CN104101675B - A kind of liquid chromatograph mass spectrography separates the method for qualification to flavonoids material in tobacco - Google Patents
A kind of liquid chromatograph mass spectrography separates the method for qualification to flavonoids material in tobacco Download PDFInfo
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Abstract
The invention discloses a kind of liquid chromatograph mass spectrography separates qualification method to flavonoids material in tobacco. Sample is ground, dry, extract with extractant by the amount of 0.1ml/mg sample, centrifugal, collect supernatant, by the amount of 0.5ml/ml, 1ml/ml supernatant add water and chloroform centrifugal, get supernatant analysis. With 153+、151-For characteristic ion scans, result is extracted to parent ion and carry out second order ms scanning, the spectrogram obtaining is verified with reference material with the definite chemical constitution of spectrum storehouse contrast again. Scan as characteristic ion using the above-mentioned molecular ion of having identified material and myricetin, dibydro myricetrin, catechin/epicatechin, Daidzein, Glycitein and glycyrrhizin/isoliquiritigenin, the molecular ion of the doubtful glycosylation flavonoids material searching is carried out to second order ms scanning as parent ion, obtain spectrogram and verify with reference material again with the definite chemical constitution of spectrum storehouse contrast. The sensitive and accurate even analyzing and testing of trace flavonoids material of trace that is applicable to of described method.
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 pairIn tobacco, flavonoids material separates the method for qualification.
Background technology
Flavonoids material, claims again flavone compound, refers to that basic parent nucleus is 2-phenyl chromone compounds, general reference twoThe individual phenyl ring with phenolic hydroxyl group is by the interconnective a series of compounds of central thricarbon atom. Flavonoids material is that plant is importantSecondary metabolites, it all plays an important role to plant itself and the mankind. Aspect plant stress-resistance, flavonoids material is plantThe aspect such as pest-resistant, antiviral, antibacterial all shows significant effect. Aspect the impact of human health, existing a large amount of reportsShow that flavonoids material has the effect of the aspect such as anticancer, anti-ageing and antiviral. Therefore, the class in separation and plant identificationFlavonoid substance has great importance to the mechanism of action of plant and human health for illustrating flavonoids material. Although peopleThrough separating and identified part flavonoids material from plant, but still have the flavonoids material of a large amount of the unknowns to wait for that we identify. BuildVertical a kind of simple, quick, highly sensitive isolation and identification method, for the even discovery of trace flavonoids material of trace in plantHave great importance.
In plant, the classical separating and extracting process of flavonoids material comprises solvent extraction, column chromatography, paper chromatography etc. Structure mirrorRule, based on 3 special ring structures of flavonoids material, adopts the methods such as ultraviolet spectra, mass spectrum, nuclear magnetic resonance to carry out separately or connectionClose qualification. Along with the development of chromatogram and mass-spectrometric technique, liquid chromatogram-ultraviolet spectra-mass spectrometric hyphenated technique becomes flavonoids materialThe main method of Structural Identification. This method utilizes liquid chromatogram to classify to flavonoids material conventionally, utilizes feature ultravioletUptake zone classification flavonoid substance and other compound, finally utilize mass spectrum to carry out Structural Identification to the flavonoids material of finding. ButBe, because UV-detector sensitivity is limited, can only be used for the analyzing and testing of the flavonoids material that content is higher, and much micro-Even trace flavonoids material cannot be found by UV-detector. Therefore, develop a kind of high sensitivity and to flavonoidsCompound has specific isolation and identification method will have very important realistic meaning.
Summary of the invention
The object of the present invention is to provide a kind of liquid chromatograph mass spectrography to separate mirror to flavonoids material in tobaccoFixed method.
