CN106706788A - Qualitative and quantitative analysis method for flavonoid effective components in persimmon leaf extract - Google Patents
Qualitative and quantitative analysis method for flavonoid effective components in persimmon leaf extract Download PDFInfo
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Abstract
The invention discloses a qualitative and quantitative analysis method for flavonoid effective components in a persimmon leaf extract. A high-performance liquid chromatography-quadrupole time-of-flight mass spectrometer is adopted for detection. The invention further discloses a quantitative analysis method for the flavonoid effective components in the persimmon leaf extract. A high-performance liquid chromatography- triple quadrupole mass spectrometer is adopted for detection. Qualitative and quantitative analysis can be carried out on the flavonoid effective components in the persimmon leaf extract, so that fundamental research on effective materials in the persimmon leaf extract is facilitated; and meanwhile, a scientific basis is also provided for improvement of the quality standard of the persimmon leaf extract and effective monitoring of the quality of Chinese patent medicine products employing the persimmon leaf extract as a raw material. A quality evaluation system of the persimmon leaf extract is completed, and the qualitative and quantitative analysis method is stable, reliable, sensitive, accurate, short in detection period and simple in sample pretreatment, and can be used for quality monitoring of the persimmon leaf extract and a preparation thereof in practical production.
Description
Technical field
The present invention relates to Chinese medical extract analysis method technical field, more particularly to there be flavonoids in a kind of extractive from leaves of persimmon
Imitate the quantification and qualification method of composition.
Background technology
Persimmon leaf dries leaf for Ebenaceae plant persimmon Diospyros kaki Thunb.'s, and flavones is mainly contained in persimmon leaf
The chemical compositions such as class, terpene, phenolic acid class and aliphatic acid, so far, domestic and foreign literature is reported and one isolated from persimmon leaf plant
Obtain more than 90 compounds.Persimmon leaf has removes free radical, anti-oxidant, neuroprotection, antiallergy, inhibition thrombosis, anti-dynamic
The pharmacological actions such as pulse atherosclerosis, are mainly used in hypertension, cerebral arteriosclerosis, coronary heart disease, angina pectoris (Chinese herbal medicine, 2014,45
(21):3195-3203)。
At present, isolated 24 flavone compounds from persimmon leaf have been reported, the compound with Quercetin as aglycon point
It is not：Quercetin (quercetin), rutin (rutin), Quercetin -3-O- β-D- glucopyranosides
(isoquercitrin), Hyperoside (hyperoside) etc.；Compound with Kaempferol as aglycon is respectively：Kaempferol
(kampferol), Kaempferol-O- β-D- glucopyranosides (astragalin), Kaempferol-O- β-D- galactopyranoses
Glycosides (trifolin), Kaempferol-O- α-L- rhamnopyranosyloxyhies glucosides (kaempferol-3-O- α-L-rhamnopyranoside)
Deng；Compound with myricetin as aglycon is respectively：Myricetin (myricetin), annulatin, myricetin -3-O- α-L- pyrroles
Mutter rhamnoside (myricetin-3-O- α-L-rhamnopyranoside), myricetin -3-O- β-D- glucopyranosides
(myricetin-3-O- β-D-glucopyranoside) etc.；Compound with Vitexin as aglycon is respectively：Vitexin
(vitexin), 2 "-O- rhamnoses Vitexin (2 "-O-rhamnosyl vitexin) etc..
The quality standard of extractive from leaves of persimmon now record in《Chinese Pharmacopoeia》Version one in 2015, is reflected with thin-layer chromatography
Not, determination of total flavonoids is carried out with high performance liquid chromatography (HPLC) method.On the document report master that persimmon leaf component content is determined
Have：With rutin and ursolic acid as standard items, with spectrophotometry general flavone and total triterpene contentses；Surveyed using HPLC methods
It is fixed, quality × 2.59 of the quality × 2.55+ Kaempferols of the quality=Quercetin of general flavone；Wang Lifeng etc. establishes a kind of anti-phase
High performance liquid chromatography, while determining flavones (rutin, Hyperoside, Quercetin) and triterpene (oleanolic acid, black bearberry in persimmon leaf
Acid) compounds content (assay office, 2008,27 supplementary issues:176-179)；Fan etc. uses reversed-phased high performace liquid chromatographic, together
When determine persimmon leaf in 3 kinds of triterpenoid barbinervic acid, rotungenic acid and 24-hydroxy ursolic
Acid contents (Journal of Pharmaceutical and Biomedical Analysis, 2006,41:950-956)；
Liu Jun etc. uses RP-HPLC methods, using rutin, Kaempferol and Quercetin as reference substance, to flavonoids in 12 batches of persimmon leaf samples into
Point carry out finger-print research, determine that 12 peaks are total peak, account for total peak area more than 90% (CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2008,
33(6):694-695)。
At present, raw material of the extractive from leaves of persimmon mainly as Chinese patent drug NAOXINQING PIAN/capsule.Persimmon leaf is extracted in the domestic market
There are many families in the manufacturing enterprise of thing, and extracting method has various, and such as water extraction, alcohol extracting, buck carries acid precipitation method, each not phase of extraction process
Together, quality is uneven, in order to prevent the generation of the adverse events of similar ginkgo biloba p.e and its preparation, it is ensured that carried with persimmon leaf
Stable and controllable for quality, the clinical effective and safe of the related preparations that thing is raw material is taken, it is necessary to many to the flavonoids in extractive from leaves of persimmon
Individual composition carries out qualitative and quantitative analysis.
