CN104865337B - 14 kinds of detection methods of flavonoid substances in a kind of quantitative analysis tobacco petal - Google Patents
14 kinds of detection methods of flavonoid substances in a kind of quantitative analysis tobacco petal Download PDFInfo
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Abstract
The invention discloses 14 kinds of detection methods of flavonoid substances in a kind of quantitative analysis tobacco petal, including the preparation of sample pre-treatments, need testing solution, the preparation of reference substance solution and determination step.The present invention identifies 17 kinds of flavonoid substances based on inventor's leaf from tobacco in 2014 and in spending, wherein 9 kinds be tobacco in first be accredited out, so that establishing the wherein 14 kinds quantitative analysis methods of flavonoid substances, the correlative study of the foundation for carrying out flavonoid substances in tobacco petal of the inventive method is significant.
Description
Technical field
The invention belongs to technical field of analysis and detection, and in particular to 14 kinds of flavonoids things in a kind of quantitative analysis tobacco petal
The detection method of matter.
Background technology
Flavonoid substances (flavonoids) refer to that two phenyl ring pass through a series ofization that central thricarbon atom is connected with each other
Compound, they are distributed widely in plant kingdom, are the important secondary metabolites of plant.Flavonoid substances are in many plants
In be proved to pest-resistant and disease-resistant related to plant.Meanwhile, flavone compound is the very strong compound of a class bioactivity, is
A kind of natural antioxidant, has a wide range of applications in fields such as medicine, food.For example, Quercetin -3-O- rutinosides
（Rutin sophorin, vitamin p）It is clinically to be used to prevent and treat cerebral hemorrhage, hypertension, retinal hemorrhage, purple scar and acute hemorrhagic ephritis
Etc. the important drugs of disease.
Containing abundant flavonoid substances in tobacco petal, but research of the people to flavonoid substances in tobacco petal at present
It is less with the report for utilizing.With the increase that people are paid close attention to flavonoid substances in tobacco, a kind of flavonoid substances are set up comprehensive
Quantitative analysis method becomes very necessary.
The content of the invention
It is an object of the invention to provide 14 kinds of detection methods of flavonoid substances in a kind of quantitative analysis tobacco petal.
The object of the present invention is achieved like this, including the preparation of sample pre-treatments, need testing solution, reference substance solution
Prepare and determination step, specifically include：
A, sample pre-treatments：Saved backup being placed in 1 ~ 4 DEG C after the grinding of measuring samples tobacco petal, freeze-drying；
The preparation of B, need testing solution：Sample after the accurate pre-treatment for weighing 10.0mg, add the methanol-chloroform of 1ml-
The inner mark solution of water extraction solution and 200 μ l, inner mark solution is the aqueous solution of Daidzein, genistin and aurantiamarin, concentration
It is 1.0 μ g/mL, supernatant is taken as need testing solution after sonicated, centrifugation；
The preparation of C, reference substance solution：The accurately weighed each n-compound of difference, i.e., 14 standard samples and 3 internal standard compounds
Daidzein, genistin and aurantiamarin, plus methyl alcohol are made solution of every 1ml containing 1mg, and the flavonoids list mark for obtaining 1mg/ml is molten
Liquid, pipette respectively 100 μ l 1mg/ml low concentration flavonoids singly mark solution and 10mg/ml high concentration flavonoids list mark it is molten
It is molten that liquid prepares standard mixing in 100ml volumetric flasks, with sample extraction solution, i.e. methanol-chloroform-water extraction solution constant volume
Liquid, with sample extraction solution stepwise dilution standard mixed solution, be made low concentration flavonoid substances concentration for 1000,600,400,
100th, 60,40,10,6,4,1ng/mL, volume is the standard liquid gradient of 1mL；The corresponding high standard solution ladder of this gradient
It is 100,60,40,10,6,4,1,0.6,0.4,0.1 μ g/mL to spend；Add 200 μ l's in the solution of above-mentioned all concentration gradients
Inner mark solution obtains reference substance solution, and inner mark solution is the aqueous solution of Daidzein, genistin and aurantiamarin, and concentration is 1.0 μ
g/mL；
D, measure：Through chromatograph mass spectrum analysis, the content with 14 flavonoids to be measured is corresponding with its as abscissa
Chromatographic peak area is ordinate with the ratio of the chromatographic peak area of internal standard compound, sets up working curve, and correction data is carried out
Linear regression, tries to achieve the regression equation of flavonoid substances, and the content of flavonoid substances is calculated as the following formula in sample：
In formula：C is the content of flavonoid substances in tobacco petal sample, unit μ g/g；
The concentration of the extract that x is measured for instrument, unit is μ g/mL.
