CN105954450A - HPLC fingerprint spectrum of pouzolzia zeylanica var. microphylla medicinal material or general flavonoids of pouzolzia zeylanica var. microphylla and building method and application of HPLC fingerprint spectrum - Google Patents
HPLC fingerprint spectrum of pouzolzia zeylanica var. microphylla medicinal material or general flavonoids of pouzolzia zeylanica var. microphylla and building method and application of HPLC fingerprint spectrum Download PDFInfo
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- CN105954450A CN105954450A CN201610171641.2A CN201610171641A CN105954450A CN 105954450 A CN105954450 A CN 105954450A CN 201610171641 A CN201610171641 A CN 201610171641A CN 105954450 A CN105954450 A CN 105954450A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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Abstract
The invention relates to the technical field of detection of a traditional Chinese medicinal material, and particularly discloses an HPLC fingerprint spectrum of a pouzolzia zeylanica var. microphylla medicinal material or general flavonoids of pouzolzia zeylanica var. microphylla and a building method and application of the HPLC fingerprint spectrum. The building method comprises the following steps: S1, precisely weighing a pouzolzia zeylanica var. microphylla medicinal material to prepare a to-be-tested solution; and S2, enabling the to-be-tested solution to be subjected to high performance liquid chromatography to obtain a fingerprint spectrum. The fingerprint spectrum can overall reflect information of characteristic peaks of a reaction sample, and the method is good in stability and reproducibility. The fingerprint spectrum can be used for true and false or quality identification of the pouzolzia zeylanica var. microphylla medicinal material or general flavonoids of pouzolzia zeylanica var. microphylla.
Description
Technical field
The present invention relates to Chinese crude drug detection technique field, be specifically related to a kind of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material or racemosus mist
The HPLC finger printing of water Pueraria lobota total flavones and method for building up thereof and application.
Background technology
Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang (formal name used at school: Pouzolzia zeylanica var.microphylla) is Urticaceae Pouzolzia
Under a mutation.Tropical Asian area blazons, and originates in the Chinese yunnan southeast, Guangxi, Guangdong, Fujian,
South Jiangxi, Taiwan；There is detoxicating, relieving inflammation effect；Swell for treating furuncle inflammation.Flavones ingredient is racemosus mist
One of effective ingredient of water Pueraria lobota.At present, lack a kind of accurate detection Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material true and false and quality,
And the method for Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones quality.
Chinese medicine fingerprint refer to certain (or certain place of production) Chinese crude drug or Chinese patent medicine appropriately processed after, adopt
With certain analysis means, the figure at the total peak that can indicate this Chinese crude drug or Chinese patent medicine medicinal property obtained
Spectrum.Finger printing goes out from traditional Chinese medical science organic conception and herbal medicine efficacy from the viewpoint of multiple chemical substance comprehensive function
Send out, it is possible to set up more objective, the overall and overall evaluation system of multi objective, and by the overall spy of collection of illustrative plates
Levy, it is possible to than more fully reflecting chemical composition contained by Chinese medicine.
Summary of the invention
The technical problem to be solved is, in order to overcome above-mentioned deficiency present in prior art, it is provided that
A kind of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material or the HPLC finger printing of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones.
Above-mentioned technical problem to be solved by this invention, is achieved by the following technical programs:
A kind of method for building up of the HPLC finger printing of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material or Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones, bag
Containing following steps:
S1. precision weighs Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material, prepares need testing solution；
S2. need testing solution is used high performance liquid chromatograph analysis, obtains finger printing.
Preferably, described need testing solution is prepared by the method comprised the steps of:
S11. weigh Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material, add 5～15 times amount (ml/g) petroleum ether reflux, extract, 1～5 times,
Each 0.5～3h, filter, discard petroleum ether extract, obtain filtering residue；
S12. by filtering residue 5～15 times amount (ml/g) alcohol reflux 1～5 times, each 0.5～3h, filter,
Merging filtrate, is concentrated into without alcohol taste, is dissolved in water, is centrifuged, and takes supernatant and adds water adjustment concentration to 0.1～0.5g
Crude drug/ml also regulates pH to 4.5～5.5, obtains sample solution；
S13. the sample solution that aspiration step S12 prepares, is enriched with total flavones by AB-8 macroporous resin column,
Loading flow velocity 0.5～3BV/h, after loading, with the water elution remove impurity of 1～5BV, with 2～5BV volumes
Mark be 50～70% ethanol with 1～3BV/h flow velocity eluting, collect ethanol elution, with filter membrane filter,
Obtain need testing solution.