The content of the invention
Based on the technical problem that background technology is present, the present invention proposes flavonoids effective constituent in a kind of extractive from leaves of persimmon
Quantification and qualification method, the perfect quality of persimmon leaf extract appraisement system of the present invention, qualitative and quantitative analysis method is steady
Fixed reliable, sensitive and accurate, detection cycle is short, and sample pre-treatments are simple, can be used for extractive from leaves of persimmon and its system in actual production
The quality monitoring of agent.
The present inventor selects methanol-water, acetonitrile-water and the aqueous formic acid of acetonitrile -0.1% as flow phase system respectively,
Screening is compared, good separating effect of the extractive from leaves of persimmon in the formic acid water flow phase system of acetonitrile -0.1% is found, energy is eluted
Power is appropriate, and the elution time than methanol-water flow phase system is short, and the good separation between each chromatographic peak, background values
It is relatively low, make component to be measured that there is response higher, and a certain amount of formic acid for containing can improve the chromatographic peak of component to be measured
Peak type, therefore, from the aqueous formic acid of acetonitrile -0.1% as flow phase system.
The method for qualitative analysis of flavonoids effective constituent in a kind of extractive from leaves of persimmon proposed by the present invention, using efficient liquid phase
Chromatographic tandem quadrupole rod time of-flight mass spectrometer detects that the high-efficient liquid phase chromatogram condition is：Chromatographic column is Dikma
Diamonsil C18Chromatographic column, mobile phase A is acetonitrile, and Mobile phase B is that volume fraction is 0.09-0.11% aqueous formic acids, stream
Speed is 0.75-0.85mL/min, and column temperature is 20-30 DEG C, carries out gradient elution, and the gradient elution program is：In 0-10min,
The volume ratio of mobile phase A and Mobile phase B is 22：78；In 10-20min, the volume ratio of mobile phase A and Mobile phase B is from 22：78 is even
Fast gradual change is to 30：70；In 20-22min, the volume ratio of mobile phase A and Mobile phase B is from 30：70 at the uniform velocity gradual changes are to 45：55；22-
In 50min, the volume ratio of mobile phase A and Mobile phase B is from 45：55 at the uniform velocity gradual changes are to 90：10；
The Mass Spectrometry Conditions are：Ion gun is ESI ion guns, and detection mode is anionic textiles, and sweep limits is 50-
1500m/z, End Plate offset are -500V, and capillary voltage is -3800V, and atomization gas pressure is 1.2bar, dries gas
Flow is 8.0mL/min.
Preferably, in high-efficient liquid phase chromatogram condition, chromatographic column is Dikma Diamonsil C18(4.6×200mm,5μm)
Chromatographic column.
Preferably, in high-efficient liquid phase chromatogram condition, Mobile phase B is that volume fraction is 0.1% aqueous formic acid.
Preferably, in high-efficient liquid phase chromatogram condition, flow velocity is 0.8mL/min.
Preferably, in high-efficient liquid phase chromatogram condition, column temperature is 25 DEG C.
Preferably, in high-efficient liquid phase chromatogram condition, sample size is 5-20 μ L.
Preferably, in high-efficient liquid phase chromatogram condition, sample size can for 5,6,7,8,9,10,11,12,13,14,15,16,
17th, 18,19 or 20 μ L.
Preferably, in high-efficient liquid phase chromatogram condition, sample size is 10 μ L.
Preferably, it is concretely comprised the following steps：Qualitative use reference substance solution and qualitative need testing solution and sample introduction are prepared respectively,
Compared by the HPLC-UV detection and mass spectrogram with reference substance, flavonoids effective constituent in identification test sample, wherein, test sample
It is extractive from leaves of persimmon, reference substance is Vitexin, Hyperoside, astragalin, trifolin, myricetin, Quercetin and Kaempferol.
The compound method of above-mentioned solution is：
It is qualitative to use reference substance solution：Vitexin, Hyperoside, astragalin, trifolin, myricetin, Quercetin are taken respectively
It is appropriate with Kaempferol reference substance powder, it is placed in same volumetric flask, methyl alcohol dissolved dilution is used, 0.22 μm of miillpore filter is crossed, take continuous
Filtrate obtains qualitative using reference substance solution.
It is qualitative to use need testing solution：It is the molten of 0.1mg/ml that accurately weighed extractive from leaves of persimmon adds methyl alcohol dissolving to be configured to concentration
Liquid, crosses 0.22 μm of miillpore filter, takes subsequent filtrate and obtains qualitative using need testing solution.
According to the above method, qualitative analysis is carried out to flavonoids effective constituent in extractive from leaves of persimmon the results are shown in Table 1 and figure
1。
The high performance liquid chromatography of table 1 series connection quadrupole rod time of-flight mass spectrometer (HPLC-QTOF-MS) anion
Under detection mode in extractive from leaves of persimmon flavonoids effective constituent the qualitative analysis
Qualitative analysis discovery is carried out to flavonoids effective constituent in extractive from leaves of persimmon：In extractive from leaves of persimmon flavonoids effectively into
Dividing has Vitexin, Hyperoside, astragalin, trifolin, myricetin, Quercetin and Kaempferol.
