CN107132292B - Towards the method for building up and its finger-print of medicine obovateleaf ainsliaea herb finger-print - Google Patents
Towards the method for building up and its finger-print of medicine obovateleaf ainsliaea herb finger-print Download PDFInfo
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- CN107132292B CN107132292B CN201710277566.2A CN201710277566A CN107132292B CN 107132292 B CN107132292 B CN 107132292B CN 201710277566 A CN201710277566 A CN 201710277566A CN 107132292 B CN107132292 B CN 107132292B
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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
- G01N30/06—Preparation
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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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
Abstract
The invention discloses the method for building up towards medicine obovateleaf ainsliaea herb finger-print, includes the following steps: to take Schaftoside, orientoside, Vitexin reference substance, add methanol to dissolve, constant volume, as reference substance solution；Obovateleaf ainsliaea herb medicinal material is taken, is extracted with ethanol solution, constant volume, as test solution；Through efficient liquid phase separation detection, with 0.4% acetic acid solution of volume fraction for A phase, 100% acetonitrile solution of volume fraction is B phase, forms mobile phase, using gradient elution, fingerprint characteristic is demarcated with relative retention time and relative peak area, to obtain the finger-print.The invention also discloses the finger-prints that the method for building up towards medicine obovateleaf ainsliaea herb finger-print obtains.
Description
Technical field
The present invention relates to Ethnic crude drugs field of quality control, and in particular to towards medicine obovateleaf ainsliaea herb finger-print method for building up and
Its finger-print.
Background technique
One of the national medicine in China be towards medicine China's historical and cultural heritage rarity, be that this is special prolonging side
Under geographical environment, Korean nationality physician struggles against long-term with disease on grasping ancient Korea's east theory of medicine and technical foundation
Formation and development is not only different from Korea's east medicine but also is different from the more complete medical system of traditional Chinese medicine during striving.It is rich
The medicine and pharmacology treasure-house of the Fu Liao Chinese nation provides a huge resources bank for ethnic drug new drug research.Towards medicine research with
Exploitation has apparent regional characteristics and representativeness in China's Development of pharmaceutical industry.
The quality standard of ethnic drug is the fundamental of ethnic drug product core competitiveness, is the weight of development of national medicine industry
Technology is wanted to guarantee.In the case where State Administration of Traditional Chinese Medicine etc. supports energetically, in recent years towards the theory of medicine, material base, clinical application
Etc. achieve very big achievement, but still have serious shortage of standard in the arrangement of kind and quality control, restrict
Towards the development of medicine.Work out comprehensively, it is system, scientific and reasonable towards medicine quality standard, not only promote the standardization produced towards medicine, mark
Standardization, modernization generate actively and far-reaching influence, and to control towards medicine quality, guarantee clinical application safely and effectively has extremely
Important meaning.
It is that pinkwort light calyx silene aprica (Melandrium firmum Rohrb.) and its modification are dredged towards medicine obovateleaf ainsliaea herb
The dry aerial parts of hair silene aprica (Melandrium pubescens Makino.), are distributed widely in Changbaishan area, plant
Object is resourceful, also there is a large amount of cultivations.Towards medicine obovateleaf ainsliaea herb be towards positive people's key medicine few in medicine, " Dong-eui-bo-gam ", " towards medicine ",
It is on the books in the ethnic drugs ancient book document such as " Korea's pharmacopeia ", " practical east pharmacy ", " China National medicine will ", " towards medicine will ",
And there is clearing heat and detoxicating, diuresis of blood circulation promoting, blood-arresting catamenia-regulating, wind-expelling pain-stopping and other effects is used for acute and chronic ephritis, urinary tract infections, the moon
Through uncomfortable, arthritis, the diseases such as uterine hemorrhage.
So far, both at home and abroad to the quality standard research of the ethnic drug there is not yet any report, therefore it is carried out efficiently
The research of liquid phase (HPLC) finger-print, makes up the blank under obovateleaf ainsliaea herb quality standard item, is the quality mark for establishing obovateleaf ainsliaea herb comprehensively
Standard provides scientific basis.
Summary of the invention
The present invention has designed and developed the method for building up towards medicine obovateleaf ainsliaea herb finger-print, and goal of the invention of the invention is to pass through court
The method for building up of medicine obovateleaf ainsliaea herb high-efficiency liquid-phase fingerprint, can for towards the identification of medicine obovateleaf ainsliaea herb and quality control provide reliably according to
According to.