Preferably, the method for building up of described HPLC finger printing, any in following A～M item
One or multinomial:
A., in step S11., 8～12 times amount (such as 8,9,10,11 or 12 times amount) petroleum ether is added；
B. in step S11., reflux, extract, 2～4 times (such as 2,3 or 4 times)；
C., in step S11., the time of each reflux, extract, is 0.5～2h (such as 0.5,1,1.5 or 2h)；
D., in step S12., 8～12 times amount (such as 8,9,10,11 or 12 times amount) ethanol is added；
E. in step S12., the volume fraction of ethanol be 50～95% (such as 50%, 60%, 70%, 80%,
90% or 95%)；
F. in step S12., reflux, extract, 2～4 times (such as 2,3 or 4 times)；
G., in step S12., the time of each reflux, extract, is 0.5～2h (such as 0.5,1,1.5 or 2h)；
H. in step S12., the centrifugal time is 5～50min, and (the such as time is for rotating speed 1500～5000rpm
5～20min, rotating speed is 2000～4000rpm；Or the time is 10min, rotating speed is 3000rpm)；
I., in step S12., take supernatant and add water adjustment concentration to 0.2～0.4g crude drug/ml (such as 0.2,0.3
Or 0.4g crude drug/ml)；
J., in step S12., pH to 4.5,4.8,5.0,5.2 or 5.5 are regulated.
K., in step S13., loading flow velocity is 1～3BV/h (such as 1BV/h, 2BV/h or 3BV/h)；
L. in step S13., with the water elution remove impurity of 2～4BV (such as 2BV, 3BV or 4BV)；
M. in step S13., with 2～4BV (such as 2BV, 3BV or 4BV) volume fraction be 50%,
The ethanol of 60% or 70% is with the flow velocity eluting of 1BV/h or 1BV/h.
Above-mentioned times amount represents addition petroleum ether or the milliliter number of ethanol in every gram of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material.
Above-mentioned unit 1BV is volume unit, represents that volume is equivalent to 1 times of macroporous resin column volume.
Preferably, in step S13. before being enriched with total flavones by AB-8 macroporous resin column, accurate aspiration step S12
The sample solution prepared；Before filtering with filter membrane, ethanol elution will be collected, be 50～70% by volume fraction
The ethanol constant volume of (such as 50%, 60% or 70%).
Preferably, in step S2, the chromatographic condition of high performance liquid chromatograph analysis is: use ACQUITY
UPLC BEH SHIELD RP18 chromatographic column；Gradient elution is carried out mutually for flowing with acetonitrile-0.2% formic acid water；
Flow velocity is 0.2～0.5mL/min, and detection wavelength is 30～330nm；Sample size is 0.2～10 μ l；
Phase gradient elution requirement is: 0～22min, and the volume fraction of acetonitrile is changed to 15%～19%；22～26min,
The volume fraction of acetonitrile is changed to 19%～24%；26～32min, the volume fraction of acetonitrile is changed to 24%～24%；
32～40min, the volume fraction of acetonitrile is changed to 24%～38%.
It is highly preferred that the chromatographic condition of high performance liquid chromatograph analysis is in step S2: employing specification is
21mm × 150mm, the ACQUITY UPLC BEH SHIELD RP18 chromatographic column of 1.7 μm；With acetonitrile
-0.2% formic acid water carries out gradient elution mutually for flowing；Flow velocity is 0.3mL/min, and detection wavelength is 330nm；
Sample size is 0.2 μ l；
Phase gradient elution requirement is: 0～22min, and the volume fraction of acetonitrile is changed to 15%～19%；22～26min,
The volume fraction of acetonitrile is changed to 19%～24%；26～32min, the volume fraction of acetonitrile is changed to 24%～24%；
32～40min, the volume fraction of acetonitrile is changed to 24%～38%.
Above-mentioned 0.2% formic acid water meter shows that containing volume fraction in water is the formic acid of 0.2%.
The present invention also provides for the HPLC finger printing of a kind of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material or Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones,
It is to be set up by the method for building up described in any of the above-described item to obtain.