After qualitative analysis being carried out to flavonoids effective constituent in extractive from leaves of persimmon, in addition it is also necessary to which flavonoids effective constituent is carried out
Quantitative analysis, to determine the content of each active ingredient of flavonoids in extractive from leaves of persimmon.
The invention allows for a kind of quantitative analysis method of flavonoids effective constituent in extractive from leaves of persimmon, using efficient liquid
The triple level Four bar mass spectrographs of phase chromatographic tandem detect that the high-efficient liquid phase chromatogram condition is：Chromatographic column is Dikma Diamonsil
C18Chromatographic column, mobile phase A is acetonitrile, and Mobile phase B is that volume fraction is 0.09-0.11% aqueous formic acids, and flow velocity is 0.75-
0.85mL/min, column temperature is 20-30 DEG C, carries out gradient elution, and the gradient elution program is：In 0-5min, mobile phase A and stream
The volume ratio of dynamic phase B is 18：82；In 5-10min, the volume ratio of mobile phase A and Mobile phase B is from 18：82 at the uniform velocity gradual changes are to 33：
67；In 10-15min, the volume ratio of mobile phase A and Mobile phase B is from 33：67 at the uniform velocity gradual changes are to 90：10；In 15-20min, flowing
The volume ratio of phase A and Mobile phase B is from 90：10 at the uniform velocity gradual changes are to 100：0；In 20-21min, the volume of mobile phase A and Mobile phase B
Than from 100：0 at the uniform velocity gradual change is to 18：82；In 21-23min, the volume ratio 18 of mobile phase A and Mobile phase B：82；
The Mass Spectrometry Conditions are：Ion gun is ESI ion guns, and detection mode is anionic textiles, and scan mode is multiple
Reaction monitoring, desolventizing temperature is 650 DEG C, and capillary voltage is -4500V, and spray pressure power is 50psi, goes the solvent gas to be
N2, N2Flow velocity is 20L/min.
Preferably, in high-efficient liquid phase chromatogram condition, chromatographic column is Dikma Diamonsil C18(4.6×200mm,5μm)
Chromatographic column.
Preferably, in high-efficient liquid phase chromatogram condition, Mobile phase B is that volume fraction is 0.1% aqueous formic acid.
Preferably, in high-efficient liquid phase chromatogram condition, flow velocity is 0.8mL/min.
Preferably, in high-efficient liquid phase chromatogram condition, column temperature is 25 DEG C.
Preferably, in high-efficient liquid phase chromatogram condition, sample size is 5-20 μ L.
Preferably, in high-efficient liquid phase chromatogram condition, sample size can for 5,6,7,8,9,10,11,12,13,14,15,16,
17th, 18,19 or 20 μ L.
Preferably, in high-efficient liquid phase chromatogram condition, sample size is 10 μ L.
Preferably, it is concretely comprised the following steps：Prepare quantitatively with reference substance solution, inner mark solution and molten quantitatively with test sample respectively
Liquid and sample introduction, the content of flavonoids effective constituent in regression equation calculation test sample is pressed by internal standard method, wherein, test sample is persimmon
Leaf extract, reference substance is Vitexin, Hyperoside, astragalin, trifolin, myricetin, Quercetin and Kaempferol, internal standard compound
It is Puerarin.
The compound method of above-mentioned solution is：
Inner mark solution：Accurately weighed Puerarin reference substance is appropriate, and it is in 5000ng/mL to be configured to concentration with methyl alcohol dissolving
Mark solution.
Quantitatively use reference substance solution：Vitexin, Hyperoside, astragalin, trifolin, myricetin, Quercetin are taken respectively
It is appropriate with Kaempferol reference substance powder that scale is dissolved and be diluted to methyl alcohol in 7 10mL measuring bottles, shake up, it is respectively prepared one
Determine the quantitative of concentration and use reference substance storing solution；Precision measures above-mentioned 7 and is quantitatively measured in 50mL with reference substance storing solution is appropriate respectively
In bottle, with methanol dilution to scale, shake up, be configured to every 1mL 500ng containing Vitexin, Hyperoside 10000ng, astragalin
6250ng, trifolin 6250ng, myricetin 500ng, Quercetin 1250ng, Kaempferol 6250ng quantitative use reference substance it is molten
Liquid.
Quantitatively use need testing solution：Accurately weighed extractive from leaves of persimmon 0.1g, puts in 100mL volumetric flasks, adds methyl alcohol abundant
Scale is diluted to after dissolving, precision measures 1.0mL in 10mL volumetric flasks, adds 1.0mL inner mark solutions, with methanol dilution to quarter
Degree, with 0.22 μm of filtering with microporous membrane, takes subsequent filtrate and is quantitatively used need testing solution.
Matter of the present inventor to high performance liquid chromatography triple level Four bar mass spectrum (HPLC-QQQ-MS/MS) GC-MSs of series connection
Spectral condition has carried out preferably, the scan mode of cation and anion having been investigated respectively.Result shows that most of components to be measured exist
It is corresponding stronger under the conditions of anion, it is easy to lose a proton, generate [M-H]-Quasi-molecular ion peak.Respectively to multiple reaction
Detect that the mass spectrometry parameters such as cluster voltage (DP) and collision induced dissociation voltage (CE) that go of the ion pair of (MRM) are optimized, most
This experiment Mass Spectrometry Conditions used are determined eventually.
