tobacco free state flavonoids material 153
+, 151
-parent ion full scan chromatogram, sweep limit 250 ~ 320;
Fig. 3 is the second order ms comparison diagram (stain mark is spectrogram overlapping position) of Isorhamnetin in actual sample and standard spectrum storehouse;
Fig. 4 is that tobacco sample is with 315
-for the parent ion full scan chromatogram of characteristic ion;
Fig. 5 is the second order ms comparison diagram (stain mark is spectrogram overlapping position) of Isorhamnetin-3-O-rutinose in actual sample and standard spectrum storehouse;
Fig. 6 is that tobacco sample is with 301
-for the parent ion full scan chromatogram of characteristic ion;
Fig. 7 is the second order ms comparison diagram (stain mark is spectrogram overlapping position) of isoquercitin-3-O-rutinose in actual sample and standard spectrum storehouse.
Embodiment
The present invention is further illustrated below, but never in any form the present invention is limited, and any conversion or the replacement done based on training centre of the present invention, all belong to protection scope of the present invention.
Liquid chromatograph mass spectrography of the present invention separates the method for qualification to flavonoids material in tobacco, it is characterized in that comprising sample preparation, instrumental analysis and separate qualification operation, specifically comprises:
Described sample preparation is that the tobacco plant tissue of collection is ground, freeze drying, take dry sample, add extraction agent by the consumption of 0.1ml/mg sample, ultrasonic extraction, centrifugal, collect supernatant liquor, add water by the consumption of 0.5ml/ml supernatant liquor, add chloroform to carry out vortex by the consumption of 1ml/ml supernatant liquor and mix, centrifugal, then get supernatant liquor as stratographic analysis sample.
Described instrumental analysis comprises liquid-phase chromatographic analysis and mass spectrophotometry, and the liquid-phase chromatographic analysis condition adopting is: m) chromatographic column of Waters BEH C18(15cm × 2.1mm × 1.7 μ; Mobile phase A is water, and Mobile phase B is acetonitrile, and two-phase is all added 0.1% formic acid and the ammonium acetate of 0.2 mmol/L; Eluent gradient elution program is as follows: 0 ~ 1min, 10%B; 1 ~ 9min, 10%B-90%B; 9 ~ 11min, 90%B-100%B; 11 ~ 11.1min, 100%B-10%B; 11.1 ~ 13min, 10%B; 30 DEG C of column temperatures, sample size 2 μ l, flow velocity 0.25 mL/min.
The mass spectrophotometry condition adopting is: ion gun is electric spray ion source; In the time doing parent ion scanning, negative ions scans simultaneously, and result is verified mutually; Electron spray voltage ± 4000 V; Curtain atmospheric pressure 40 psi; 700 DEG C of ion source temperatures; Assisted gas 1,60 psi; Assisted gas 2,50 psi; Remove bunch voltage-100 V.
Described Structural Identification comprises the Structural Identification of free state flavonoids material and the Structural Identification of glycosylation flavonoids material.Known according to the architectural feature of the flavonoids material having identified in the concept of flavonoids material and tobacco, flavonoids material has the structure shown in Fig. 1 mostly.
Kaempferol Kaempferol R1=H, R2=H, R3=OH molecular weight MW=286
Quercetin Quercetin R1=OH, R2=H, R3=OH molecular weight MW=302
Myricetin Myricetin R1=OH, R2=OH, R3=OH molecular weight MW=318
Isorhamnetin Isorhamnetin R1=OMe, R2=H, R3=OH molecular weight MW=316
This structure makes these materials can produce 153 in the time that mass spectrophotometry fragment ruptures
+(151
-) characteristic ion.
The Structural Identification of described free state flavonoids material is with 153
+, 151
-for characteristic ion carries out parent ion full scan, scanning voltage 30v, sweep limit 250 ~ 320, searches the free state flavonoids not replaced by glycosyl.Scanning result under negative ion mode is carried out to the ion extraction, under positive ion mode, under corresponding retention time, also can find the corresponding hydrogen ion that adds, choose these ions and carry out second order ms scanning as parent ion, scanning voltage 30v, by the second order ms obtaining and the contrast of spectrum storehouse, determine the chemical constitution of flavonoids material, then carry out retention time and second order ms checking with n-compound.