The present invention identifies 17 kinds of flavonoid substances based on inventor's leaf from tobacco in 2014 and in spending, wherein 9 kinds are
It is accredited out first in tobacco so that establish the wherein 14 kinds quantitative analysis methods of flavonoid substances, the inventive method
The correlative study set up for carrying out flavonoid substances in tobacco petal is significant.
Brief description of the drawings
Fig. 1 is that the flavonoid substances of tobacco petal sample of the present invention separate TIC.
Specific embodiment
With reference to embodiment and accompanying drawing, the present invention is further illustrated, but the present invention is subject to never in any form
Limitation, based on present invention teach that any conversion or replacement made, belong to protection scope of the present invention.
14 kinds of detection methods of flavonoid substances in quantitative analysis tobacco petal of the present invention, including locate before sample
Reason, the preparation of need testing solution, the preparation of reference substance solution and determination step, specifically include：
A, sample pre-treatments：Saved backup being placed in 1 ~ 4 DEG C after the grinding of measuring samples tobacco petal, freeze-drying；
The preparation of B, need testing solution：Sample after the accurate pre-treatment for weighing 10.0mg, add the methanol-chloroform of 1ml-
The inner mark solution of water extraction solution and 200 μ l, inner mark solution is the aqueous solution of Daidzein, genistin and aurantiamarin, concentration
It is 1.0 μ g/mL, supernatant is taken as need testing solution after sonicated, centrifugation；
The preparation of C, reference substance solution：The accurately weighed each n-compound of difference, i.e., 14 standard samples and 3 internal standard compounds
Daidzein, genistin and aurantiamarin, plus methyl alcohol are made solution of every 1ml containing 1mg, and the flavonoids list mark for obtaining 1mg/ml is molten
Liquid, pipette respectively 100 μ l 1mg/ml low concentration flavonoids singly mark solution and 10mg/ml high concentration flavonoids list mark it is molten
It is molten that liquid prepares standard mixing in 100ml volumetric flasks, with sample extraction solution, i.e. methanol-chloroform-water extraction solution constant volume
Liquid, with sample extraction solution stepwise dilution standard mixed solution, be made low concentration flavonoid substances concentration for 1000,600,400,
100th, 60,40,10,6,4,1ng/mL, volume is the standard liquid gradient of 1mL；The corresponding high standard solution ladder of this gradient
It is 100,60,40,10,6,4,1,0.6,0.4,0.1 μ g/mL to spend；Add 200 μ l's in the solution of above-mentioned all concentration gradients
Inner mark solution obtains reference substance solution, and inner mark solution is the aqueous solution of Daidzein, genistin and aurantiamarin, and concentration is 1.0 μ
g/mL；
D, measure：Through chromatograph mass spectrum analysis, the content with 14 flavonoids to be measured is corresponding with its as abscissa
Chromatographic peak area is ordinate with the ratio of the chromatographic peak area of internal standard compound, sets up working curve, and correction data is carried out
Linear regression, tries to achieve the regression equation of flavonoid substances, and the content of flavonoid substances is calculated as the following formula in sample：
In formula：C is the content of flavonoid substances in tobacco petal sample, unit μ g/g；
The concentration of the extract that x is measured for instrument, unit is μ g/mL.
Grinding described in step A is that measuring samples tobacco petal is put into mortar, and liquid feeding chilled nitrogen grinds.
The volume proportion of the methanol-chloroform-water extraction solution described in step A is 5:2:2.
Chromatographiccondition described in D steps is：Chromatographic column, Waters BEH C18（15cm × 2.1mm, 1.7 μm of grains
Footpath）；A phases, water；B phases, acetonitrile, two-phase adds 0.1% formic acid and the ammonium acetate of 0.2mmol/L；Mobility gradient, 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, keeps 10%B；30 DEG C of column temperature, sample size 2 μ L, flow velocity 0.25mL/min.