The object of the present invention is achieved like this, and described liquid chromatograph mass spectrography carries out flavonoids material in tobaccoSeparate the method for qualification, comprise sample preparation, Instrumental Analysis and separate qualification operation, specifically comprise:
A, sample preparation: the tobacco plant tissue of collection is ground, and freeze drying, takes dry sample, by 0.1ml/The consumption of mg sample adds extractant, and ultrasonic extraction is centrifugal, collects supernatant liquor, adds by the consumption of 0.5ml/ml supernatant liquorEnter water, add chloroform to carry out vortex by the consumption of 1ml/ml supernatant liquor and mix, centrifugal, then get supernatant liquor as chromatographySample.
B, Instrumental Analysis: liquid-phase chromatographic analysis condition: m) chromatographic column of WatersBEHC18(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 0.2mmol/L; Eluent gradientElution 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 0.25mL/min.
Mass spectral analysis condition: ion gun is electric spray ion source; In the time doing parent ion scanning, negative ions scans simultaneously, knotFruit is checking mutually; Electron spray voltage ± 4000V; Curtain atmospheric pressure 40psi; 700 DEG C of ion source temperatures; Assisted gas 1,60psi;Assisted gas 2,50psi; Remove a bunch voltage-100V.
C, Structural Identification: the Structural Identification of free state flavonoids material: with 153+、151-For characteristic ion carries out parent ionFull scan, scanning voltage 30v, sweep limits 250 ~ 320, searches the free state flavonoids not replaced by glycosyl; To anion mouldScanning result under formula carries out the ion extraction, under positive ion mode, under corresponding retention time, also can find corresponding hydrogenationIon, chooses these ions and carries out second order ms scanning as parent ion, and scanning voltage 30v, by the second order ms obtaining and spectrumStorehouse contrast, determines the chemical constitution of flavonoids material, then carries 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, with theseThe molecular ion of having identified material is that characteristic ion carries out parent ion scanning, simultaneously by myricetin, dibydro myricetrin, catechin/The molecular ion of epicatechin, Daidzein, Glycitein and glycyrrhizin/isoliquiritigenin also carries out parent ion as characteristic ionScanning, scanning voltage 30v, sweep limits 250 ~ 1500; In scanning result, search possible glycosylation flavonoids material, chooseThe molecular ion of these glycosylation flavonoids carries out second order ms scanning as parent ion, and scanning voltage 30v, by the secondary obtainingMass spectrum and the contrast of spectrum storehouse, determine the chemical constitution of flavonoids material, then carry out retention time and second order ms with n-compoundChecking.
Isolation and identification method of the present invention is highly sensitive, accuracy good, is specially adapted to even trace class Huang of traceThe analyzing and testing of ketone material, and sample preparation is simple and quick, has effectively improved detection efficiency, has the higher valency of applyingValue.
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 limits 250 ~ 320;
Fig. 3 is that (stain mark is spectrogram weight for the second order ms comparison diagram of Isorhamnetin in actual sample and standard spectrum storehouseClose place);
Fig. 4 is that tobacco sample is with 315-For the parent ion full scan chromatogram of characteristic ion;
Fig. 5 is second order ms comparison diagram (the stain mark of Isorhamnetin-3-O-rutinose in actual sample and standard spectrum storehousePlace is spectrogram overlapping position);
Fig. 6 is that tobacco sample is with 301-For the parent ion full scan chromatogram of characteristic ion;
Fig. 7 is second order ms comparison diagram (the stain mark of isoquercitin-3-O-rutinose in actual sample and standard spectrum storehousePlace is spectrogram overlapping position).
Detailed description of the invention
The present invention is further illustrated below, but never in any form the present invention is limited, based on the present inventionAny conversion or replacement that training centre is done, 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, itsBe characterised in that and comprise sample preparation, Instrumental Analysis and separate qualification operation, specifically comprise:
Described sample preparation is that the tobacco plant tissue of collection is ground, and freeze drying takes dry sample, pressesThe consumption of 0.1ml/mg sample adds extractant, and ultrasonic extraction is centrifugal, collects supernatant liquor, by 0.5ml/ml supernatant liquorConsumption adds water, adds chloroform to carry out vortex mix by the consumption of 1ml/ml supernatant liquor, centrifugal, then gets supernatant liquor as lookAnalysis of spectrum sample.