The present invention has designed and developed the finger-print obtained towards the method for building up of medicine obovateleaf ainsliaea herb finger-print, hair of the invention
Improving eyesight is by obtaining towards medicine obovateleaf ainsliaea herb high-efficiency liquid-phase fingerprint, can be the identification and quality control towards medicine obovateleaf ainsliaea herb
Reliable basis is provided.
Technical solution provided by the invention are as follows:
Towards the method for building up of medicine obovateleaf ainsliaea herb finger-print, include the following steps:
Schaftoside, orientoside, Vitexin reference substance are taken, methanol is added to dissolve, constant volume, as reference substance solution；
Obovateleaf ainsliaea herb medicinal material is taken, is extracted with ethanol solution, constant volume, as test solution；
Through efficient liquid phase separation detection, with 0.4% acetic acid solution of volume fraction for A phase, 100% acetonitrile solution of volume fraction
For B phase, mobile phase is formed, using gradient elution, fingerprint characteristic is demarcated with relative retention time and relative peak area, to obtain
The finger-print.
Preferably, the efficient liquid phase separation detection condition are as follows: use chromatographic column with octadecylsilane chemically bonded silica
For stationary phase, column temperature is 50 DEG C, Detection wavelength 253nm, flow velocity 1mL/min, analysis time 70min, using sample volume
High performance liquid chromatograph is injected for 10 μ L.
Preferably, the elution program of the gradient elution is as follows:
At 0 minute, the acetic acid solution that mobile phase A is 90%, the acetonitrile solution that Mobile phase B is 10%；
At 35 minutes, the acetic acid solution that mobile phase A is 75%, the acetonitrile solution that Mobile phase B is 25%；
At 50 minutes, the acetic acid solution that mobile phase A is 70%, the acetonitrile solution that Mobile phase B is 30%；
At 60 minutes, the acetic acid solution that mobile phase A is 60%, the acetonitrile solution that Mobile phase B is 40%；
At 70 minutes, the acetic acid solution that mobile phase A is 10%, the acetonitrile solution that Mobile phase B is 90%；
Column temperature is 50 DEG C；Flow velocity: 1mL/min；Sample volume: 10 μ L.
It preferably, include: to take obovateleaf ainsliaea herb medicinal powder 1.0g towards the preparation process of medicine obovateleaf ainsliaea herb test solution, it is accurate
It is weighed, it sets and 70% ethanol solution is added in the volumetric flask of 25mL, weighed initial weight, ultrasound lh, is cooled to room temperature at room temperature, uses
Ethanol solution supplies less loss weight, shakes up, with 0.45 μm of filtering with microporous membrane to get test sample.
Preferably, the preparation process of reference substance solution includes: accurately weighed Schaftoside, orientoside, Vitexin control
Product weigh 5mg, 5mg respectively, and 2mg is sequentially placed into 10mL, 25mL, 10mL volumetric flask, add methanol dilution to scale, shake up, determine
Hold, it is respectively 500ppm that concentration, which is made, and the solution of 200ppm, 200ppm are molten to get reference substance through 0.45 μm of miillpore filter filtration
Liquid is spare.
Preferably, the finger-print shares 21 shared fingerprint peaks, and relative retention time is respectively as follows:
No. 1 peak: 0.281；No. 2 peaks: 0.368；No. 3 peaks: 0.444；No. 4 peaks: 0.603；No. 5 peaks: 0.824；No. 6 peaks are ginseng
According to peak: 1.000；No. 7 peaks: 1.053；No. 8 peaks: 1.079；No. 9 peaks: 1.150；No. 10 peaks: 1.185；No. 11 peaks: 1.206；No. 12
Peak: 1.225；No. 13 peaks: 1.271；No. 14 peaks: 1.332；No. 15 peaks: 1.362；No. 16 peaks: 1.380；No. 17 peaks: 1.413；18
Number peak: 1.498；No. 19 peaks: 1.559；No. 20 peaks: 1.741；No. 21 peaks: 1.768.
Preferably, the finger-print shares 21 shared fingerprint peaks, and relative peak area is respectively as follows:
No. 1 peak: 0.662；No. 2 peaks: 0.744；No. 3 peaks: 0.303；No. 4 peaks: 0.092；No. 5 peaks: 0.242；No. 6 peaks are ginseng
According to peak: 1.000；No. 7 peaks: 0.152；No. 8 peaks: 0.061；No. 9 peaks: 0.398；No. 10 peaks: 1.015；No. 11 peaks: 0.136；No. 12
Peak: 0.370；No. 13 peaks: 1.671；No. 14 peaks: 0.547；No. 15 peaks: 0.189；No. 16 peaks: 0.318；No. 17 peaks: 0.133；18
Number peak: 0.177；No. 19 peaks: 0.107；No. 20 peaks: 0.159；No. 21 peaks: 1.907.