Mass spectral analysis condition described in D steps is：Ion gun, electron spray ionisation ion gun；Spray voltage, -4000V；
Curtain atmospheric pressure, 40psi；Ion source temperature, 700 DEG C；Auxiliary gas 1,60psi；Auxiliary gas 2,50psi；Remove cluster voltage, -100V；Change
Compound quota ion and energy are shown in Table 1.
The flavonoid substances of table 1 analyze mass spectrum quota ion and collision energy
With embodiment, the invention will be further described below：
Experiment reagent and device：
Acetonitrile（Chromatographic grade）, methyl alcohol（Chromatographic grade）, ethanol（Chromatographic grade）Buy in German Merck companies.Ultra-pure water by
It is prepared by Millipore purification systems.Dihydrokaempferol, dihydroquercetin, Kaempferol, Quercetin, naringenin, rutin sophorin, kaempferia galamga
Phenol -3-O- rutinosides, Isorhamnetin, Quercitrin-3-O-glucoside, cyanidenon, Isorhamnetin -3-O- glucosides,
Standard substance and the Huangs such as Isorhamnetin -3-O- rutinosides, Kaempferol -3-O- glucosides, eriodictyol-7- O -glucoside
Beans aglycon, genistin（Glucosyl group）, aurantiamarin（Rue glycosyl）Bought in Sigma-Aldrich, Alfa etc. internal standard substance
Aesar and lark prestige company.Waters UPLC liquid chromatographs（The U.S.）, the triple quadrupole mass spectrometers of AB SCIEX 5500
（The U.S.).SB-50D ultrasonic wave extraction instrument（NingBo XinZhi Biology Science Co., Ltd）；Waters BEH C18（15 cm
× 2.1 mm, 1.7 μm of particle diameters）Reverse-phase chromatographic column（Waters, USA）, MILLI-Q water purification machines（MILLIPORE companies）,
LD5-2A centrifuges（System in Beijing Jing founds centrifuge Co., Ltd）, vortex vortex mixer（Dutch Breda companies）；CP2245 analyzes day
It is flat（Sensibility reciprocal 0.0001g, German Sartorious companies）.
Embodiment 1
--- the quantitative analysis of tobacco bred tobacco bred N. alata petal flavonoid substances
Experiment material：Fresh lyophilized N. alata maturity periods middle part fireworks valve
Experimental technique：
The accurate lyophilized tobacco petal sample for weighing 10.0mg, adds 1mL extractants（Methanol-chloroform-water, 5:2:2, body
Product ratio）With 200 μ L inner mark solutions（1.0μg/mL）, ultrasonic 30min, centrifugation takes supernatant and is transferred to liquid chromatogram sample introduction bottle point
Analysis.
Chromatographiccondition condition：Chromatographic column, Waters BEH C18（15cm × 2.1mm, 1.7 μm of particle diameters）；A phases, water；
B phases, acetonitrile, two-phase adds 0.1% formic acid and the ammonium acetate of 0.2 mmol/L；Mobility gradient, 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, keeps 10%B；
30 DEG C of column temperature, sample size 2 μ L, flow velocity 0.25mL/min.
Mass spectral analysis condition：Ion gun, electron spray ionisation ion gun；Spray voltage, -4000V；Curtain atmospheric pressure, 40psi；
Ion source temperature, 700 DEG C；Auxiliary gas 1,60psi；Auxiliary gas 2,50psi；Remove cluster voltage, -100V.Compound quota ion and
Energy is shown in Table 1.