Described Instrumental Analysis comprises liquid-phase chromatographic analysis and mass spectral analysis, and the liquid-phase chromatographic analysis condition adopting is:M) chromatographic column of WatersBEHC18(15cm × 2.1mm × 1.7 μ; Mobile phase A is water, and Mobile phase B is acetonitrile, and two-phase is all added0.1% formic acid and the ammonium acetate of 0.2mmol/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 0.25mL/min.
The mass spectral analysis condition adopting is: ion gun is electric spray ion source; Negative ions in the time doing parent ion scanningScanning simultaneously, result is verified mutually; Electron spray voltage ± 4000V; Curtain atmospheric pressure 40psi; 700 DEG C of ion source temperatures; AuxiliaryGas 1,60psi; Assisted gas 2,50psi; Remove a bunch voltage-100V.
Described Structural Identification comprises the structure of Structural Identification and the glycosylation flavonoids material of free state flavonoids materialQualification. Known according to the architectural feature of the flavonoids material having identified in the concept of flavonoids material and tobacco, flavonoids thingMatter has the structure shown in Fig. 1 mostly.
Kaempferol KaempferolR1=H, R2=H, R3=OH molecular weight MW=286
Quercetin QuercetinR1=OH, R2=H, R3=OH molecular weight MW=302
Myricetin MyricetinR1=OH, R2=OH, R3=OH molecular weight MW=318
Isorhamnetin IsorhamnetinR1=OMe, R2=H, R3=OH molecular weight MW=316
This structure makes these materials can produce 153 in the time that mass spectral analysis fragment ruptures+（151-) characteristic ion.
The Structural Identification of described free state flavonoids material is with 153+、151-For carrying out parent ion, entirely sweeps characteristic ionRetouch, scanning voltage 30v, sweep limits 250 ~ 320, searches the free state flavonoids not replaced by glycosyl. Under negative ion modeScanning result carry out the ion extraction, under positive ion mode, under corresponding retention time, also can find corresponding hydrogenation fromSon, chooses these ions and carries out second order ms scanning as parent ion, and scanning voltage 30v, by the second order ms obtaining and spectrum storehouseContrast, determines the chemical constitution of flavonoids material, then carries 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 molecular ion of having identified material taking these carries out parent ion scanning as characteristic ion, considers part glycosylation flavonoids simultaneouslyMay not there is not the form of free state in material, by myricetin (molecular weight MW=318), dibydro myricetrin (molecular weight MW=320), youngsterTheine/epicatechin (molecular weight MW=290), Daidzein (molecular weight MW=254), Glycitein (molecular weight MW=284) and sweetThe molecular ion of the common flavonoids aglycon of grass element/isoliquiritigenin (molecular weight MW=256) etc. also as characteristic ion carry out mother fromSon scanning, scanning voltage 30v, sweep limits 250 ~ 1500. In scanning result, search possible glycosylation flavonoids material, choosingThe molecular ion of getting these glycosylation flavonoids carries out second order ms scanning as parent ion, scanning voltage 30v, by obtain twoLevel mass spectrum and the contrast of spectrum storehouse, determine the chemical constitution of flavonoids material, then carry out retention time and secondary matter with n-compoundSpectrum checking.
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,Add liquid nitrogen frozen, then grind.
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 extractant to carry out ultrasonic extraction and centrifugal in dry sample, the extraction adoptingTechnological parameter is supersonic frequency 20KHz, ultrasonic time 30min, and temperature is controlled at 25 degrees Celsius. Ultrasonic complete with 10000rpmCentrifugal 10min.
Sample preparation described in operation A, adds water and chloroform to carry out vortex supernatant liquor and mixes, centrifugal, the technique adoptingParameter is the centrifugal 10min of 10000rpm.
The extractant that sample preparation described in operation A adopts is that 70 ~ 85% alcohol is the aqueous solution.