The present inventor is to 7 reference substance Vitexins, Hyperoside, astragalin, trifolin, myricetin, Quercetin, Kaempferia galangas
The ion pair of the multiple ion reaction monitoring (MRM) of the anion reaction of phenol and internal standard compound Puerarin is removed cluster voltage (DP), touches
Hit induction solution ionization voltage (CE), exit potential (CXP) to optimize, the relevant parameter after optimization is shown in Table 2.
The polyion reaction monitoring ion pair and associated voltage parameter of the reference substance of table 2 and internal standard compound
Quantitative analysis method of the present inventor to flavonoids effective constituent in above-mentioned extractive from leaves of persimmon has carried out methodology and has tested
Card, comprises the following steps that：
Specificity is tested：By blank solvent methyl alcohol, quantitative use with reference substance solution, inner mark solution and quantitatively need testing solution
Sample introduction, records collection of illustrative plates, as a result sees Fig. 3-9, by Fig. 3-9 as can be seen that reference substance Vitexin, Hyperoside, astragalin, Russian red clover
The retention time of glycosides, myricetin, Quercetin, Kaempferol and internal standard compound Puerarin respectively may be about 14.02,14.48,15.17,
15.41st, 16.87,18.21,19.45,8.52min, blank solvent methyl alcohol not interference measurement, illustrates quantitative analysis method of the present invention
Selectivity it is good.
Linear test：Precision measures quantitative use reference substance solution 0.5,1.0,2.0,4.0,8.0,20.0mL, inner mark solution
2mL, is respectively placed in 50mL volumetric flasks, plus methanol dilution is to scale, shakes up, and is made into internal standard concentration for 200ngmL-1Series
Standard liquid, sample introduction and records collection of illustrative plates, with the concentration X (ng/mL) of each reference substance as abscissa, with each reference substance peak area successively
It is ordinate with internal standard compound peak area ratio Y, with weighting (1/X2) least square method carries out recurrence calculating, the linear regression tried to achieve
Equation and coefficient correlation, the results are shown in Table 3.
The equation of linear regression and coefficient correlation of each reference substance of table 3
As can be seen from Table 3, each reference substance is presented good linear pass under the conditions of quantitative analysis method of the present invention
System.
Precision test：Precision measures quantitative use reference substance solution 4.0mL and inner mark solution 2.0mL, is placed in 50mL measuring bottles
In, plus methanol dilution is to scale, repeats sample introduction 6 times and records collection of illustrative plates, statistics Vitexin, Hyperoside, astragalin, Russian red clover
Glycosides, myricetin, Quercetin, Kaempferol peak area respectively with the ratio of puerarin peak area, be calculated Vitexin, Hypericum Chinense
Glycosides, astragalin, trifolin, myricetin, Quercetin, Kaempferol respectively with the relative standard deviation of the peak area ratio of Puerarin
RSD is respectively 2.0%, 1.8%, 1.9%, 1.9%, 1.2%, 2.0% and 1.2%, meets Method validation requirement, shows instrument
The precision of device is good.
Replica test：Same extractive from leaves of persimmon is taken, 6 parts is configured to respectively and is quantitatively used need testing solution, sample introduction simultaneously to record
Collection of illustrative plates, by Vitexin, Hyperoside, astragalin, trifolin, myricetin, Quercetin in regression equation calculation extractive from leaves of persimmon
With the content of Kaempferol, count and obtain Vitexin, Hyperoside, astragalin, trifolin, myricetin, Quercetin and Kaempferol
Content relative standard deviation RSD be respectively 2.11%, 1.47%, 1.90%, 1.91%, 1.93%, 1.13% and 0.99%,
Meet Method validation requirement, show that the repeatability of quantitative analysis method of the present invention is good.
Stability test：Prepare and quantitatively use need testing solution, in 0 after preparation, 2,4,8,12 and 24h sample introductions and record figure
Spectrum, Vitexin, Hyperoside, astragalin, trifolin, myricetin, Quercetin and Kaempferol peak area point in statistics test sample
Not with the ratio of puerarin peak area, Vitexin, Hyperoside, astragalin, trifolin, red bayberry in test sample are calculated
Element, Quercetin, Kaempferol are respectively 1.9%, 2.1% with the relative standard deviation RSD of the peak area ratio of Puerarin respectively,
1.3%, 1.6%, 1.4%, 2.6%, 1.8%.Result shows that need testing solution is stable in 24h.
Average recovery is tested：Precision weighs known Vitexin, Hyperoside, astragalin, trifolin, myricetin, Mongolian oak
The extractive from leaves of persimmon of Pi Su and Kaempferol content, it is accurate respectively to add quantitatively with reference substance storing solution in right amount, add appropriate internal standard
Solution is prepared and obtains quantitative use need testing solution, and 6 parts are prepared altogether, sample introduction and records collection of illustrative plates successively, by regression equation calculation for trying
The content of Vitexin, Hyperoside, astragalin, trifolin, myricetin, Quercetin and Kaempferol in product, and the rate of recovery is calculated,
The results are shown in Table 4.
