CN106918667A - The micro- extraction equipment of one kind pressurization and the micro- extracting method of pressurization and its application - Google Patents
The micro- extraction equipment of one kind pressurization and the micro- extracting method of pressurization and its application Download PDFInfo
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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
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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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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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
- G01N2030/062—Preparation extracting sample from raw material
Abstract
The present invention provides a kind of micro- extraction equipment of pressurization, including extraction column and pressue device, and the extraction column and pressue device are connected by pipeline；The extraction column is the pre-column core of the pre- column sleeve of liquid chromatograph and being filled with of being placed in one sample to be measured and inert filler.The present invention also provides application of the described micro- extraction equipment of pressurization in plant chemical ingredient extraction, qualitative detection and/or quantitative analysis, the particularly application in the composition detection of safflower and saline cistanche.The micro- extraction equipment of pressurization of the present invention, can realize quick to plant sample, micro, accurate qualitative and quantitative determination, more more environmentally friendly than traditional extracting mode, quick, be particularly suitable for the detection of precious Chinese medicine.
Description
Technical field
The invention belongs to analytical chemistry and field of natural product chemistry, and in particular to the micro- extraction of one kind pressurization
Equipment and the micro- extracting method of pressurization based on the equipment and its composition detection and quality in medicinal plant
The application of control aspect.
Background technology
The active component of medicinal plant (including Chinese medicine) is all present in plant tissue, only by activity
Composition is extracted from plant tissue, could carry out Identification of chemical structure, Activity determination, quality control
The research of the aspects such as system.Therefore, extractive technique is medicinal plants study, especially constituent analysis and matter
The basis of amount control and key technology.
In traditional Chinese medicine ingredients analysis and field of quality control, traditional extracting method has soxhlet extraction, surpasses
Sound extraction method, reflux extraction, infusion process etc..Although these extracting modes are widely used, one
It is secondary to extract short then dozens of minutes, long then several hours；The medicinal material of extraction is also in grams.Above-mentioned biography
The extracting mode of system not only takes time and effort, and all larger to the usage amount of medicinal material and organic solvent,
Especially it is unsuitable for the analysis of rare medicinal herbs, is unfavorable for environmental protection.In addition, the knot of many active components
Structure is unstable, and long-time high temperature is extracted can cause these ingredient degradations or conversion, and influence even misleads inspection
Survey result.In the case where detection means reaches its maturity, it is clearly to realize fast trace analysis to extract
It is crucial.Obviously, traditional extracting method cannot have been met with quick, micro, accurate etc. as target
The requirement that traditional Chinese medicine quality is controlled and studied.
Occur in that some new extractive techniques successively in recent years, such as pressurized solvent extraction, overcritical
Fluid Chromatography technology etc..As publication number CN104857739A (publication date 2015 on August 26),
The Chinese invention patent application of entitled " a kind of new pressurized extraction element " discloses a kind of pressurization and carries
Device, including small-sized extractor, flat-top cover are taken, the cover is bolted in small-sized extractor
Top, sealing ring is provided between the cover and tank body mouth, and cover is provided with pressure gauge, safety valve, pressure
Contracting air intlet and control valve, sensor for measuring temperature, sampling catheter, sampling control valve, liquid feeding
Conduit and add hydraulic control valve, tank body outside have oil bath or water bath heating device, heater is externally provided with
Heat-insulation layer, tank bottom has electric heating tube, temperaturecontrol sensor and PID temperature adjusting apparatus.This sets
Although standby can be sampled at any time during pressurised extraction, however it is necessary that special pressurised extraction sets
It is standby, and miniaturization can not be realized.
Therefore, it is necessary to developing, consumption medicinal material is smaller, it is shorter to take, especially it is that by online
The method extracted and detect linkage.
The content of the invention
Regarding to the issue above, it is an object of the present invention to provide one kind micro- extraction equipment of pressurization and
Using a kind of micro- extraction of pressurization of the equipment and the method for on-line checking.Equipment of the present invention and side
Method is to be capable of achieving based on existing liquid chromatograph, and easy, quick, completion is once extracted and only needs milligram
The sample of rank, takes a few minutes；The consumption of medicinal material and solvent is not only drastically reduce the area, is especially fitted
Quality control and the composition Study of rare medicinal herbs are closed, and is significantly reduced chemical composition degraded and is tied
The probability of structure change, so that testing result is truer.