The finger-print obtained towards the method for building up of medicine obovateleaf ainsliaea herb finger-print will be prepared into ethanol solution towards medicine obovateleaf ainsliaea herb
Afterwards, by efficient liquid phase separation detection, the finger-print towards medicine obovateleaf ainsliaea herb is obtained.
Preferably, the efficient liquid phase separation detection condition are as follows: use chromatographic column with octadecylsilane chemically bonded silica
For stationary phase, with 0.4% acetic acid solution of volume fraction for A phase, 100% acetonitrile solution of volume fraction is B phase, forms mobile phase,
Using gradient elution, column temperature is 50 DEG C, Detection wavelength 253nm, flow velocity 1mL/min, analysis time 70min, using into
Sample amount is that 10 μ L inject high performance liquid chromatograph；And
The elution program of the gradient elution is as follows:
At 0 minute, the acetic acid solution that mobile phase A is 90%, the acetonitrile solution that Mobile phase B is 10%；
At 35 minutes, the acetic acid solution that mobile phase A is 75%, the acetonitrile solution that Mobile phase B is 25%；
At 50 minutes, the acetic acid solution that mobile phase A is 70%, the acetonitrile solution that Mobile phase B is 30%；
At 60 minutes, the acetic acid solution that mobile phase A is 60%, the acetonitrile solution that Mobile phase B is 40%；
At 70 minutes, the acetic acid solution that mobile phase A is 10%, the acetonitrile solution that Mobile phase B is 90%.
Preferably, the finger-print shares 21 shared fingerprint peaks；
The relative retention time of the shared fingerprint peaks is respectively as follows: No. 1 peak: 0.281；No. 2 peaks: 0.368；No. 3 peaks:
0.444；No. 4 peaks: 0.603；No. 5 peaks: 0.824；No. 6 peaks are referring to peak: 1.000；No. 7 peaks: 1.053；No. 8 peaks: 1.079；No. 9
Peak: 1.150；No. 10 peaks: 1.185；No. 11 peaks: 1.206；No. 12 peaks: 1.225；No. 13 peaks: 1.271；No. 14 peaks: 1.332；15
Number peak: 1.362；No. 16 peaks: 1.380；No. 17 peaks: 1.413；No. 18 peaks: 1.498；No. 19 peaks: 1.559；No. 20 peaks: 1.741；
No. 21 peaks: 1.768；
The relative peak area of the shared fingerprint peaks is respectively as follows: No. 1 peak: 0.662；No. 2 peaks: 0.744；No. 3 peaks: 0.303；
No. 4 peaks: 0.092；No. 5 peaks: 0.242；No. 6 peaks are referring to peak: 1.000；No. 7 peaks: 0.152；No. 8 peaks: 0.061；No. 9 peaks:
0.398；No. 10 peaks: 1.015；No. 11 peaks: 0.136；No. 12 peaks: 0.370；No. 13 peaks: 1.671；No. 14 peaks: 0.547；No. 15
Peak: 0.189；No. 16 peaks: 0.318；No. 17 peaks: 0.133；No. 18 peaks: 0.177；No. 19 peaks: 0.107；No. 20 peaks: 0.159；21
Number peak: 1.907.
The present invention is possessed compared with prior art the utility model has the advantages that establishing criteria medicinal material of the present invention and Yan Bian difference produce
9 batches, ground test sample testing result has selected its retention time and the stable 21 shared peaks of peak area to be demarcated as obovateleaf ainsliaea herb chemistry
The finger-print of ingredient, and shared peak peak area is greater than the 90% of total peak area, non-shared peak peak area is less than total peak area
10%, show that there is good systematicness, characteristic and repeatability.It is selected to prolong side difference counties and cities by similarity evaluation
Obovateleaf ainsliaea herb fingerprint similarity is both greater than 0.90 substantially, has good correlation, and completes and prolongs side real estate obovateleaf ainsliaea herb medicinal material
The foundation of finger-print.This method is reliable, easy, can be used as the effective ways of identification and the quality control of obovateleaf ainsliaea herb.
Detailed description of the invention
Fig. 1 is the high-efficient liquid phase chromatogram of obovateleaf ainsliaea herb under DAD detector different wave length.
Fig. 2 is the high-efficient liquid phase chromatogram of reference substance test liquid.