Prepare all n-compounds（It is shown in Table 1,14 standard specimens and 3 internal standards）1mg/mL singly mark solution（Methyl alcohol is
Solvent）.The low concentration flavonoids singly mark solution of 100 μ L 1mg/mL and the high concentration flavonoids of 10mL 1mg/mL are pipetted respectively
Singly mark solution in 100mL volumetric flasks, with sample extraction solution (methanol-chloroform-water, 5:2:2, volume ratio) constant volume, it is made standard
Mixed solution.With sample extraction solution stepwise dilution standard mixed solution, be made low concentration flavonoid substances concentration for 1000,
600th, 400,100,60,40,10,6,4,1ng/mL, volume is the standard liquid gradient of 1mL.The corresponding highly concentrated scale of this gradient
Quasi- solution gradient is 100,60,40,10,6,4,1,0.6,0.4,0.1 μ g/mL.200 are added in the solution of all concentration gradients
μ L internal standard mixed solutions.Internal standard mixed liquor is Daidzein, genistin, the aqueous solution of aurantiamarin, and concentration is 1.0 μ g/mL.It is yellow
Beans aglycon, genistin, aurantiamarin are respectively used to correction sugar-free base class flavone aglycone, glucosyl group substitution flavonoids and rutinose
Base replaces flavonoids.Under the conditions of selected chromatographic mass spectrometry, the content with 12 flavonoids to be measured as abscissa, with it
Corresponding chromatographic peak area is ordinate with the ratio of the chromatographic peak area of internal standard compound, sets up working curve.To correction number
According to linear regression is carried out, the regression equation of flavonoid substances is tried to achieve.The content of flavonoid substances, is counted as the following formula in sample
Calculate：
In formula：C is the content of flavonoid substances in tobacco petal sample, unit μ g/g；
The concentration of the extract that x is measured for instrument, unit is μ g/mL.
Experimental result is shown in Table 2,
The content of the tobacco bred N. alata petal flavonoid substances of table 2
| Flavonoid substances | Content（μg/g） |
| Quercetin -3-O- rutinosides | 132.87±0.01 |
| Quercitrin-3-O-glucoside | 70.21±25.91 |
| FNS | 444.39±0.03 |
| Isorhamnetin -3-O- glucosides | 3.46±0.18 |
| Kaempferol -3-O- glucosides | 74.41±0.32 |
| Isorhamnetin -3-O- rutinosides | 6.11±0.25 |
| Dihydroquercetin | 253.99±0.00 |
| Eriodictyol-7- O -glucoside | 3.05±0.02 |
| Dihydrokaempferol | 28.19±0.50 |
| Cyanidenon | 42.22±0.02 |
| Quercetin | 37.44±0.05 |
| Naringenin | 4.79±0.01 |
| Kaempferol | 39.68±0.06 |
| Isorhamnetin | 43.47±10.64 |
| Summation | 1184.28±38 |
Embodiment 2
--- the quantitative analysis of tobacco bred N. rustica petal flavonoid substances
Experiment material：Fresh lyophilized N. rustica maturity periods middle part fireworks valve
Experimental technique：
The accurate lyophilized tobacco petal sample for weighing 10.0mg, adds 1mL extractants（Methanol-chloroform-water, 5:2:2, body
Product ratio）With 200 μ L inner mark solutions（1.0μg/mL）, ultrasonic 30min, centrifugation takes supernatant and is transferred to liquid chromatogram sample introduction bottle point
Analysis.
Chromatographiccondition condition：Chromatographic column, Waters BEH C18（15cm × 2.1mm, 1.7 μm of particle diameters）；A phases, water；
B phases, acetonitrile, two-phase adds 0.1% formic acid and the ammonium acetate of 0.2 mmol/L；Mobility gradient, 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, keeps 10%B；
30 DEG C of column temperature, sample size 2 μ L, flow velocity 0.25mL/min.
Mass spectral analysis condition：Ion gun, electron spray ionisation ion gun；Spray voltage, -4000V；Curtain atmospheric pressure, 40psi；
Ion source temperature, 700 DEG C；Auxiliary gas 1,60psi；Auxiliary gas 2,50psi；Remove cluster voltage, -100V.Compound quota ion and
Energy is shown in Table 1.