The Structural Identification of described glycosylation flavonoids material is according to the Structural Identification result of free state flavonoids material, the molion of having identified material taking these carries out parent ion scanning as characteristic ion, consider that part glycosylation flavonoids material may not exist the form of free state simultaneously, by myricetin (molecular weight MW=318), dibydro myricetrin (molecular weight MW=320), catechin/epicatechin (molecular weight MW=290), Daidzein (molecular weight MW=254), the molion of the common flavonoids aglycon such as Glycitein (molecular weight MW=284) and glycyrrhizin/isoliquiritigenin (molecular weight MW=256) also carries out parent ion scanning as characteristic ion, scanning voltage 30v, sweep limit 250 ~ 1500.In scanning result, search possible glycosylation flavonoids material, the molion of choosing these glycosylation flavonoids carries out second order ms scanning as parent ion, scanning voltage 30v, by the second order ms obtaining and the contrast of spectrum storehouse, determine the chemical constitution of flavonoids material, then carry out retention time and second order ms checking with n-compound.
Sample preparation described in operation A, the tobacco plant adopting is organized the ripe middle leaf of preferred cigarette strain and/or petal.
Sample preparation described in operation A, grinds tobacco plant tissue to refer to tobacco plant tissue is put into mill, adds liquid nitrogen frozen, then grinds.
Sample preparation described in operation A, the sample after freeze drying can be placed in-80 DEG C and preserve.
Sample preparation described in operation A, adds extraction agent to carry out ultrasonic extraction and centrifugal in dry sample, and the Extraction technique adopting is ultrasonic frequency 20 KHz, ultrasonic time 30 min, and temperature is controlled at 25 degrees Celsius.Ultrasonic complete with centrifugal 10 min of 10000 rpm.
Sample preparation described in operation A, adds water and chloroform to carry out vortex supernatant liquor and mixes, centrifugal, and the technological parameter adopting is centrifugal 10 min of 10000 rpm.
The extraction agent that sample preparation described in operation A adopts is that 70 ~ 85% alcohol is aqueous solution.
The extraction agent that sample preparation described in operation A adopts is 75% methanol aqueous solution.
In Structural Identification described in operation C, the Structural Identification of free state flavonoids material is under negative ion mode, there is obvious chromatographic peak at retention time 4.90 ± 0.02 min, 5.39 ± 0.02 min, 5.83 ± 0.02 min, 6.38 ± 0.02 min, 6.48 ± 0.02 min places, there is weak chromatographic peak at retention time 5.77 min, 6.29 min, 6.42 min, as shown in Figure 2.
In Structural Identification described in operation C, the scanning result under negative ion mode is carried out to the ion extraction, be respectively 303 at the parent ion at retention time 4.90 ± 0.02 min, 5.39 ± 0.02 min, 5.77 ± 0.02 min, 5.83 ± 0.02 min, 6.29 ± 0.02 min, 6.38 ± 0.02 min, 6.42 ± 0.02 min, 6.48 ± 0.02 min places
-, 287
-, 285
-, 301
-, 269
-, 271
-, 285
-, 315
-.
In Structural Identification described in operation C, the scanning result of the Structural Identification of glycosylation flavonoids material shows, searches possible glycosylation flavonoids at the molion taking Quercetin, Kaempferol/cyanidenon, Isorhamnetin, naringenin, dihydroquercetin and dihydrokaempferol in the parent ion full scan of characteristic ion.