The present invention a kind of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material or the standard HPLC finger printing of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones,
Any one in (1)～(3):
(1) single batch or multiple batches of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang genuine medicinal materials are used and according to described in any of the above-described item
The Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang HPLC-FPS that method for building up obtains；
(2) multiple batches of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang genuine medicinal materials will be used and according to the foundation side described in any of the above-described item
The HPLC finger printing that method obtains makes comparison spectrogram by average method or median method；
(3) it has 11 characteristic peaks, and retention time is respectively 6.814 ± 0.05min, 11.737 ± 0.05min,
12.307 ± 0.05min, 12.987 ± 0.05min, 18.821 ± 0.05min, 19.888 ± 0.05min,
21.115 ± 0.05min, 24.684 ± 0.05min, 26.924 ± 0.05min, 27.643 ± 0.05min and
30.297±0.05min。
The method provided according to the present invention, determines Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material, it is determined that above-mentioned 11 characteristic peaks.
The present invention also provides for a kind of side detecting Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang quality of medicinal material or Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones quality
Method, comprises the steps: the method for building up of first HPLC finger printing as described in any of the above-described item, sets up
Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material to be detected or the HPLC finger printing of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones；Then with above-mentioned racemosus
The standard HPLC finger printing of Herba Pouzolziae Zeylanicae medical material or Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones compares, and finally judges racemosus
The quality of Herba Pouzolziae Zeylanicae medical material or Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones is the most qualified.
Described judgement can judge according to the similarity of finger printing, if the similarity of the two finger printing is not
Less than 0.85, then it is up-to-standard；If less than 0.85, being then defective；Described similarity is by country's medicine
The fingerprint similarity evaluation software that prison office promulgates obtains.
The present invention also provides for a kind of above-mentioned Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material or the HPLC fingerprint of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones
Collection of illustrative plates, or the standard HPLC finger printing of above-mentioned Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material or Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones is in discriminating
Application in Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material or the true and false of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones or quality.
Beneficial effect: (1) present invention establishes Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material or Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones first
The standard HPLC finger printing of HPLC finger printing and Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material or Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones；
(2) this finger printing and standard finger-print can the characteristic peak information of more fully response sample, and should
Method stability and repeatability are preferable；(3) utilize this finger printing and the standard finger-print can be to racemosus mist
Water Pueraria lobota medical material or Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones carry out the true and false or quality discernment.
Accompanying drawing explanation
Fig. 1 is the HPLC standard finger-print of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material or Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones.
Fig. 2 is the precision of the HPLC standard finger-print of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material or Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones
Test Drawing.
Fig. 3 is the repeatability of the HPLC standard finger-print of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material or Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones
Lab diagram.
Fig. 4 is the stability of the HPLC standard finger-print of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material or Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones
Experiment.
Fig. 5 is HPLC standard finger-print and the liquid-matter of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material or Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones
(composing) combination total ion current figure comparison diagram；Wherein A is total ion current figure, and B is fingerprint image spectrogram.
Detailed description of the invention
The present invention is explained further below in conjunction with specific embodiment, but the present invention is not done any form by embodiment
Restriction.
Unreceipted actual conditions person in embodiment, the condition advised according to normal condition or manufacturer is carried out.Wherein,
Agents useful for same or instrument unreceipted production firm person, be can by city available from conventional products.
The foundation of the HPLC finger printing of embodiment 1 Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material or Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones
1, instrument and reagent
Waters SYNAPT G2-S Q-TOF mass spectrograph (Waters, US)
ACQUITY UPLC type Ultra Performance Liquid Chromatography instrument (waters company of the U.S.)
ACQUITY UPLC BEH SHIELD RP18 chromatographic column (21mm × 150mm, 1.7 μm)
AY120 type pallet electronic analytical balance (Shimadzu Corporation of Japan)
DK-S24 type electric-heated thermostatic water bath (above Nereid's grand experimental facilities company limited)
LABOROTA4000 Rotary Evaporators (Germany Heidolph).
Acetonitrile is chromatographically pure (Ou Pusen board), and liquid phase water is Watson distilled water, and ethanol is industrial alcohol,
Other reagent is analytical pure.
Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material is purchased from Guangxi Yulin, identifies being Urticaceae mist through Guangdong Pharmaceutical University professor Liu Jizhu
Water Pueraria lobota belongs to Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang Pouzolzia zeylanica (L.) Benn.var.microphylla (Wedd.) W.T.
The herb of Wang.
2, experimental technique
The preparation of 2.1 need testing solutions
Take Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material to pulverize, weigh medicinal material coarse powder 25g, be placed in round-bottomed flask, add respectively
Enter 80 DEG C of reflux, extract, of 8 times amount petroleum ether 2 times, each 1h.After filtering residue flings to petroleum ether, add 8 times amount 70%
Alcohol reflux 3 times, each 1h, merge 3 filtrates, be evaporated to without alcohol taste, add water and make fully to dissolve,
Centrifugal (3000r/min) 10min, takes the supernatant adjustment concentration that adds water and to 0.3g crude drug/ml and regulates pH to 5.