Prepare all n-compounds（It is shown in Table 1,14 standard specimens and 3 internal standards）1mg/mL singly mark solution（Methyl alcohol is
Solvent）.The low concentration flavonoids singly mark solution of 100 μ L 1mg/mL and the high concentration flavonoids of 10mL 1mg/mL are pipetted respectively
Singly mark solution in 100mL volumetric flasks, with sample extraction solution (methanol-chloroform-water, 5:2:2, volume ratio) constant volume, it is made standard
Mixed solution.With sample extraction solution stepwise dilution standard mixed solution, be made low concentration flavonoid substances concentration for 1000,
600th, 400,100,60,40,10,6,4,1ng/mL, volume is the standard liquid gradient of 1mL.The corresponding highly concentrated scale of this gradient
Quasi- solution gradient is 100,60,40,10,6,4,1,0.6,0.4,0.1 μ g/mL.200 are added in the solution of all concentration gradients
μ L internal standard mixed solutions.Internal standard mixed liquor is Daidzein, genistin, the aqueous solution of aurantiamarin, and concentration is 1.0 μ g/mL.It is yellow
Beans aglycon, genistin, aurantiamarin are respectively used to correction sugar-free base class flavone aglycone, glucosyl group substitution flavonoids and rutinose
Base replaces flavonoids.Under the conditions of selected chromatographic mass spectrometry, the content with 12 flavonoids to be measured as abscissa, with it
Corresponding chromatographic peak area is ordinate with the ratio of the chromatographic peak area of internal standard compound, sets up working curve.To correction number
According to linear regression is carried out, the regression equation of flavonoid substances is tried to achieve.The content of flavonoid substances, is counted as the following formula in sample
Calculate：
In formula：C is the content of flavonoid substances in tobacco petal sample, unit μ g/g；
The concentration of the extract that x is measured for instrument, unit is μ g/mL.
Experimental result is shown in Table 3,
The content of the tobacco bred N. rustica petal flavonoid substances of table 3
| Flavonoid substances | Content（μg/g） |
| Quercetin -3-O- rutinosides | 3021.18±0.21 |
| Quercitrin-3-O-glucoside | 58.85±2.00 |
| FNS | 898.98±0.00 |
| Isorhamnetin -3-O- glucosides | 2.86±0.16 |
| Kaempferol -3-O- glucosides | 11.54±0.02 |
| Isorhamnetin -3-O- rutinosides | 144.22±1.19 |
| Dihydroquercetin | 262.01±0.00 |
| Eriodictyol-7- O -glucoside | 0.13±0.01 |
| Dihydrokaempferol | 3.77±0.12 |
| Cyanidenon | 44.46±0.01 |
| Quercetin | 42.06±0.08 |
| Naringenin | 4.00±0.01 |
| Kaempferol | 41.50±0.09 |
| Isorhamnetin | 45.77±207.49 |
| Summation | 4581.33±211.39 |
Test example 1
Specification Curve of Increasing is carried out according to described method, the linearly dependent coefficient of all compounds is found（r 2 ）Exist
More than 0.99, illustrate that set up method has good linear response, it is adapted to quantitative analysis.These compounds are quantified
Limit and test limit assessment, it is found that test limit and the quantitative limit difference of different compounds are larger, wherein Isorhamnetin -3- glucose
Glycosides, dihydroquercetin, naringenin -7- glucose, dihydrokaempferol, Isorhamnetin -3-O- rutinosides, Kaempferol -3-O- Portugals
Polyglycoside, the quantitative limit of Quercitrin-3-O-glucoside can reach 1.2 ~ 5.6ng/mL, and Quercetin -3-O- rutin sophorins, kaempferia galamga
The quantitative limit of phenol -3-O- rutin sophorins, Quercetin, naringenin, Kaempferol, cyanidenon, Isorhamnetin etc. is in 0.065 ~ 0.4 μ g/
Between mL（It is shown in Table 4）.Flavonoid substances are carried out with repeated investigation, it is found that the in a few days repeatability of investigated flavonoid substances exists
Between 3.2% ~ 6.8%, repeatability is between 5.3% ~ 11.2% in the daytime（It is shown in Table 5）.Determined in this experiment according to flavonoid substances
11 grow tobacco in mean concentration, add mean concentration about 50% and 100% standard sample to actual sample in assay method
The rate of recovery, as a result finds the rate of recovery of this method between 86.7% ~ 103.4%（It is shown in Table 5）.