The average recovery (n=6) of each flavones ingredient in the extractive from leaves of persimmon of table 4
As can be seen from Table 4, the rate of recovery of quantitative analysis method of the present invention is good, and test sample preparation process does not influence Vitex negundo var cannabifolia
The content of element, Hyperoside, astragalin, trifolin, myricetin, Quercetin and Kaempferol.
Sample determination：Take extractive from leaves of persimmon preparation and quantitatively use need testing solution, sample introduction simultaneously records collection of illustrative plates, based on regression equation
The content of Vitexin, Hyperoside, astragalin, trifolin, myricetin, Quercetin and Kaempferol in test sample is calculated, is as a result seen
Table 5.
7 kinds of flavone component assays result (n=3) identified in the extractive from leaves of persimmon of table 5
As can be seen from Table 5, the content sequence of 7 kinds of Main Flavonoids effective constituents is in extractive from leaves of persimmon：Hyperoside>
Astragalin>Trifolin>Kaempferol>Quercetin>Myricetin>Vitexin, also reflects flavonoids in persimmon leaf to a certain extent
Composition is and less with myricetin aglycon and its glycosides based on Kaempferol, quercetin aglycon and its glycosides, consistent with document report.
Compared with HPLC technologies, HPLC/Q-TOF-MS (high performance liquid chromatography series connection quadrupole rod flight time mass spectrum) connection
With technology separation function it is strong, sensitivity is high, good resolution, analyze speed are fast, can provide the accurate average molecular matter of compound
Amount, effectively can carry out qualitative analysis to extractive from leaves of persimmon；HPLC-QQQ-MS/MS (the triple level Four bars of high performance liquid chromatography series connection
Mass spectrum) GC-MS has sensitivity higher and specificity, and being kept completely separate for chromatographic peak need not be reached, persimmon leaf can be carried
Multiple compounds are quickly and accurately quantitative in taking thing.It is qualitative fixed that the present invention can be carried out to flavonoids effective constituent in extractive from leaves of persimmon
Amount analysis, is conducive to the basic research to effective substance in extractive from leaves of persimmon, meanwhile, also it is the quality mark of raising extractive from leaves of persimmon
Accurate, effective monitoring provides scientific basis as the Chinese patent drug product quality of raw material.The perfect extractive from leaves of persimmon matter of the present invention
Amount appraisement system, qualitative and quantitative analysis method is reliable and stable, sensitive and accurate, and detection cycle is short, and sample pre-treatments are simple, can
For the quality monitoring of extractive from leaves of persimmon in actual production and its preparation.
Brief description of the drawings
Fig. 1 is the qualitative analysis chromatogram of extractive from leaves of persimmon.
Fig. 2 is the structural formula of reference substance and internal standard compound, wherein, a is Vitexin, b is Hyperoside, c is astragalin, d is
Trifolin, e are myricetin, f is Quercetin, g is Kaempferol, h is Puerarin.
Fig. 3 be quantitative analysis specificity experiment multiple ion reaction monitoring chromatogram, wherein, a be blank solvent methyl alcohol,
B is Vitexin, c is extractive from leaves of persimmon.
Fig. 4 be quantitative analysis specificity experiment multiple ion reaction monitoring chromatogram, wherein, a be blank solvent methyl alcohol,
B is Hyperoside, c is extractive from leaves of persimmon.
Fig. 5 be quantitative analysis specificity experiment multiple ion reaction monitoring chromatogram, wherein, a be blank solvent methyl alcohol,
B is astragalin, c is extractive from leaves of persimmon.
Fig. 6 be quantitative analysis specificity experiment multiple ion reaction monitoring chromatogram, wherein, a be blank solvent methyl alcohol,
B is trifolin, c is extractive from leaves of persimmon.
Fig. 7 be quantitative analysis specificity experiment multiple ion reaction monitoring chromatogram, wherein, a be blank solvent methyl alcohol,
B is myricetin, c is extractive from leaves of persimmon.
Fig. 8 be quantitative analysis specificity experiment multiple ion reaction monitoring chromatogram, wherein, a be blank solvent methyl alcohol,
B is Quercetin, c is extractive from leaves of persimmon.
Fig. 9 be quantitative analysis specificity experiment multiple ion reaction monitoring chromatogram, wherein, a be blank solvent methyl alcohol,
B is Kaempferol, c is extractive from leaves of persimmon.
Specific embodiment
Below, technical scheme is described in detail by specific embodiment.