The second order ms obtaining and spectrum storehouse are contrasted described in operation C, the chemical constitution of determining flavonoids material is the second order ms obtaining to be uploaded to mass bank website (www.massbank.jp) compose storehouse comparison, tentatively determines the chemical constitution of these flavonoids materials.
embodiment 1
---separation and the qualification of Isorhamnetin in tobacco
(1) experiment reagent and device
Acetonitrile (chromatographic grade), methyl alcohol (chromatographic grade), ethanol (chromatographic grade) are bought the company in German Merck.Ultrapure water is prepared by Millipore purification system.Other standard model is bought in Sigma-Aldrich, Alfa Aesar and lark prestige company.Waters UPLC liquid chromatograph (U.S.), AB SCIEX 5500 triple quadrupole bar mass spectrometers (U.S.).SB-50D ultrasound wave extraction apparatus (NingBo XinZhi Biology Science Co., Ltd), m) reverse-phase chromatographic column (Waters of Waters BEH C18(15 cm × 2.1 mm × 1.7 μ, the U.S.), MILLI-Q water purification machine (MILLIPORE company), LD5-2A hydro-extractor (the vertical hydro-extractor of system in Beijing Jing company limited), vortex vortex mixer (Dutch Breda company), CP2245 analytical balance (sensibility reciprocal 0.0001g, German Sartorius company).
(2) experiment material
Ripe middle leaf and the petal of cloud and mist 97, the large gold dollar of safflower, K326, flavonoids separating substances is the equal amount of mixture of ripe middle leaf and the equal amount of mixture of petal that these three product grow tobacco with the tobacco plant tissue that qualification adopts, each 5 g.
(3) experimental technique
The fresh tobacco plant tissue gathering is put into mortar, add liquid nitrogen frozen, and grind rapidly.Sample after grinding is put into the Refrigerator store of-80 DEG C.Take the fresh sample that grinds of 400 mg, add the methanol aqueous solution of 4ml 75%, ultrasonic frequency 20 KHZ, ultrasonic time 30 min, temperature is controlled at 25 degrees Celsius.Ultrasonic complete with centrifugal 10 min of 10000 rpm.Collect 2ml supernatant liquor, add 1ml water and 2ml chloroform to carry out vortex and mix 30 s, with centrifugal 10 min of 10000 rpm, then get supernatant liquor as stratographic analysis sample, proceed to chromatogram sample introduction bottle to be analyzed.
The liquid-phase chromatographic analysis condition adopting is: m) chromatographic column of Waters BEH C18(15cm × 2.1mm × 1.7 μ; Mobile phase A is water, and Mobile phase B is acetonitrile, and two-phase is all added 0.1% formic acid and the ammonium acetate of 0.2 mmol/L; Eluent gradient elution program is as follows: 0 ~ 1min, 10%B; 1 ~ 9min, 10%B-90%B; 9 ~ 11min, 90%B-100%B; 11 ~ 11.1min, 100%B-10%B; 11.1 ~ 13min, 10%B; 30 DEG C of column temperatures, sample size 2 μ l, flow velocity 0.25 mL/min.
The mass spectrophotometry condition adopting is: ion gun is electric spray ion source; In the time doing parent ion scanning, negative ions scans simultaneously, and result is verified mutually; Electron spray voltage ± 4000 V; Curtain atmospheric pressure 40 psi; 700 DEG C of ion source temperatures; Assisted gas 1,60 psi; Assisted gas 2,50 psi; Remove bunch voltage-100 V.
(4) experimental result
With 151
-while carrying out parent ion scanning for characteristic ion, be that 6.48 min places have obvious chromatographic peak at chromatographic retention, its parent ion detecting is 315
-.Gather 6.48 min places 315
-the secondary fragmention of ion, and be committed to mass bank website (www.massbank.jp) and compose storehouse comparison, (in figure, stain mark is spectrogram overlapping position) as shown in Figure 3.Found that it is very similar to the second order ms figure of Isorhamnetin in spectrum storehouse, so tentatively judge that it is Isorhamnetin.Buy the standard substance of Isorhamnetin and carry out comparing of retention time and second order ms with compound to be identified, being confirmed that it is Isorhamnetin.Isorhamnetin be one grow tobacco in before have no the sequestered flavonoids material of report.
embodiment 2
---separation and the qualification of Isorhamnetin-3-O-rutinoside in tobacco
(1) experiment reagent and device
With embodiment 1.
(2) experiment material
With embodiment 1.