The extractant 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, under negative ion mode, is retainingTime 4.90 ± 0.02min, 5.39 ± 0.02min, 5.83 ± 0.02min, 6.38 ± 0.02min, 6.48 ± 0.02There is obvious chromatographic peak at min place, has weak chromatographic peak, as figure at retention time 5.77min, 6.29min, 6.42minShown in 2.
In Structural Identification described in operation C, the scanning result under negative ion mode is carried out to the ion extraction, in retention time4.90±0.02min、5.39±0.02min、5.77±0.02min、5.83±0.02min、6.29±0.02min、The parent ion at 6.38 ± 0.02min, 6.42 ± 0.02min, 6.48 ± 0.02min place is respectively 303-、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, with Mongolian oakThe molecular ion of Pi Su, Kaempferol/cyanidenon, Isorhamnetin, naringenin, dihydroquercetin and dihydrokaempferol be feature fromIn the parent ion full scan of son, search possible glycosylation flavonoids.
The second order ms obtaining and spectrum storehouse are contrasted described in operation C, the chemical constitution of determining flavonoids material is mustTo second order ms upload to massbank website (www.massbank.jp) and compose storehouse comparison, tentatively determine these class HuangsThe chemical constitution of ketone material.
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. Ultra-pure water byThe preparation of Millipore purification system. Other standard sample is bought in Sigma-Aldrich, AlfaAesar and lark prestige company.WatersUPLC liquid chromatograph (U.S.), ABSCIEX5500 triple quadrupole bar mass spectrograph (U.S.). SB-50D ultrasonic waveExtraction apparatus (NingBo XinZhi Biology Science Co., Ltd), WatersBEHC18(15cm × 2.1mm × 1.7 μ is m) anti-Phase chromatographic column (Waters, the U.S.), MILLI-Q water purification machine (MILLIPORE company), (system in Beijing Jing is vertical centrifugal for LD5-2A centrifugeMachine Co., Ltd), vortex vortex mixer (Dutch Breda company), CP2245 assay balance (sensibility reciprocal 0.0001g, GermanySartorius 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 adopts with qualificationTobacco plant tissue is the equal amount of mixture of ripe middle leaf and the equal amount of mixture of petal that these three product grow tobacco, each 5g.
(3) experimental technique
The fresh tobacco plant tissue gathering is put into mortar, add liquid nitrogen frozen, and grind rapidly. Sample after grindingOriginally put into the Refrigerator store of-80 DEG C. Take the fresh sample that grinds of 400mg, add the methanol aqueous solution of 4ml75%, supersonic frequency20KHZ, ultrasonic time 30min, temperature is controlled at 25 degrees Celsius. Ultrasonic complete with the centrifugal 10min of 10000rpm. Collect 2mlSupernatant liquor, adds 1ml water and 2ml chloroform to carry out vortex and mixes 30s, and with the centrifugal 10min of 10000rpm, then it is clear to get upper strataLiquid, as chromatography sample, proceeds to chromatogram sample introduction bottle to be analyzed.
The liquid-phase chromatographic analysis condition adopting is: m) chromatographic column of WatersBEHC18(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 0.2mmol/L; Eluent gradientElution 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 0.25mL/min.
The mass spectral analysis condition adopting is: ion gun is electric spray ion source; Negative ions in the time doing parent ion scanningScanning simultaneously, result is verified mutually; Electron spray voltage ± 4000V; Curtain atmospheric pressure 40psi; 700 DEG C of ion source temperatures; AuxiliaryGas 1,60psi; Assisted gas 2,50psi; Remove a bunch voltage-100V.