Fig. 3 is the high-efficient liquid phase chromatogram towards medicine obovateleaf ainsliaea herb standard medicinal material solution.
Fig. 4 is 10 batches of high-efficiency liquid-phase fingerprints towards medicine obovateleaf ainsliaea herb；Wherein, R: shared map；S1: standard medicinal material；S2:
Longjing City；S3: Wangqing County；S4: Antu County；S5: Hunchun City；S6: Tumen City；S7: Dunhua City；S8: Yanji 1；S9: Yanji
2；The county S10: He Long.
Fig. 5 is to share peak finger-print towards medicine obovateleaf ainsliaea herb.
Fig. 6 is towards medicine obovateleaf ainsliaea herb Precision Experiment high-efficient liquid phase color map.
Fig. 7 is towards medicine obovateleaf ainsliaea herb repeated experiment high-efficient liquid phase color map.
Fig. 8 is towards medicine obovateleaf ainsliaea herb stability experiment high-efficient liquid phase color map.
Specific embodiment
Present invention will be described in further detail below with reference to the accompanying drawings, to enable those skilled in the art referring to specification text
Word can be implemented accordingly.
As shown in Fig. 1~8, the present invention provides the method for building up towards medicine obovateleaf ainsliaea herb finger-print, includes the following steps:
Schaftoside, orientoside, Vitexin reference substance are taken, methanol is added to dissolve, constant volume, as reference substance solution；
Obovateleaf ainsliaea herb medicinal material is taken, is extracted with ethanol solution, constant volume, as test solution；
Through efficient liquid phase separation detection, with 0.4% acetic acid solution of volume fraction for A phase, 100% acetonitrile solution of volume fraction
For B phase, mobile phase is formed, using gradient elution, fingerprint characteristic is demarcated with relative retention time and relative peak area, to obtain
The finger-print.
In another embodiment, efficient liquid phase separation detection condition are as follows: chromatographic column is used to be bonded with octadecylsilane
Silica gel is stationary phase, and column temperature is 50 DEG C, Detection wavelength 253nm, flow velocity 1mL/min, analysis time 70min, using into
Sample amount is that 10 μ L inject high performance liquid chromatograph.
In another embodiment, the elution program of gradient elution is as follows:
At 0 minute, the acetic acid solution that mobile phase A is 90%, the acetonitrile solution that Mobile phase B is 10%；
At 35 minutes, the acetic acid solution that mobile phase A is 75%, the acetonitrile solution that Mobile phase B is 25%；
At 50 minutes, the acetic acid solution that mobile phase A is 70%, the acetonitrile solution that Mobile phase B is 30%；
At 60 minutes, the acetic acid solution that mobile phase A is 60%, the acetonitrile solution that Mobile phase B is 40%；
At 70 minutes, the acetic acid solution that mobile phase A is 10%, the acetonitrile solution that Mobile phase B is 90%；
The column temperature is 50 DEG C；The flow velocity: 1mL/min；The sample volume: 10 μ L.
It in another embodiment, include: to take obovateleaf ainsliaea herb standard medicinal material towards the preparation process of medicine obovateleaf ainsliaea herb test solution
Powder 1.0g, it is accurately weighed, it sets and 70% ethanol solution is added in the volumetric flask of 25mL, weighed initial weight, ultrasound lh at room temperature,
It is cooled to room temperature, supplies less loss weight with ethanol solution, shake up, with 0.45 μm of filtering with microporous membrane to get test sample.
In another embodiment, the preparation process of reference substance solution include: accurately weighed Schaftoside, it is orientoside, male
Jing Su reference substance weighs 5mg, 5mg respectively, and 2mg is sequentially placed into 10mL, 25mL, 10mL volumetric flask, adds methanol dilution to quarter
Degree, shakes up, constant volume, and it is respectively 500ppm that concentration, which is made, and the solution of 200ppm, 200ppm filter, i.e., through 0.45 μm of miillpore filter
It is spare to obtain reference substance solution.