In order to realize foregoing invention purpose, present invention employs following technical scheme：
The micro- extraction equipment of one kind pressurization, including extraction column and pressue device, the extraction column and pressurization are filled
Put and connected by pipeline；The extraction column is that the pre- column sleeve of liquid chromatograph and being filled with of being placed in one are to be measured
Sample and inert filler pre-column core.
Preferably, the inert filler be selected from diatomite, purification on normal-phase silica gel, octadecyl silane,
One or more in octane bonded silica gel and cellulose.
Preferably, testing sample is crushed, particle diameter≤0.5mm.
Preferably, the pressue device is the high pressure pump of liquid chromatograph.
Preferably, the micro- extraction equipment of pressurization also includes temperature control system；It is described to carry during extraction
Post is taken to be placed in the temperature control system.
It is furthermore preferred that column oven of the temperature control system for liquid chromatograph.
It is another object of the present invention to provide the micro- extraction equipment of pressurization in plant chemical ingredient
Application in extraction, qualitative detection and/or quantitative analysis, including plant sample to be measured is filled out with inert filler
Fill in the extraction column, Extraction solvent enters in the extraction column through the pressurized equipment, collect stream
Go out liquid, obtain final product plant extraction liquid to be measured, then carry out chemical composition separation, qualitative detection and/or quantify
Analysis.
Preferably, qualitative detection and/or the detector of quantitative analysis are selected from UV-detector, fluoroscopic examination
Device, electrochemical detector, differential refraction detector, PDAD, evaporative light-scattering inspection
Survey the conventional detector in this areas such as device and mass detector.
As one preferred embodiment, the present invention provides a kind of micro- extracting method of the pressurization of safflower,
Concrete operations are：
I. extraction column is loaded：Flos carthami is crushed, and crosses 60~80 mesh sieves, takes 1.0mg loaded on removal
It is filled in the pre-column core of filler and with 4.0~6.0mg of inert material diatomite, is then charged into pre-
It is placed in column sleeve in liquid chromatograph column oven；
II. pressurize micro- extraction：Column oven temperature is 55~65 DEG C, with the first of concentration expressed in percentage by volume 25%
The aqueous formic acid of alcohol solution or concentration expressed in percentage by volume 0.01% is Extraction solvent, opens phase autoclave
Pump, 3~5mL/min of flow velocity, 20~120 seconds extraction times；Collect efflux.
Preferably, the methanol aqueous solution with volumetric concentration 25% is as Extraction solvent, flow velocity 5mL/min,
27 seconds extraction times.
It is also an object of the present invention to provide a kind of micro- extraction of pressurization of safflower and offline inspection hydroxyl
The method of base carthamus tinctorius yellow colour A, concrete operations are：
I. extraction column is loaded：Flos carthami is crushed, and crosses 60~80 mesh sieves, takes 1.0mg loaded on removal
It is filled in the pre-column core of filler and with 4.0~6.0mg of inert material diatomite, is then charged into pre-
It is placed in column sleeve in liquid chromatograph column oven；
II. pressurize micro- extraction：Column oven temperature is 55~65 DEG C, with the first of concentration expressed in percentage by volume 25%
Alcohol solution is Extraction solvent, opens phase autoclave pump, 3~5mL/min of flow velocity, extraction time 20~120
Second；Efflux is collected, is well mixed, that is, obtain Flos Carthami extract；
III. the μ L of Flos Carthami extract 5 that step II is obtained are taken, hydroxyl is detected in injection high performance liquid chromatograph
The content of base carthamus tinctorius yellow colour A.
Preferably, the chromatographic condition in above-mentioned steps III is：
Chromatographic column：UPLC HSS T3,100mm × 2.1mm, 1.8 μm；
Column temperature：35℃；
Mobile phase：0.01% formic acid (concentration of volume percent) water is A phases, 0.01% formic acid (body
Product percent concentration) acetonitrile be B phases, carry out gradient elution according to following program：
0-5min, 0% → 23%B (percent by volume), balance of A；
5-8min, 23% → 100%B (percent by volume), balance of A；
Flow velocity：0.4mL/min；
Detector：Photodiode array detector, Detection wavelength is 403nm.