The accurate above-mentioned sample solution of 75ml of drawing is added in AB-8 macroporous resin column (Φ 1.5 × 8cm), loading flow velocity 2BV/h,
Add water 2BV remove impurity, adds 60% ethanol 2BV with 2BV/h eluting, collects eluent, add 60% ethanol and be settled to
In 100ml volumetric flask, shaking up, 0.22 μm filter membrane filters, and to obtain final product.Described ethanol percentage composition is volume
Percentage composition.
2.2 chromatographic condition
Employing specification is 21mm × 150mm, the ACQUITY UPLC BEH SHIELD RP18 of 1.7 μm
Chromatographic column；Gradient elution is carried out mutually for flowing with acetonitrile-0.2 (volume) % formic acid water；Flow velocity is 0.3mL/min,
Detection wavelength is 330nm；Sample size is 0.2 μ l；
Phase gradient elution requirement is: 0～22min, and the volume fraction of acetonitrile is changed to 15%～19%；22～26min,
The volume fraction of acetonitrile is changed to 19%～24%；26～32min, the volume fraction of acetonitrile is changed to 24%～24%；
32～40min, the volume fraction of acetonitrile is changed to 24%～38%.
By need testing solution, detect according to the chromatographic condition described in 2.2, the Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material or many obtained
The HPLC standard finger-print of branch Herba Pouzolziae Zeylanicae total flavones is as shown in Figure 1.Fig. 1 shows that this finger printing has 11
Individual characteristic peak, retention time is respectively 6.814min, 11.737min, 12.307min, 12.987min, 18.821
Min, 19.888min, 21.115min, 24.684min, 26.924min, 27.643min and 30.297min.
3, methodological study
3.1 Precision Experiment
Precision weighing medicinal material coarse powder 25g, prepares need testing solution by the method under 2.1, by the chromatograph of 2.2
Condition is measured, continuous sample introduction 6 times, and the RSD of the relative peak area of each chromatographic peak is less than 2.0%, relatively
Retention time RSD is less than 2.0%, calculates the similarity of finger printing by 2.4 lower methods, respectively 0.993,
0.996,0.992,0.997,0.995,0.996 (see Fig. 2), shows that instrument precision is good.
3.2 repeated experiment
Precision weighing medicinal material coarse powder 25g, prepares need testing solution 6 parts, by 2.2 by the method under 2.1 is parallel
Chromatographic condition be measured, result shows, the RSD of the relative retention time of each chromatographic peak is respectively less than 7%,
The RSD of relative peak area is respectively less than 5%, calculates the similarity of finger printing by 2.4 lower methods, is respectively
0.997,0.998,0.998,0.995,0.997 (see Fig. 3), illustrates that the repeatability of the method is good.
3.3 stability experiment
Precision weighing medicinal material coarse powder 25g, by the parallel need testing solution of preparing of the method under 2.1, ambient temperatare
Putting, when 0,2,4,12,24, chromatographic condition by 2.2 is measured respectively, the relative guarantor of each chromatographic peak
Stay the RSD of time less than 3%；The RSD of relative peak area is less than 5%, calculates by 2.4 lower methods and refers to
The similarity of stricture of vagina collection of illustrative plates, respectively 0.993,0.987,0.997,0.994,0.997,0.996 (see Fig. 4),
Show that need testing solution is stable in 24 hours.
The HPLC Fingerprints peak of embodiment 2 Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material or Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones
Identify
Need testing solution preparation is with reference to 2.1 in embodiment 1.
Chromatographic condition is with reference to 2.2 in embodiment 1.
Prepared by reference substance solution: precision weighs 6,7-dimethyl-1,4-dihydro-2,3-Quinoxalinediones, apigenin
-6-C-a-L-arabinose 8-C-β-D-Glucose glycosides, apigenin-6-C-[α-L-arabinose (1 → 6)-β-D-Portugal
Grape sugar], kaempferol-3-O-[α-L-rhamnose (1 → 6)-β-D-Glucose]-7-O-α-L-glucoside, Quercetin
-3-O-[α-L-rhamnose (1 → 6)-β-D-Glucose]-7-O-alpha-L-rhamnoside, Quercetin-3,7-2-O-α-L-pyrrole
Mutter rhamnoside, kaempferol-3-O-[α-L-rhamnose (1 → 6)-β-D-Glucose]-7-O-alpha-L-rhamnoside and mountain
How phenol-3,7-2-O-α-L-rhamnopyranosyloxyhy glucosides reference substance is appropriate, dissolves by proper amount of methanol and is configured to reference substance solution,
Obtain.