(3) experimental technique
With embodiment 1.
(4) experimental result
Owing to having found Isorhamnetin in tobacco sample, therefore suppose also to exist in tobacco the glycosyl of Isorhamnetin in conjunction with state compound.With the molion (315 of Isorhamnetin
-) carry out parent ion full scan as characteristic ion, found that retention time is that 4.55 min places have obvious chromatographic peak (Fig. 4), its parent ion detecting is 623
-.According to 623
-with 315
-between exist 308 neutrality to lose, tentatively judge that this compound is Isorhamnetin-rutinoside.Gather 4.55 min places 623
-the secondary fragmention of ion, and be committed to mass bank website (www.massbank.jp) and composed storehouse comparison, found that it is very similar to the second order ms figure of Isorhamnetin-3-O-rutinoside in spectrum storehouse, (in figure, stain mark is spectrogram overlapping position) as shown in Figure 5, so judge that it is Isorhamnetin-3-O-rutinoside.This compound, owing to not obtaining standard items, does not therefore carry out standard specimen checking.Isorhamnetin-3-O-rutinoside be one grow tobacco in before have no the glycosylation flavonoids material of report.
embodiment 3
---separation and the qualification of Quercetin-3-O rutinoside in tobacco
(1) experiment reagent and device
?with embodiment 1.
(2) experiment material
With embodiment 1.
(3) experimental technique
With embodiment 1.
(4) experimental result
With 151
-while carrying out parent ion scanning for characteristic ion, be that 5.80 min places have obvious chromatographic peak at chromatographic retention, its parent ion detecting is 301
-.Gather 5.80 min places 301
-the secondary fragmention of ion, and be committed to mass bank website (www.massbank.jp) and composed storehouse comparison, found that it is very similar to the second order ms figure of Quercetin in spectrum storehouse, buy the standard substance of Quercetin and carry out comparing of retention time and second order ms with compound to be identified, being confirmed that it is Quercetin.With the molion 301 of Quercetin
-carry out parent ion full scan as characteristic ion, found that retention time is that 4.28 min places have obvious chromatographic peak (Fig. 6), its parent ion detecting is 609
-.According to 609
-with 301
-between exist 308 neutrality to lose, tentatively judge that this compound is Quercetin-rutinoside.Gather 4.28 min places 609
-the secondary fragmention of ion, and be committed to mass bank website (www.massbank.jp) and composed storehouse comparison, found that it is very similar to the second order ms figure of Quercetin-3-O-rutinoside in spectrum storehouse, (in figure, stain mark is spectrogram overlapping position) as shown in Figure 7, so tentatively judge that it is Quercetin-3-O-rutinoside.Buy the standard substance of Quercetin-3-O-rutinoside and carry out comparing of retention time and second order ms with compound to be identified, being confirmed that it is Quercetin-3-O-rutinoside.Quercetin-3-O-rutinoside be one grow tobacco in important glycosylation flavonoids material.