It is also preferred that above-mentioned steps III also includes for the μ L of reference substance solution 5 injecting high performance liquid chromatography
In instrument, wherein the reference substance solution is prepared via a method which：
Hydroxyl radical carthamin yellow carthamus A standard items are weighed, plus it is 6.925 that the dissolving of 25% methyl alcohol is configured to concentration
The stock solution of mg/mL；Take the storing solution and be diluted to Sydroxy carthamin with 25% methanol solution
A concentration is the titer of 35 μ g/mL, is obtained final product.
Additionally, it is also an object of the present invention to provide a kind of side of pressurize micro- extraction and on-line checking
Method, including the micro- extraction equipment of above-mentioned pressurization and detecting system are connected, make Extraction solvent through the pressurization
Equipment enter the extraction column in, efflux directly or through after treatment enter the detecting system.
Preferably, the detecting system includes liquid-phase chromatographic analysis post and detector.
Preferably, the micro- extraction equipment of pressurization passes through multi-channel valve and the liquid-phase chromatographic analysis post
Connection；During extraction, the extraction column is connected with the liquid-phase chromatographic analysis post, open the pressurization
After device, Extraction solvent flows into the extraction column, and the chemical composition in efflux is in the liquid chromatogram
Analytical column front end is enriched with；After extraction terminates, the pressue device is directly connected with the liquid-phase chromatographic column,
Mobile phase is set to be directly entered the liquid-phase chromatographic column, the chemical composition to being enriched with is eluted and separated,
Eluent is detected with detector.
It is also preferred that also including SPE between the micro- extraction equipment of the pressurization and the detecting system
Pillar, pressurised extraction liquid enters back into the detecting system after purification by the solid phase extraction column.
As one preferred embodiment, a kind of pressurization of safflower of present invention offer is micro- extracts and online
The method of detection, specific operation is：
I. extraction column is loaded：Flos carthami is crushed, and crosses 60~80 mesh sieves, takes 1.0mg loaded on removal
It is filled in the pre-column core of filler and with 4.0~6.0mg of inert material diatomite, is then charged into pre-
It is placed in column sleeve in liquid chromatograph column oven；
II. the micro- extraction equipment of pressurization and detecting system are connected：The extraction column, phase autoclave pump, point
Analysis chromatographic column is connected on six-way valve, and the analysis chromatographic column and detector are connected；
III. pressurize micro- extraction：Switching six-way valve, makes the phase autoclave pump, extraction column and analysis color
Spectrum post sequential series；Column oven temperature is 50~60 DEG C, with the formic acid water of concentration expressed in percentage by volume 0.01%
Solution is Extraction solvent, opens phase autoclave pump, flow velocity 1mL/min, 3 minutes extraction times；
IV. on-line checking：After extraction terminates, six-way valve is switched immediately to be made the phase autoclave pump and divides
Analysis chromatographic column is directly connected, and is eluted and is detected.
Preferably, in the step I, diatomaceous loading is 6.0mg.
Preferably, in the step II, column oven temperature is 55 DEG C.
Preferably, in the step IV, chromatographic condition is：
Chromatographic column：Waters CSH chromatographic columns, (150mm × 4.6mm, 3.5 μm),
Column temperature：35 DEG C,
Mobile phase：It is A phases with the aqueous formic acid of concentration expressed in percentage by volume 0.01%, it is dense with volume basis
The formic acid acetonitrile solution of degree 0.01% is B phases；Gradient elution is carried out according to following program：
0-3min, 0% → 12%B (percent by volume), balance of A,
3-20min, 12% → 30%B (percent by volume), balance of A,
23-26min, 30% → 100%B (percent by volume), balance of A；
Flow velocity：1mL/min.
It is also preferred that in the step IV, the detector is mass detector, Mass Spectrometer Method bar
Part is：
Ion gun：Electrospray ion sources,
Detection pattern：Negative ion mode detection,
Ion injection electric：- 4500V,
Temperature：650 DEG C,
Gas 1 and gas 2 in source：All it is nitrogen, pressure is all 65psi,
Curtain gas：Nitrogen, pressure is 40.0psi,
Scan mode：Multiple reaction is detected.