(4) experimental result
With 151-While carrying out parent ion scanning for characteristic ion, be that 6.48min place has obviously at chromatographic retentionChromatographic peak, its parent ion detecting is 315-. Gather 6.48min place 315-The secondary fragment ion of ion, and carriedHand over to massbank website (www.massbank.jp) and compose storehouse comparison, (in figure, stain mark is spectrogram as shown in Figure 3Overlapping position). Found that it is very similar to the second order ms figure of Isorhamnetin in spectrum storehouse, so tentatively judge that it is different sandlwoodElement. Buy the standard substance of Isorhamnetin and carry out comparing of retention time and second order ms with compound to be identified, confirming itFor 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 combination of IsorhamnetinState compound. With the molecular ion (315 of Isorhamnetin-) carry out parent ion full scan as characteristic ion, found that reservationTime is that 4.55min place has obvious chromatographic peak (Fig. 4), and its parent ion detecting is 623-. According to 623-With 315-BetweenThere is 308 neutrality loss, tentatively judge that this compound is Isorhamnetin-rutinoside. Gather 4.55min place 623-IonSecondary fragment ion, and be committed to massbank website (www.massbank.jp) and composed storehouse comparison, result is sent outNow it is very similar to the second order ms figure of Isorhamnetin-3-O-rutinoside in spectrum storehouse, (stain mark in figure as shown in Figure 5For spectrogram overlapping position), so judge that it is Isorhamnetin-3-O-rutinoside. This compound is not owing to obtaining standard items, thereforeDo not carry out standard specimen checking. Isorhamnetin-3-O-rutinoside be one grow tobacco in before have no the glycosylation flavonoids thing of reportMatter.
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 5.80min place has obviously at chromatographic retentionChromatographic peak, its parent ion detecting is 301-. Gather 5.80min place 301-The secondary fragment ion of ion, and carriedHand over to massbank website (www.massbank.jp) and compose storehouse comparison, found that itself and the secondary of composing Quercetin in storehouseMass spectrogram is very similar, buys the standard substance of Quercetin and carries out the ratio of retention time and second order ms with compound to be identifiedRight, be confirmed that it is Quercetin. With the molecular ion 301 of Quercetin-Carry out parent ion full scan as characteristic ion, result is sent outExisting retention time is that 4.28min place has obvious chromatographic peak (Fig. 6), and its parent ion detecting is 609-. According to 609-With301-Between exist 308 neutrality to lose, tentatively judge that this compound is Quercetin-rutinoside. Gather 4.28min place609-The secondary fragment ion of ion, and be committed to massbank website (www.massbank.jp) and compose storehouse ratioRight, found that it is very similar to the second order ms figure of Quercetin-3-O-rutinoside in spectrum storehouse, (stain in figure as shown in Figure 7Mark is spectrogram overlapping position), so tentatively judge that it is Quercetin-3-O-rutinoside. Buy Quercetin-3-O-rutinoseThe standard substance of glycosides also carries out comparing of retention time and second order ms with compound to be identified, is confirmed that it is Quercetin-3-O-Rutinoside. Quercetin-3-O-rutinoside be one grow tobacco in important glycosylation flavonoids material.