Claims (9)
1. liquid chromatograph mass spectrography separates a method for qualification to flavonoids material in tobacco, it is characterized in that comprising sample preparation, instrumental analysis and separates qualification operation, specifically comprises:
A, sample preparation: the tobacco plant tissue of collection is ground, freeze drying, take dry sample, add extraction agent by the consumption of 0.1ml/mg sample, ultrasonic extraction, centrifugal, collect supernatant liquor, add water by the consumption of 0.5ml/ml supernatant liquor, add chloroform to carry out vortex by the consumption of 1ml/ml supernatant liquor and mix, centrifugal, then get supernatant liquor as stratographic analysis sample;
B, instrumental analysis: liquid-phase chromatographic analysis condition: specification is the Waters BEH C18 chromatographic column of 15cm × 2.1mm × 1.7 μ m; Mobile phase A is water, and Mobile phase B is acetonitrile, and two-phase is all added 0.1% formic acid and the ammonium acetate of 0.2 mmol/L; Eluent gradient elution program is as follows: 0 ~ 1min, 10%B; 1 ~ 9min, 10%B-90%B; 9 ~ 11min, 90%B-100%B; 11 ~ 11.1min, 100%B-10%B; 11.1 ~ 13min, 10%B; 30 DEG C of column temperatures, sample size 2 μ l, flow velocity 0.25 mL/min;
Mass spectrophotometry condition: ion gun is electric spray ion source; In the time doing parent ion scanning, negative ions scans simultaneously, and result is verified mutually; Electron spray voltage ± 4000 V; Curtain atmospheric pressure 40 psi; 700 DEG C of ion source temperatures; Assisted gas 1,60 psi; Assisted gas 2,50 psi; Remove bunch voltage-100 V;
C, Structural Identification: the Structural Identification of free state flavonoids material: with 153
+, 151
-for characteristic ion carries out parent ion full scan, scanning voltage 30v, sweep limit 250 ~ 320, searches the free state flavonoids not replaced by glycosyl; Scanning result under negative ion mode is carried out to the ion extraction, under positive ion mode, under corresponding retention time, also can find the corresponding hydrogen ion that adds, choose these ions and carry out second order ms scanning as parent ion, scanning voltage 30v, by the second order ms obtaining and the contrast of spectrum storehouse, determine the chemical constitution of flavonoids material, then carry out retention time and second order ms checking with n-compound;
The Structural Identification of glycosylation flavonoids material: according to the Structural Identification result of free state flavonoids material, the molion of having identified material taking these carries out parent ion scanning as characteristic ion, the molion of myricetin, dibydro myricetrin, catechin/epicatechin, Daidzein, Glycitein and glycyrrhizin/isoliquiritigenin is also carried out to parent ion scanning as characteristic ion simultaneously, scanning voltage 30v, sweep limit 250 ~ 1500; In scanning result, search possible glycosylation flavonoids material, the molion of choosing these glycosylation flavonoids carries out second order ms scanning as parent ion, scanning voltage 30v, by the second order ms obtaining and the contrast of spectrum storehouse, determine the chemical constitution of flavonoids material, then carry out retention time and second order ms checking with n-compound.
2. isolation and identification method according to claim 1, is characterized in that the sample preparation described in operation A, tobacco plant tissue is ground to refer to tobacco plant tissue is put into mill, adds liquid nitrogen frozen, then grinds.
3. isolation and identification method according to claim 1, is characterized in that the sample preparation described in operation A, and the sample after freeze drying can be placed in-80 DEG C and preserve.
4. isolation and identification method according to claim 1, it is characterized in that the sample preparation described in operation A, add extraction agent to carry out ultrasonic extraction in dry sample, centrifugal, the Extraction technique adopting is ultrasonic frequency 20 KHz, ultrasonic time 30 min, temperature is controlled at 25 degrees Celsius, ultrasonic complete with centrifugal 10 min of 10000 rpm.
5. isolation and identification method according to claim 1, is characterized in that the alcohol that extraction agent that the sample preparation described in operation A adopts is 70 ~ 85% is aqueous solution.
6. isolation and identification method according to claim 5, the methanol aqueous solution that the extraction agent that the sample preparation described in it is characterized in that adopts is 75%.
7. isolation and identification method according to claim 1, it is characterized in that the Structural Identification of free state flavonoids material in the Structural Identification described in operation C is under negative ion mode, there is obvious chromatographic peak at retention time 4.90 ± 0.02 min, 5.39 ± 0.02 min, 5.83 ± 0.02 min, 6.38 ± 0.02 min, 6.48 ± 0.02 min places, have weak chromatographic peak at retention time 5.77 ± 0.02 min, 6.29 ± 0.02 min, 6.42 ± 0.02 min.
8. isolation and identification method according to claim 1, it is characterized in that, in the Structural Identification described in operation C, the scanning result under negative ion mode is carried out to the ion extraction, be respectively 303 at the parent ion at retention time 4.90 ± 0.02 min, 5.39 ± 0.02 min, 5.77 ± 0.02 min, 5.83 ± 0.02 min, 6.29 ± 0.02 min, 6.38 ± 0.02 min, 6.42 ± 0.02 min, 6.48 ± 0.02 min places
-, 287
-, 285
-, 301
-, 269
-, 271
-, 285
-, 315
-.