In another embodiment, finger-print shares 21 shared fingerprint peaks, and relative retention time is respectively as follows: No. 1 peak:
0.281；No. 2 peaks: 0.368；No. 3 peaks: 0.444；No. 4 peaks: 0.603；No. 5 peaks: 0.824；No. 6 peaks are referring to peak: 1.000；No. 7
Peak: 1.053；No. 8 peaks: 1.079；No. 9 peaks: 1.150；No. 10 peaks: 1.185；No. 11 peaks: 1.206；No. 12 peaks: 1.225；No. 13
Peak: 1.271；No. 14 peaks: 1.332；No. 15 peaks: 1.362；No. 16 peaks: 1.380；No. 17 peaks: 1.413；No. 18 peaks: 1.498；19
Number peak: 1.559；No. 20 peaks: 1.741；No. 21 peaks: 1.768；Relative peak area is respectively as follows: No. 1 peak: 0.662；No. 2 peaks:
0.744；No. 3 peaks: 0.303；No. 4 peaks: 0.092；No. 5 peaks: 0.242；No. 6 peaks are referring to peak: 1.000；No. 7 peaks: 0.152；No. 8
Peak: 0.061；No. 9 peaks: 0.398；No. 10 peaks: 1.015；No. 11 peaks: 0.136；No. 12 peaks: 0.370；No. 13 peaks: 1.671；No. 14
Peak: 0.547；No. 15 peaks: 0.189；No. 16 peaks: 0.318；No. 17 peaks: 0.133；No. 18 peaks: 0.177；No. 19 peaks: 0.107；20
Number peak: 0.159；No. 21 peaks: 1.907.
The present invention also provides the finger-prints that the method for building up towards medicine obovateleaf ainsliaea herb finger-print obtains, will be towards medicine obovateleaf ainsliaea herb
After being prepared into ethanol solution, by efficient liquid phase separation detection, the finger-print towards medicine obovateleaf ainsliaea herb is obtained.
In another embodiment, the efficient liquid phase separation detection condition are as follows: use chromatographic column with octadecylsilane
Bonded silica gel is stationary phase, and with 0.4% acetic acid solution of volume fraction for A phase, 100% acetonitrile solution of volume fraction is B phase, composition
Mobile phase, using gradient elution, column temperature is 50 DEG C, Detection wavelength 253nm, flow velocity 1mL/min, analysis time 70min,
Sample volume is used to inject high performance liquid chromatograph for 10 μ L；The elution program of gradient elution is as follows:
At 0 minute, the acetic acid solution that mobile phase A is 90%, the acetonitrile solution that Mobile phase B is 10%；
At 35 minutes, the acetic acid solution that mobile phase A is 75%, the acetonitrile solution that Mobile phase B is 25%；
At 50 minutes, the acetic acid solution that mobile phase A is 70%, the acetonitrile solution that Mobile phase B is 30%；
At 60 minutes, the acetic acid solution that mobile phase A is 60%, the acetonitrile solution that Mobile phase B is 40%；
At 70 minutes, the acetic acid solution that mobile phase A is 10%, the acetonitrile solution that Mobile phase B is 90%.
In another embodiment, finger-print shares 21 shared fingerprint peaks；
The relative retention time of the shared fingerprint peaks is respectively as follows: No. 1 peak: 0.281；No. 2 peaks: 0.368；No. 3 peaks:
0.444；No. 4 peaks: 0.603；No. 5 peaks: 0.824；No. 6 peaks are referring to peak: 1.000；No. 7 peaks: 1.053；No. 8 peaks: 1.079；No. 9
Peak: 1.150；No. 10 peaks: 1.185；No. 11 peaks: 1.206；No. 12 peaks: 1.225；No. 13 peaks: 1.271；No. 14 peaks: 1.332；15
Number peak: 1.362；No. 16 peaks: 1.380；No. 17 peaks: 1.413；No. 18 peaks: 1.498；No. 19 peaks: 1.559；No. 20 peaks: 1.741；
No. 21 peaks: 1.768；The relative peak area of shared fingerprint peaks is respectively as follows: No. 1 peak: 0.662；No. 2 peaks: 0.744；No. 3 peaks:
0.303；No. 4 peaks: 0.092；No. 5 peaks: 0.242；No. 6 peaks are referring to peak: 1.000；No. 7 peaks: 0.152；No. 8 peaks: 0.061；No. 9
Peak: 0.398；No. 10 peaks: 1.015；No. 11 peaks: 0.136；No. 12 peaks: 0.370；No. 13 peaks: 1.671；No. 14 peaks: 0.547；15
Number peak: 0.189；No. 16 peaks: 0.318；No. 17 peaks: 0.133；No. 18 peaks: 0.177；No. 19 peaks: 0.107；No. 20 peaks: 0.159；
No. 21 peaks: 1.907.
1, material
1.1 test sample medicinal materials
Standard medicinal material (S1): it is provided by prolonging inhabitant of a border area race institute of Pharmaceutical Research；S2: Longjing City；S3: Wangqing County；S4: Antu County；
S5: Hunchun City；S6: Tumen City；S7: Dunhua City；S8: Yanji 1；S9: Yanji 2；The county S10: He Long.