Embodiment 1
The method for qualitative analysis of flavonoids effective constituent in a kind of extractive from leaves of persimmon, using high performance liquid chromatography series connection quadrupole
Bar time of-flight mass spectrometer detects that the high-efficient liquid phase chromatogram condition is：Chromatographic column is that chromatographic column is Dikma Diamonsil
C18(4.6 × 200mm, 5 μm) chromatographic column, mobile phase A is acetonitrile, and Mobile phase B is that volume fraction is 0.1% aqueous formic acid, stream
Speed is 0.8mL/min, and column temperature is 25 DEG C, and sample size is 10 μ L, carries out gradient elution, and the gradient elution program is：0-10min
Interior, the volume ratio of mobile phase A and Mobile phase B is 22：78；In 10-20min, the volume ratio of mobile phase A and Mobile phase B is from 22：78
At the uniform velocity gradual change is to 30：70；In 20-22min, the volume ratio of mobile phase A and Mobile phase B is from 30：70 at the uniform velocity gradual changes are to 45：55；22-
In 50min, the volume ratio of mobile phase A and Mobile phase B is from 45：55 at the uniform velocity gradual changes are to 90：10；
The Mass Spectrometry Conditions are：Ion gun is ESI ion guns, and detection mode is anionic textiles, and sweep limits is 50-
1500m/z, End Plate offset are -500V, and capillary voltage is -3800V, and atomization gas pressure is 1.2bar, dries gas
Flow is 8.0mL/min.
Solution is prepared：
It is qualitative to use reference substance solution：Vitexin, Hyperoside, astragalin, trifolin, myricetin, Quercetin are taken respectively
It is appropriate with Kaempferol reference substance powder, it is placed in same volumetric flask, methyl alcohol dissolved dilution is used, 0.22 μm of miillpore filter is crossed, take continuous
Filtrate obtains qualitative using reference substance solution.
It is qualitative to use need testing solution：It is the molten of 0.1mg/ml that accurately weighed extractive from leaves of persimmon adds methyl alcohol dissolving to be configured to concentration
Liquid, crosses 0.22 μm of miillpore filter, takes subsequent filtrate and obtains qualitative using need testing solution.
Specific steps：Prepare qualitative use reference substance solution and qualitative need testing solution and sample introduction respectively, by with compare
The HPLC-UV detection and mass spectrogram of product compare, flavonoids effective constituent in identification test sample.
Typical collection of illustrative plates is shown in Fig. 1.
Embodiment 2
The method for qualitative analysis of flavonoids effective constituent in a kind of extractive from leaves of persimmon, using high performance liquid chromatography series connection quadrupole
Bar time of-flight mass spectrometer detects that the high-efficient liquid phase chromatogram condition is：Chromatographic column is that chromatographic column is Dikma Diamonsil
C18(4.6 × 200mm, 5 μm) chromatographic column, mobile phase A is acetonitrile, and Mobile phase B is that volume fraction is 0.09% aqueous formic acid, stream
Speed is 0.85mL/min, and column temperature is 30 DEG C, and sample size is 5 μ L, carries out gradient elution, and the gradient elution program is with embodiment 1；
The Mass Spectrometry Conditions are with embodiment 1.
Solution is prepared with embodiment 1.
Specific steps are with embodiment 1.
Embodiment 3
The method for qualitative analysis of flavonoids effective constituent in a kind of extractive from leaves of persimmon, using high performance liquid chromatography series connection quadrupole
Bar time of-flight mass spectrometer detects that the high-efficient liquid phase chromatogram condition is：Chromatographic column is that chromatographic column is Dikma Diamonsil
C18(4.6 × 200mm, 5 μm) chromatographic column, mobile phase A is acetonitrile, and Mobile phase B is that volume fraction is 0.11% aqueous formic acid, stream
Speed is 0.75mL/min, and column temperature is 20 DEG C, and sample size is 20 μ L, carries out gradient elution, the same embodiment of gradient elution program
1；
The Mass Spectrometry Conditions are with embodiment 1.
Solution is prepared with embodiment 1.
Specific steps are with embodiment 1.
Embodiment 4
The quantitative analysis method of flavonoids effective constituent in a kind of extractive from leaves of persimmon, is connected triple using high performance liquid chromatography
Level Four bar mass spectrograph detects that the high-efficient liquid phase chromatogram condition is：Chromatographic column is Dikma DiamonsilC18(4.6×200mm,
5 μm) chromatographic column, mobile phase A is acetonitrile, and Mobile phase B is that volume fraction is 0.1% aqueous formic acid, and flow velocity is 0.8mL/min,
Column temperature is 25 DEG C, and sample size is 10 μ L, carries out gradient elution, and the gradient elution program is：In 0-5min, mobile phase A and stream
The volume ratio of dynamic phase B is 18：82；In 5-10min, the volume ratio of mobile phase A and Mobile phase B is from 18：82 at the uniform velocity gradual changes are to 33：
67；In 10-15min, the volume ratio of mobile phase A and Mobile phase B is from 33：67 at the uniform velocity gradual changes are to 90：10；In 15-20min, flowing
The volume ratio of phase A and Mobile phase B is from 90：10 at the uniform velocity gradual changes are to 100：0；In 20-21min, the volume of mobile phase A and Mobile phase B
Than from 100：0 at the uniform velocity gradual change is to 18：82；In 21-23min, the volume ratio 18 of mobile phase A and Mobile phase B：82；
The Mass Spectrometry Conditions are：Ion gun is ESI ion guns, and detection mode is anionic textiles, and scan mode is multiple
Reaction monitoring, desolventizing temperature is 650 DEG C, and capillary voltage is -4500V, and spray pressure power is 50psi, goes the solvent gas to be
N2, N2Flow velocity is 20L/min.
Solution is prepared：
Inner mark solution：Accurately weighed Puerarin reference substance is appropriate, and it is in 5000ng/mL to be configured to concentration with methyl alcohol dissolving
Mark solution.