9. isolation and identification method according to claim 1, is characterized in that the Structural Identification of glycosylation flavonoids material in the Structural Identification described in operation C searches possible glycosylation flavonoids in the parent ion full scan of characteristic ion at the molion taking Quercetin, Kaempferol/cyanidenon, Isorhamnetin, naringenin, dihydroquercetin and dihydrokaempferol.
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| CN104844615A (en) * | 2015-04-11 | 2015-08-19 | 云南中烟工业有限责任公司 | Flavone compound in Yunnan local sun cured tobacco, preparation method and use thereof |
| CN104865337A (en) * | 2015-06-10 | 2015-08-26 | 云南省烟草农业科学研究院 | Detection method for quantitatively analyzing 14 kinds of flavonoids in tobacco petals |
| CN105136946A (en) * | 2015-06-10 | 2015-12-09 | 云南省烟草农业科学研究院 | Detection method for quantitative analysis of 12 kinds of flavonoid substances in tobacco leaves |
| CN105241982A (en) * | 2015-09-30 | 2016-01-13 | 广东研捷医药科技有限公司 | Chiral chromatographic separation and analysis method of dihydromyricetin enantiomer |
| CN105699579A (en) * | 2016-04-27 | 2016-06-22 | 中国烟草总公司郑州烟草研究院 | Metabonomics method for detecting multiple flavonoids in fresh tobacco leaves |
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| CN104844615A (en) * | 2015-04-11 | 2015-08-19 | 云南中烟工业有限责任公司 | Flavone compound in Yunnan local sun cured tobacco, preparation method and use thereof |
| CN104844615B (en) * | 2015-04-11 | 2017-07-18 | 云南中烟工业有限责任公司 | A kind of Yunnan local dries flavone compound and its production and use in cigarette |
| CN104865337B (en) * | 2015-06-10 | 2017-05-31 | 云南省烟草农业科学研究院 | 14 kinds of detection methods of flavonoid substances in a kind of quantitative analysis tobacco petal |
| CN104865337A (en) * | 2015-06-10 | 2015-08-26 | 云南省烟草农业科学研究院 | Detection method for quantitatively analyzing 14 kinds of flavonoids in tobacco petals |
| CN105136946A (en) * | 2015-06-10 | 2015-12-09 | 云南省烟草农业科学研究院 | Detection method for quantitative analysis of 12 kinds of flavonoid substances in tobacco leaves |
| CN105241982A (en) * | 2015-09-30 | 2016-01-13 | 广东研捷医药科技有限公司 | Chiral chromatographic separation and analysis method of dihydromyricetin enantiomer |
| CN105241982B (en) * | 2015-09-30 | 2017-05-10 | 广东研捷医药科技有限公司 | Chiral chromatographic separation and analysis method of dihydromyricetin enantiomer |
| CN105699579A (en) * | 2016-04-27 | 2016-06-22 | 中国烟草总公司郑州烟草研究院 | Metabonomics method for detecting multiple flavonoids in fresh tobacco leaves |
| CN107505405A (en) * | 2017-07-06 | 2017-12-22 | 北京林业大学 | The efficient rapid extraction and assay method of flavonoids pigment in Chinese rose petal |
| CN107505405B (en) * | 2017-07-06 | 2020-02-21 | 北京林业大学 | Method for efficiently and rapidly extracting and measuring flavonoid pigment in Chinese rose petals |
| CN108132317A (en) * | 2018-02-27 | 2018-06-08 | 吉林农业大学 | A kind of method of flavones ingredient content in measure corn stigma |
| CN109828019A (en) * | 2019-02-21 | 2019-05-31 | 南昌大学 | The method that electron spray extraction ionization mass spectrometry quickly detects Citrus Huanglongbing pathogen |
| CN111366653A (en) * | 2020-04-01 | 2020-07-03 | 上海中科新生命生物科技有限公司 | Method for detecting flavonoid substances in red wine |
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