1.2 reference substance
Schaftoside reference substance (51938-32-0, upper Hiroad standing grain Pharmaceutical Technology Co., Ltd), orientoside reference substance
(28608-75-5, upper Hiroad standing grain Pharmaceutical Technology Co., Ltd), Vitexin reference substance (3681-93-4, upper Hiroad standing grain medical sci-tech
Co., Ltd).
1.3 reagent
Acetonitrile (chromatographic grade, Fisher Scientific, the U.S.), methanol (chromatographic grade, Fisher Scientific, beauty
State), ethyl alcohol (analyzes pure, Liaoning Quan Rui reagent Co., Ltd), and methanol (analyzes pure, Liaoning Quan Rui reagent Co., Ltd), formic acid
(analyzing pure, Tianjin Kermel Chemical Reagent Co., Ltd.), trifluoroacetic acid (chromatographic grade, Shanghai Aladdin biochemical technology share
Co., Ltd), acetic acid (glacial acetic acid analyzes pure, Gongzhuling City chemical reagent factory), distilled water, ultrapure water (Yanbian University's self-control).
1.4 instrument
CP214 type electronic analytical balance (Ohaus Instrument (Changzhou) Co., Ltd.), filtering vacuum plant, (sunrise Shanghai, Shanghai is real
Test equipment Co., Ltd), liquid-transfering gun (100 μ L, Henan brother's experimental instruments and equipment limited), liquid-transfering gun (200 μ L, France),
HITACHI high performance liquid chromatograph (Hitachi, Japan), Primaide-1410 UV detector (Hitachi, Japan),
Primaide-5430 second level array tube (DAD) detector (Hitachi, Japan), work station: Primaide chromatographic work station (day
This Hitachi, Ltd).
2, method and result
2.1 chromatographic condition
2.1.1 the selection of Detection wavelength
As shown in Figure 1, utilizing diode battle array to obtain the most abundant obovateleaf ainsliaea herb chemical component information in finger-print
Column detector (DAD) detector is to progress all band scanning within the scope of 200~400nm.The finger-print at 253nm as the result is shown
Chromatographic peak number it is most, the chemical component information content of representative is maximum, and the separating degree at peak is also preferable, so finally determining 253nm
For Detection wavelength, as shown in Figure 1.
2.1.2 the selection of mobile phase
Gradient elution is carried out using acetonitrile-water and methanol-water system as mobile phase respectively, as the result is shown acetonitrile-water solvent system
It is more preferable that system elutes resulting spectrogram effect, but separating degree is still not ideal enough, in order to improve its peak shape, has attempted to add in water phase
Formic acid, acetic acid and trifluoroacetic acid solution is added to improve separating degree.0.4% acetic acid solution is as the water phase in mobile phase as the result is shown
When peak shape it is best, so choose -0.4% acetic acid solution of acetonitrile it is as shown in table 1 as mobile phase elution time program.
1. mobile phase elution system of table
2.1.3 the determination of experiment condition
Chromatographic column is SunFire C18 (5 μm, 4.6 × 250mm)；B is acetonitrile solution (ACN) in mobile phase solvent system,
A is 0.4% acetic acid solution (HAc) (A+B=100%)；Flow velocity 1mL/min；50 DEG C of column temperature；Detection wavelength 253nm；Sample volume 10
μl；Chromatography records the time for 70min, shown in table 2.
The chromatographic condition of table 2.HPLC
The preparation of 2.2 sample solutions
2.2.1 the preparation of reference substance solution
Accurately weighed Schaftoside, orientoside, Vitexin reference substance, respectively 5mg, 5mg, 2mg be sequentially placed into 10mL,
In 25mL, 10mL volumetric flask, methanol dilution is added to shake up, constant volume to scale, it is respectively 500ppm, 200ppm that concentration, which is made,
The solution of 200ppm is filtered through 0.45 μm of miillpore filter, spare.
2.2.2 the preparation of test solution
Obovateleaf ainsliaea herb medicinal material and 9 batches of test sample medicinal powders about 1.0g are taken, it is accurately weighed, it sets in the volumetric flask of 25mL and is added
70% ethanol solution, weighed initial weight, ultrasound l hours, are cooled to room temperature at room temperature, supply less loss weight with ethanol solution,
It shakes up, with 0.45 μm of filtering with microporous membrane to get test sample.