Quantitatively use reference substance solution：Vitexin, Hyperoside, astragalin, trifolin, myricetin, Quercetin are taken respectively
It is appropriate with Kaempferol reference substance powder that scale is dissolved and be diluted to methyl alcohol in 7 10mL measuring bottles, shake up, it is respectively prepared one
Determine the quantitative of concentration and use reference substance storing solution；Precision measures above-mentioned 7 and is quantitatively measured in 50mL with reference substance storing solution is appropriate respectively
In bottle, with methanol dilution to scale, shake up, be configured to every 1mL 500ng containing Vitexin, Hyperoside 10000ng, astragalin
6250ng, trifolin 6250ng, myricetin 500ng, Quercetin 1250ng, Kaempferol 6250ng quantitative use reference substance it is molten
Liquid.
Quantitatively use need testing solution：Accurately weighed extractive from leaves of persimmon 0.1g, puts in 100mL volumetric flasks, adds methyl alcohol abundant
Scale is diluted to after dissolving, precision measures 1.0mL in 10mL volumetric flasks, adds 1.0mL inner mark solutions, with methanol dilution to quarter
Degree, with 0.22 μm of filtering with microporous membrane, takes subsequent filtrate and is quantitatively used need testing solution.
Concretely comprise the following steps：Prepare respectively and quantitatively use reference substance solution, inner mark solution and quantitative need testing solution and sample introduction,
The content of flavonoids effective constituent in regression equation calculation test sample is pressed by internal standard method, wherein, test sample is extractive from leaves of persimmon,
Reference substance is Vitexin, Hyperoside, astragalin, trifolin, myricetin, Quercetin and Kaempferol, and internal standard compound is Puerarin.
Testing result is shown in above-mentioned table 5, the content of Vitexin, Hyperoside, astragalin, trifolin, myricetin, Quercetin and Kaempferol
Respectively 0.06,20.10,12.00,11.10,0.09,4.46,9.51mg/g.
Embodiment 5
The quantitative analysis method of flavonoids effective constituent in a kind of extractive from leaves of persimmon, is connected triple using high performance liquid chromatography
Level Four bar mass spectrograph detects that the high-efficient liquid phase chromatogram condition is：Chromatographic column is Dikma DiamonsilC18(4.6×200mm,
5 μm) chromatographic column, mobile phase A is acetonitrile, and Mobile phase B is that volume fraction is 0.09% aqueous formic acid, and flow velocity is 0.85mL/
Min, column temperature is 30 DEG C, and sample size is 5 μ L, carries out gradient elution, and the gradient elution program is with embodiment 4；
The Mass Spectrometry Conditions are with embodiment 4.
Solution is prepared with embodiment 4.
Specific steps are with embodiment 4.
Embodiment 6
The quantitative analysis method of flavonoids effective constituent in a kind of extractive from leaves of persimmon, is connected triple using high performance liquid chromatography
Level Four bar mass spectrograph detects that the high-efficient liquid phase chromatogram condition is：Chromatographic column is Dikma DiamonsilC18(4.6×200mm,
5 μm) chromatographic column, mobile phase A is acetonitrile, and Mobile phase B is that volume fraction is 0.11% aqueous formic acid, and flow velocity is 0.75mL/
Min, column temperature is 30 DEG C, and sample size is 20 μ L, carries out gradient elution, and the gradient elution program is with embodiment 4；
The Mass Spectrometry Conditions are with embodiment 4.
Solution is prepared with embodiment 4.
Specific steps are with embodiment 4.
The above, the only present invention preferably specific embodiment, but protection scope of the present invention is not limited thereto,
Any one skilled in the art the invention discloses technical scope in, technology according to the present invention scheme and its
Inventive concept is subject to equivalent or change, should all be included within the scope of the present invention.
Claims (10)
1. in a kind of extractive from leaves of persimmon flavonoids effective constituent method for qualitative analysis, it is characterised in that use high-efficient liquid phase color
Spectrum series connection quadrupole rod time of-flight mass spectrometer detects that the high-efficient liquid phase chromatogram condition is：Chromatographic column is Dikma Diamonsil
C18Chromatographic column, mobile phase A is acetonitrile, and Mobile phase B is that volume fraction is 0.09-0.11% aqueous formic acids, and flow velocity is 0.75-
0.85mL/min, column temperature is 20-30 DEG C, carries out gradient elution, and the gradient elution program is：In 0-10min, mobile phase A and
The volume ratio of Mobile phase B is 22：78；In 10-20min, the volume ratio of mobile phase A and Mobile phase B is from 22：78 at the uniform velocity gradual change is extremely
30：70；In 20-22min, the volume ratio of mobile phase A and Mobile phase B is from 30：70 at the uniform velocity gradual changes are to 45：55；In 22-50min, stream
The volume ratio of dynamic phase A and Mobile phase B is from 45：55 at the uniform velocity gradual changes are to 90：10；
The Mass Spectrometry Conditions are：Ion gun is ESI ion guns, and detection mode is anionic textiles, and sweep limits is 50-1500m/
Z, End Plate offset are -500V, and capillary voltage is -3800V, and atomization gas pressure is 1.2bar, and dry gas stream amount is
8.0mL/min；
The flavonoids effective constituent is Vitexin, Hyperoside, astragalin, trifolin, myricetin, Quercetin and/or mountain
Naphthol.