The calibration curve of the flavonoids of table 4, test limit and quantitative limit
The repeatability and the rate of recovery of the flavonoids of table 5
Claims (2)
1. a kind of 14 kinds of detection methods of flavonoid substances in quantitative analysis tobacco petal, it is characterised in that 14 kinds of flavonoids
Material be dihydrokaempferol, dihydroquercetin, Kaempferol, Quercetin, naringenin, rutin sophorin, FNS,
Isorhamnetin, Quercitrin-3-O-glucoside, cyanidenon, Isorhamnetin -3-O- glucosides, Isorhamnetin -3-O- rues
Glucosides, Kaempferol -3-O- glucosides, eriodictyol-7- O -glucoside；The detection method is including sample pre-treatments, for examination
The preparation of product solution, the preparation of reference substance solution and determination step, specifically include：
A, sample pre-treatments：Saved backup being placed in 1 ~ 4 DEG C after the grinding of measuring samples tobacco petal, freeze-drying；
The preparation of B, need testing solution：Sample after the accurate pre-treatment for weighing 10.0mg, adds volume ratio 5:2:2 methyl alcohol-
Chloroform-water extractant 1mL and μ the L of inner mark solution 200, inner mark solution is the aqueous solution of Daidzein, genistin and aurantiamarin,
Inner mark solution concentration be 1.0 μ g/mL, ultrasonic 30min, centrifugation after take supernatant as need testing solution；
The preparation of C, reference substance solution：The accurately weighed each n-compound of difference, i.e., 14 standard samples and 3 internal standard compound soya beans
Aglycon, genistin and aurantiamarin, plus methyl alcohol are made solution of every 1mL containing 1mg, obtain the flavonoids singly mark solution of 1mg/mL,
Pipette respectively 100 μ L 1mg/mL low concentration flavonoids singly mark solution and 10mL 1mg/mL high concentration flavonoids list mark it is molten
It is molten that liquid prepares standard mixing in 100mL volumetric flasks, with sample extraction solution, i.e. methanol-chloroform-water extraction solution constant volume
Liquid, with sample extraction solution stepwise dilution standard mixed solution, be made low concentration flavonoid substances concentration for 1000,600,400,
100th, 60,40,10,6,4,1ng/mL, volume is the standard liquid gradient of 1mL；The corresponding high standard solution ladder of this gradient
It is 100,60,40,10,6,4,1,0.6,0.4,0.1 μ g/mL to spend；Add 200 μ L's in the solution of above-mentioned all concentration gradients
Inner mark solution obtains reference substance solution, and inner mark solution is the aqueous solution of Daidzein, genistin and aurantiamarin, and concentration is 1.0 μ
g/mL；
D, measure：Carry out chromatograph mass spectrum analysis, chromatographiccondition：The Waters BEH of 15cm × 2.1mm, 1.7 μm of particle diameters
C18 chromatographic columns；A phases, water；B phases, acetonitrile, two-phase adds 0.1% formic acid and the ammonium acetate of 0.2mmol/L；Mobility gradient,
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, keeps 10%B；30 DEG C of column temperature, sample size 2 μ L, flow velocity 0.25mL/min；Mass spectral analysis condition：Electron spray ionisation ion
Source；Spray voltage -4000V；Curtain atmospheric pressure 40psi；700 DEG C of ion source temperature；Auxiliary gas 1 is 60psi；Auxiliary gas 2 be
50psi；Remove cluster voltage -100V；Compound quota ion and energy are shown in Table 1,
The flavonoid substances of table 1 analyze mass spectrum quota ion and collision energy
Content with 14 flavonoids to be measured as abscissa, with the color of its corresponding chromatographic peak area and internal standard compound
The ratio of spectral peak area is ordinate, sets up working curve, and linear regression is carried out to correction data, tries to achieve returning for flavonoid substances
Return equation, the content of flavonoid substances is calculated as the following formula in sample：
In formula：C is the content of flavonoid substances in tobacco petal sample, unit μ g/g；
The concentration of the need testing solution that x is measured for instrument.
2. 14 kinds of detection methods of flavonoid substances in quantitative analysis tobacco petal according to claim 1, its feature exists
Grinding described in step A is that measuring samples tobacco petal is put into mortar, and liquid feeding chilled nitrogen grinds.
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