Mass Spectrometer Method condition: electric spray ion source (ESI), negative ion mode；Capillary voltage (capillary
voltage)3000V；Collision voltage (collision, CE) is 30～40V；Taper hole voltage (sampling cone)
40V；Removing solvent temperature (TEM) is 350 DEG C；Nitrogen is assistant spray ionization and removes solvent gas, goes
Solvent gas volume flow is 800L/h；Taper hole air-flow cone gas flow) it is 50L/h.
By UPLC-ESI-Q-TOF-MS detection obtain each chemical composition in Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material always from
Subflow figure and fingerprint image spectrogram (see Fig. 5), and combine extraction ion flow graph and and the reference substance of each chemical composition
In finger printing 11 characteristic peaks are carried out further chemical composition by chromatogram, pertinent literature Data Comparison
Confirm, the results are shown in Table 1.
The discriminating of table 1 chromatographic peak and qualification
Table 1 result shows, contains in Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang in the finger printing of the present invention and standard finger-print
Known multiple compounds, have the reliability of height.
Embodiment 3 utilizes the method for the standard finger-print detection Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material true and false
The most a collection of random Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material detects its true and false.
First the method for building up of the HPLC finger printing as described in embodiment 1, sets up Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang to be detected
The HPLC finger printing of medical material；Then with the Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material obtained by the foundation of embodiment 1 method
HPLC standard finger-print compares；The fingerprint similarity evaluation software promulgated by Bureau of Drugs Supervision of country
The similarity of the HPLC finger printing and standard HPLC finger printing that record this batch of medical material is 0.9, can sentence
Fixed, this batch of medical material is qualified Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material.
Claims (10)
1. a method for building up for the HPLC finger printing of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material or Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones,
It is characterized in that, comprise the steps of:
S1. precision weighs Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material, prepares need testing solution；
S2. need testing solution is used high performance liquid chromatograph analysis, obtains finger printing.
Method for building up the most according to claim 1, it is characterised in that described need testing solution passes through
The method comprised the steps of prepares:
S11. weigh Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material, add 5～15 times amount (ml/g) petroleum ether reflux, extract, 1～5 times,
Each 0.5～3h, filter, discard petroleum ether extract, obtain filtering residue；
S12. by filtering residue 5～15 times amount (ml/g) alcohol reflux 1～5 times, each 0.5～3h, filter,
Merging filtrate, is concentrated into without alcohol taste, is dissolved in water, is centrifuged, and takes supernatant and adds water adjustment concentration to 0.1～0.5g
Crude drug/ml also regulates pH to 4.5～5.5, obtains sample solution；
S13. the sample solution that aspiration step S12 prepares, is enriched with total flavones by AB-8 macroporous resin column,
Loading flow velocity 0.5～3BV/h, after loading, with the water elution remove impurity of 1～5BV, with 2～5BV volumes
Mark be 50～70% ethanol with 1～3BV/h flow velocity eluting, collect ethanol elution, with filter membrane filter,
Obtain need testing solution.
The method for building up of HPLC finger printing the most according to claim 2, it is characterised in that be selected from
The following any one in A～M item or multinomial:
A., in step S11., 8～12 times amount (such as 8,9,10,11 or 12 times amount) petroleum ether is added；
B. in step S11., reflux, extract, 2～4 times (such as 2,3 or 4 times)；
C., in step S11., the time of each reflux, extract, is 0.5～2h (such as 0.5,1,1.5 or 2h)；
D., in step S12., 8～12 times amount (such as 8,9,10,11 or 12 times amount) ethanol is added；
E. in step S12., the volume fraction of ethanol be 50～95% (such as 50%, 60%, 70%, 80%,
90% or 95%)；
F. in step S12., reflux, extract, 2～4 times (such as 2,3 or 4 times)；
G., in step S12., the time of each reflux, extract, is 0.5～2h (such as 0.5,1,1.5 or 2h)；
H. in step S12., the centrifugal time is 5～50min, and (the such as time is for rotating speed 1500～5000rpm
5～20min, rotating speed is 2000～4000rpm；Or the time is 10min, rotating speed is 3000rpm)；
I., in step S12., take supernatant and add water adjustment concentration to 0.2～0.4g crude drug/ml (such as 0.2,0.3
Or 0.4g crude drug/ml)；
J., in step S12., pH to 4.5,4.8,5.0,5.2 or 5.5 are regulated.