Claims (7)
1. liquid chromatograph mass spectrography separates a method for qualification to flavonoids material in tobacco, it is characterized in that bagDraw together sample preparation, Instrumental Analysis and Structural Identification operation, specifically comprise:
A, sample preparation: the tobacco plant tissue of collection is ground, and freeze drying, takes dry sample, by 0.1ml/mg sampleIt is the aqueous solution that this consumption adds 70 ~ 85% alcohol, ultrasonic extraction 30min under 25 DEG C, supersonic frequency 20KHz, ultrasonic complete withThe centrifugal 10min of 10000rpm, collects supernatant liquor, adds water by the consumption of 0.5ml/ml supernatant liquor, clear by 1ml/ml upper strataThe consumption of liquid adds chloroform to carry out vortex to mix, centrifugal, then gets supernatant liquor as chromatography sample;
B, Instrumental Analysis: liquid-phase chromatographic analysis condition: specification is the WatersBEHC18 chromatogram of 15cm × 2.1mm × 1.7 μ mPost; 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 0.2mmol/L; Mobile phase ladderDegree 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 0.25mL/min;
Mass spectral analysis condition: ion gun is electric spray ion source; In the time doing parent ion scanning, negative ions scans simultaneously, result phaseChecking mutually; Electron spray voltage ± 4000V; Curtain atmospheric pressure 40psi; 700 DEG C of ion source temperatures; Assisted gas 1,60psi; Assisted gas 2,50psi; Remove a bunch voltage-100V;
C, Structural Identification:
The Structural Identification of free state flavonoids material: with 153+、151-For characteristic ion carries out parent ion full scan, scanning voltage30v, sweep limits 250 ~ 320, searches the free state flavonoids not replaced by glycosyl; Scanning result under negative ion mode is enteredRow the ion extraction also can find the corresponding hydrogen ion that adds under positive ion mode under corresponding retention time, choose these fromSon carries out second order ms scanning as parent ion, and scanning voltage 30v, by the second order ms obtaining and the contrast of spectrum storehouse, determines class HuangThe chemical constitution of ketone 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, reflect with theseThe molecular ion of earnest matter is that characteristic ion carries out parent ion scanning, simultaneously by myricetin, dibydro myricetrin, catechin/table youngsterThe molecular ion of theine, Daidzein, Glycitein and glycyrrhizin/isoliquiritigenin also carries out parent ion as characteristic ion and sweepsRetouch scanning voltage 30v, sweep limits 250 ~ 1500; In scanning result, search glycosylation flavonoids material, choose these glycosylsThe molecular ion of changing flavonoids carries out second order ms scanning as parent ion, and scanning voltage 30v, by the second order ms obtaining and spectrumStorehouse contrast, determines the chemical constitution of flavonoids material, then carries out retention time and second order ms checking with n-compound.
2. isolation and identification method according to claim 1, is characterized in that described in operation A the tobacco plant tissue gatheringGrinding is that 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 after freeze drying is put described in operation APreserve in-80 DEG C.
4. isolation and identification method according to claim 1, is characterized in that described in operation A that 70 ~ 85% alcohol is that the aqueous solution is75% methanol aqueous solution.
5. isolation and identification method according to claim 1, is characterized in that described in operation C free state class Huang in Structural IdentificationThe Structural Identification of ketone material under negative ion mode, retention time 4.90 ± 0.02min, 5.39 ± 0.02min, 5.83 ±There is obvious chromatographic peak at 0.02min, 6.38 ± 0.02min, 6.48 ± 0.02min place, retention time 5.77 ± 0.02min,There is weak chromatographic peak in 6.29 ± 0.02min, 6.42 ± 0.02min.
6. isolation and identification method according to claim 1, is characterized in that described in operation C in Structural Identification anion mouldScanning result under formula carries out the ion extraction, retention time 4.90 ± 0.02min, 5.39 ± 0.02min, 5.77 ±0.02min、5.83±0.02min、6.29±0.02min、6.38±0.02min、6.42±0.02min、6.48±0.02minThe parent ion at place is respectively 303-、287-、285-、301-、269-、271-、285-、315-。
7. isolation and identification method according to claim 1, is characterized in that glycosylation class in the Structural Identification described in operation CThe Structural Identification of flavonoid substance is with Quercetin, Kaempferol/cyanidenon, Isorhamnetin, naringenin, dihydroquercetin and dihydroThe molecular ion of Kaempferol is to search glycosylation flavonoids in the parent ion full scan of characteristic ion.
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| 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 |
| CN105136946B (en) * | 2015-06-10 | 2017-06-06 | 云南省烟草农业科学研究院 | 12 kinds of detection methods of flavonoid substances in a kind of quantitative analysis tobacco leaf |
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| CN105699579B (en) * | 2016-04-27 | 2017-07-07 | 中国烟草总公司郑州烟草研究院 | Multiple types Flavone metabolism group method in one kind detection fresh tobacco leaf |
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| CN104101675A (en) | 2014-10-15 |
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