2. according to claim 1 in extractive from leaves of persimmon flavonoids effective constituent method for qualitative analysis, it is characterised in that it is high
In effect liquid phase chromatogram condition, chromatographic column is Dikma Diamonsil C18(4.6 × 200mm, 5 μm) chromatographic column.
3. in extractive from leaves of persimmon according to claim 1 or claim 2 flavonoids effective constituent method for qualitative analysis, its feature exists
In in high-efficient liquid phase chromatogram condition, Mobile phase B is that volume fraction is 0.1% aqueous formic acid.
4. in the extractive from leaves of persimmon according to claim any one of 1-3 flavonoids effective constituent method for qualitative analysis, it is special
Levy and be, in high-efficient liquid phase chromatogram condition, flow velocity is 0.8mL/min；Preferably, in high-efficient liquid phase chromatogram condition, column temperature is 25
℃；Preferably, in high-efficient liquid phase chromatogram condition, sample size is 5-20 μ L；Preferably, in high-efficient liquid phase chromatogram condition, sample size
It is 10 μ L.
5. in the extractive from leaves of persimmon according to claim any one of 1-4 flavonoids effective constituent method for qualitative analysis, it is special
Levy and be, it is concretely comprised the following steps：Prepare qualitative use reference substance solution and qualitative need testing solution and sample introduction respectively, by with it is right
HPLC-UV detection and mass spectrogram according to product compare, flavonoids effective constituent in identification test sample, wherein, test sample is carried for persimmon leaf
Thing is taken, reference substance is Vitexin, Hyperoside, astragalin, trifolin, myricetin, Quercetin and Kaempferol.
6. in a kind of extractive from leaves of persimmon flavonoids effective constituent quantitative analysis method, it is characterised in that use high-efficient liquid phase color
The triple level Four bar mass spectrographs of spectrum series connection detect that the high-efficient liquid phase chromatogram condition is：Chromatographic column is Dikma Diamonsil C18
Chromatographic column, mobile phase A is acetonitrile, and Mobile phase B is that volume fraction is 0.09-0.11% aqueous formic acids, and flow velocity is 0.75-
0.85mL/min, column temperature is 20-30 DEG C, carries out gradient elution, and the gradient elution program is：In 0-5min, mobile phase A and stream
The volume ratio of dynamic phase B is 18：82；In 5-10min, the volume ratio of mobile phase A and Mobile phase B is from 18：82 at the uniform velocity gradual changes are to 33：
67；In 10-15min, the volume ratio of mobile phase A and Mobile phase B is from 33：67 at the uniform velocity gradual changes are to 90：10；In 15-20min, flowing
The volume ratio of phase A and Mobile phase B is from 90：10 at the uniform velocity gradual changes are to 100：0；In 20-21min, the volume of mobile phase A and Mobile phase B
Than from 100：0 at the uniform velocity gradual change is to 18：82；In 21-23min, the volume ratio 18 of mobile phase A and Mobile phase B：82；
The Mass Spectrometry Conditions are：Ion gun is ESI ion guns, and detection mode is anionic textiles, and scan mode is multiple reaction
Monitoring, desolventizing temperature is 650 DEG C, and capillary voltage is -4500V, and spray pressure power is 50psi, and it is N to remove solvent gas2, N2
Flow velocity is 20L/min；
The flavonoids effective constituent is Vitexin, Hyperoside, astragalin, trifolin, myricetin, Quercetin and/or mountain
Naphthol.
7. according to claim 6 in extractive from leaves of persimmon flavonoids effective constituent quantitative analysis method, it is characterised in that it is high
In effect liquid phase chromatogram condition, chromatographic column is Dikma Diamonsil C18(4.6 × 200mm, 5 μm) chromatographic column.
8. in the extractive from leaves of persimmon according to claim 6 or 7 flavonoids effective constituent quantitative analysis method, its feature exists
In in high-efficient liquid phase chromatogram condition, Mobile phase B is that volume fraction is 0.1% aqueous formic acid.
9. in the extractive from leaves of persimmon according to claim any one of 6-8 flavonoids effective constituent quantitative analysis method, it is special
Levy and be, in high-efficient liquid phase chromatogram condition, flow velocity is 0.8mL/min；Preferably, in high-efficient liquid phase chromatogram condition, column temperature is 25
℃；Preferably, in high-efficient liquid phase chromatogram condition, sample size is 5-20 μ L；Preferably, in high-efficient liquid phase chromatogram condition, sample size
It is 10 μ L.
10. in the extractive from leaves of persimmon according to claim any one of 6-9 flavonoids effective constituent quantitative analysis method, it is special
Levy and be, it is concretely comprised the following steps：Quantitative reference substance solution, inner mark solution are prepared respectively and is quantified gone forward side by side with need testing solution
Sample, the content of flavonoids effective constituent in regression equation calculation test sample is pressed by internal standard method, wherein, test sample is that persimmon leaf is extracted
Thing, reference substance is Vitexin, Hyperoside, astragalin, trifolin, myricetin, Quercetin and Kaempferol, and internal standard compound is the root of kudzu vine
Element.
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