2.3 establish towards medicine obovateleaf ainsliaea herb finger-print
As shown in Figure 2 and Figure 3, according to the above chromatographic condition, to 1~10 batch of test sample, sample introduction is analyzed, it is determined that separating degree is greater than
The map at 21,1.0 shared peak, reference substance mixed liquor is qualitative by retention time it is found that peak 6,7,10 is distinguished in standard medicinal material
For Schaftoside, the chromatographic peak of orientoside and Vitexin；Wherein, chromatographic peak (peak 6) content of Schaftoside is higher, and when reservation
Between stablize, as referring to peak；The relative retention time and relative peak area such as table 3 at 10 batches of obovateleaf ainsliaea herbs, 21 shared peaks, table 4
It is shown；Wherein, relative retention time=each group swarming retention time/reference peak retention time, opposite reservation peak area=
The peak area of each group swarming/reference peak peak area；
The relative retention time of fingerprint peaks is shared in 3 10 batches of obovateleaf ainsliaea herb finger-prints of table
The relative peak area of fingerprint peaks is shared in 4 10 batches of obovateleaf ainsliaea herb finger-prints of table
By chart it is found that when the obovateleaf ainsliaea herb Herbal HPLC Fingerprint for prolonging Region In Yanbian different sources shares the opposite reservation at peak
Between RSD be that 0.04-0.2% less than 3% illustrates that reproducibility is fine；But relative peak area value is in the presence of more significant poor
Different (RSD 17-81%), this illustrates 10 batches of different sources medicinal materials because the difference of its production environment results in its chemical component
The difference of content.
The calculating of 2.4 similarities
Respectively by legal system available test sample solution below " 2.2 " item, sample introduction is analyzed, and utilizing " Chinese Pharmacopoeia ", " Chinese medicine chromatography refers to
Line map similarity evaluation system 2004A editions " imports 1~10 batch of obovateleaf ainsliaea herb test sample spectrum data, through Supplements sum number
According to matching, reference fingerprint is generated with median, establishes the finger-print (Fig. 5) of common pattern, according to matched data, really
Determine 21 shared peaks, the relative retention time at shared peak has been analyzed, the relative retention time at 21 shared peaks
For RSD between 0.1%~1%, the shared peak retention time of each place of production medicinal material is almost the same (Fig. 4).
Similarity-rough set between 2.5 different sources
According to above-mentioned chromatographic condition, the obovateleaf ainsliaea herb test sample for prolonging side different sources is analyzed respectively, is recorded
Chromatogram utilizes " Chinese Pharmacopoeia " " similarity evaluation 2004B editions ", by 1~10 batch of obovateleaf ainsliaea herb
The control spectrogram that medicinal material map generates imports, and calculates the similarity-rough set prolonged between side different sources sample and common pattern, knot
Fruit is as shown in table 5.
Table 5. prolongs the similarity of side different sources obovateleaf ainsliaea herb medicinal material
2.5 methodology validation
2.5.1 Precision Experiment
Precision is drawn with a test solution 1.0mL, by the above chromatographic condition continuous sample introduction 5 times, through " Chinese medicine chromatography refers to
Line map similarity evaluation system A editions " is investigated the peak area and retention time of each main chromatographic peak and is calculated, the results show that its
The peak area and retention time relative standard deviation of main chromatographic peak are respectively less than 3%, show that instrument precision is good, as Fig. 6,
Shown in table 6.
The test of 6 method precision of table
2.5.2 repeated experiment
Precision is drawn with a 5 parts of test solution, sample introduction is distinguished by the above chromatographic condition, through " Chinese medicine chromatographic fingerprint figure
Spectrum similarity evaluation system A editions " is investigated the peak area and retention time of each main chromatographic peak and is calculated, as the result is shown its main color
The peak area and retention time relative standard deviation of spectral peak are respectively less than 3%, and illustration method repeatability is good, such as Fig. 7,7 institute of table
Show.
7 method reperformance test of table
2.5.3 stability experiment
Precision is drawn with a test solution, by the above chromatographic condition, respectively 1,4,8,12, sample introduction for 24 hours, through " in
Medicine chromatographic fingerprinting similarity evaluation system A editions " is investigated the peak area and retention time of each main chromatographic peak and is calculated, as a result
The peak area and retention time relative standard deviation for showing its main chromatographic peak are respectively less than 3%, show test solution for 24 hours
It is basicly stable in hour, as shown in Fig. 8, table 8.
The test of 8 sample stability of table
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily
Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and legend shown and described herein.