K., in step S13., loading flow velocity is 1～3BV/h (such as 1BV/h, 2BV/h or 3BV/h)；
L. in step S13., with the water elution remove impurity of 2～4BV (such as 2BV, 3BV or 4BV)；
M. in step S13., with 2～4BV (such as 2BV, 3BV or 4BV) volume fraction be 50%,
The ethanol of 60% or 70% is with the flow velocity eluting of 1BV/h or 1BV/h.
The method for building up of HPLC finger printing the most according to claim 2, it is characterised in that step
S13. in before being enriched with total flavones by AB-8 macroporous resin column, the sample that accurate aspiration step S12 prepares
Solution；With filter membrane filter before, ethanol elution will be collected, with volume fraction be 50～70% (such as 50%,
60% or 70%) ethanol constant volume.
The method for building up of HPLC finger printing the most according to claim 2, it is characterised in that step
In S2, the chromatographic condition of high performance liquid chromatograph analysis is: use ACQUITY UPLC BEH SHIELD
RP18 chromatographic column；Gradient elution is carried out mutually for flowing with acetonitrile-0.2% formic acid water；Flow velocity is 0.2～0.5mL/min,
Detection wavelength is 30～330nm；Sample size is 0.2～10 μ l；
Phase gradient elution requirement is: 0～22min, and the volume fraction of acetonitrile is changed to 15%～19%；22～26min,
The volume fraction of acetonitrile is changed to 19%～24%；26～32min, the volume fraction of acetonitrile is changed to 24%～24%；
32～40min, the volume fraction of acetonitrile is changed to 24%～38%.
The method for building up of HPLC finger printing the most according to claim 5, it is characterised in that step
In S2, the chromatographic condition of high performance liquid chromatograph analysis is: employing specification is 21mm × 150mm, 1.7 μm
ACQUITY UPLC BEH SHIELD RP18 chromatographic column；Carry out mutually with acetonitrile-0.2% formic acid water for flowing
Gradient elution；Flow velocity is 0.3mL/min, and detection wavelength is 330nm；Sample size is 0.2 μ l；
Phase gradient elution requirement is: 0～22min, and the volume fraction of acetonitrile is changed to 15%～19%；22～26min,
The volume fraction of acetonitrile is changed to 19%～24%；26～32min, the volume fraction of acetonitrile is changed to 24%～24%；
32～40min, the volume fraction of acetonitrile is changed to 24%～38%.
7. a HPLC finger printing for Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material or Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones, its feature exists
In, set up by the method for building up described in any one of claim 1～6 and obtain.
8. a standard HPLC finger printing for Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material or Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones, it is special
Levy and be, any one in (1)～(3):
(1) single batch or multiple batches of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang genuine medicinal materials are used and according in claim 1 to 6
The Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang HPLC-FPS that method for building up described in any one obtains；
(2) multiple batches of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang genuine medicinal materials will be used and according to any one of claim 1 to 6
The HPLC finger printing that described method for building up obtains makes comparison spectrum by average method or median method
Figure；
(3) it has 11 characteristic peaks, and retention time is respectively 6.814 ± 0.05min, 11.737 ± 0.05min,
12.307 ± 0.05min, 12.987 ± 0.05min, 18.821 ± 0.05min, 19.888 ± 0.05min,
21.115 ± 0.05min, 24.684 ± 0.05min, 26.924 ± 0.05min, 27.643 ± 0.05min and
30.297±0.05min。
9. detect Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang quality of medicinal material or a method for Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones quality, its feature
It is, comprises the steps: the foundation of first HPLC finger printing as described in any one of claim 1～6
Method, sets up the HPLC finger printing of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material to be detected or Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones；Then
With the Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material described in claim 8 or the standard HPLC finger printing of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones
Compare, finally judge that the quality of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material or Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones is the most qualified.
10. the Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material described in claim 7 or the HPLC fingerprint image of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones
Spectrum, or the Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material described in claim 8 or the standard HPLC fingerprint of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones
Collection of illustrative plates application in differentiating Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang medical material or the true and false of Pouzolzia zeylanica (L.) Benn. Var. Micro-nhylla (Wedd) W. T. Wang total flavones or quality.
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