Claims (5)
1. towards the method for building up of medicine obovateleaf ainsliaea herb finger-print, which comprises the steps of:
Schaftoside, orientoside, Vitexin reference substance are taken, methanol is added to dissolve, constant volume, as reference substance solution；
Obovateleaf ainsliaea herb medicinal material is taken, is extracted with ethanol solution, constant volume, as test solution；
Through efficient liquid phase separation detection, with 0.4% acetic acid solution of volume fraction for A phase, 100% acetonitrile solution of volume fraction is B
Phase forms mobile phase, using gradient elution, fingerprint characteristic is demarcated with relative retention time and relative peak area, to obtain institute
State finger-print；
The efficient liquid phase separation detection condition are as follows: use chromatographic column using octadecylsilane chemically bonded silica as stationary phase, column temperature
It is 50 DEG C, Detection wavelength 253nm, flow velocity 1mL/min, analysis time 70min uses sample volume efficient for 10 μ L injection
Liquid chromatograph；
The elution program of the gradient elution is as follows:
At 0 minute, the acetic acid solution that mobile phase A is 90%, the acetonitrile solution that Mobile phase B is 10%；
At 35 minutes, the acetic acid solution that mobile phase A is 75%, the acetonitrile solution that Mobile phase B is 25%；
At 50 minutes, the acetic acid solution that mobile phase A is 70%, the acetonitrile solution that Mobile phase B is 30%；
At 60 minutes, the acetic acid solution that mobile phase A is 60%, the acetonitrile solution that Mobile phase B is 40%；
At 70 minutes, the acetic acid solution that mobile phase A is 10%, the acetonitrile solution that Mobile phase B is 90%.
2. as described in claim 1 towards the method for building up of medicine obovateleaf ainsliaea herb finger-print, which is characterized in that towards medicine obovateleaf ainsliaea herb for examination
The preparation process of product solution includes: to take obovateleaf ainsliaea herb medicinal powder 1.0g, accurately weighed, sets and 70% second is added in the volumetric flask of 20mL
Alcoholic solution, weighed initial weight, ultrasound lh, is cooled to room temperature at room temperature, supplies less loss weight with ethanol solution, shakes up, and uses
0.45 μm of filtering with microporous membrane is to get test sample.
3. as claimed in claim 2 towards the method for building up of medicine obovateleaf ainsliaea herb finger-print, which is characterized in that the system of reference substance solution
Standby technique includes: accurately weighed Schaftoside, orientoside, Vitexin reference substance, weighs 5mg, 5mg respectively, 2mg is sequentially placed into
In 10mL, 25mL, 10mL volumetric flask, methanol dilution is added to shake up, constant volume to scale, it is respectively 500ppm that concentration, which is made,
The solution of 200ppm, 200ppm, it is spare to get reference substance solution through 0.45 μm of miillpore filter filtration.
4. as claimed in claim 3 towards the method for building up of medicine obovateleaf ainsliaea herb finger-print, which is characterized in that the finger-print is total
There are 21 shared fingerprint peaks, relative retention time is respectively as follows:
No. 1 peak: 0.281；No. 2 peaks: 0.368；No. 3 peaks: 0.444；No. 4 peaks: 0.603；No. 5 peaks: 0.824；No. 6 peaks are reference
Peak: 1.000；No. 7 peaks: 1.053；No. 8 peaks: 1.079；No. 9 peaks: 1.150；No. 10 peaks: 1.185；No. 11 peaks: 1.206；No. 12
Peak: 1.225；No. 13 peaks: 1.271；No. 14 peaks: 1.332；No. 15 peaks: 1.362；No. 16 peaks: 1.380；No. 17 peaks: 1.413；18
Number peak: 1.498；No. 19 peaks: 1.559；No. 20 peaks: 1.741；No. 21 peaks: 1.768.
5. such as the method for building up of any of claims 1-4 towards medicine obovateleaf ainsliaea herb finger-print, which is characterized in that described
Finger-print shares 21 shared fingerprint peaks, and relative peak area is respectively as follows:
No. 1 peak: 0.662；No. 2 peaks: 0.744；No. 3 peaks: 0.303；No. 4 peaks: 0.092；No. 5 peaks: 0.242；No. 6 peaks are reference
Peak: 1.000；No. 7 peaks: 0.152；No. 8 peaks: 0.061；No. 9 peaks: 0.398；No. 10 peaks: 1.015；No. 11 peaks: 0.136；No. 12
Peak: 0.370；No. 13 peaks: 1.671；No. 14 peaks: 0.547；No. 15 peaks: 0.189；No. 16 peaks: 0.318；No. 17 peaks: 0.133；18
Number peak: 0.177；No. 19 peaks: 0.107；No. 20 peaks: 0.159；No. 21 peaks: 1.907.
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