As another preferred embodiment, the present invention provide a kind of micro- extraction of the pressurization of saline cistanche and
The method of on-line checking, specific operation is：
I. extraction column is loaded：Saline cistanche pulverizing medicinal materials, cross 60~80 mesh sieves, take 1.0mg loaded on going
Except being filled in the pre-column core of filler and with 4.0~6.0mg of inert material purification on normal-phase silica gel, then fill
To enter be placed in liquid chromatograph column oven in pre- column sleeve；
II. the micro- extraction equipment of pressurization and detecting system are connected：The extraction column, phase autoclave pump, C18
Chromatographic column and solid phase extraction column are connected on a six-way valve, the C18Chromatographic column, detector and
Solid phase extraction column is connected on another six-way valve；The C18Chromatographic column and detector are connected；
III. pressurize micro- extraction：Two six-way valves of regulation, make the phase autoclave pump, extraction column solid phase
Extraction pillar sequential series；Column oven temperature is 55~65 DEG C, with the formic acid water of volumetric concentration 0.1%
Solution is Extraction solvent, opens phase autoclave pump, 2~4mL/min of flow velocity, extraction time 60~180
Second；
IV. on-line checking：After extraction terminates, two six-way valves of regulation make the phase autoclave pump, institute
Solid phase extraction column and analysis chromatographic column sequential series are stated, is eluted and is detected.
Preferably, in the step I, the loading of purification on normal-phase silica gel is 6.0mg.
Preferably, in the step III, column oven temperature is 60 DEG C.
Preferably, in the step III, the flow velocity of the aqueous formic acid of volumetric concentration 0.1% is 2
ML/min, extraction time is 180 seconds.
Preferably, in the step IV, chromatographic condition is：
Column temperature：Room temperature,
Mobile phase：It is A phases with the aqueous formic acid of concentration expressed in percentage by volume 0.1%, with acetonitrile solution as B
Phase；According to following program gradient elutions：
0-32min, 10% → 30%B, balance of A；
Flow velocity：0.25mL/min,
Detector：Photodiode array detector, Detection wavelength 278nm.
Preferably, in the step IV, also including the μ L of reference substance solution 10 are injected into the height
Effect liquid phase chromatogram instrument；The reference substance solution is prepared via a method which：
Boschnaloside E, echinacoside, acteoside, different acteoside, meat desert are weighed respectively
Rong's glycosides C, 2 '-acetyl group poliumoside, different boschnaloside C, pipe flower glycosides B standard product, plus DMSO
Dissolving is configured to the stock solution that concentration is 4mg/mL；Each standard items storing solution mixing is taken,
With 50% methyl alcohol dissolved dilution into the hybrid standard storing solution that each standard concentration is 400 μ g/mL, i.e.,
.
Using the micro- extraction equipment of pressurization described in invention and pressurize micro- extraction and online test method, can
To be widely used in various medicinal plants (including Chinese medicine), micro- extraction and composition detection are carried out,
It is not limited to above-mentioned safflower and saline cistanche.And, according to the physicochemical property of medicinal plant ingredient
And extraction, analysis purpose, can neatly use suitable Extraction solvent, eluting solvent, analysis color
Spectrum post and detector.
In the prior art, medicinal plant (including Chinese medicine) is carried out into using high performance liquid chromatography
When go-on-go is surveyed and analyzed, need testing solution is generally extracted or cold using heating and refluxing extraction, sonic oscillation
Leaching method, no matter which kind of method, be required for consume several grams of testing samples and tens of supreme hundred milliliters of extraction
Solvent, expends dozens of minutes to a few hours, and actual analysis only need tens microlitres, remaining test sample
Solution can only all be discarded.The waste of testing sample and organic solvent is so not only caused, and it is easily dirty
Dye environment.In addition for precious Chinese medicine, such as Chinese caterpillar fungus, safflower etc., because one sample of analysis is needed
The medicinal material amount to be consumed is relatively large, testing cost is remained high.
The micro- extraction equipment of pressurization and the micro- extraction of the pressurization based on it and on-line checking side that the present invention is provided
Method, based on the high performance liquid chromatograph being used widely, in high pressure and higher temperature bar
Under part, extract only needs milligram level medicinal material every time, and several milliliters of Extraction solvents, extraction time is short by then tens
Second, only a few minutes long.And seen with the extraction example of safflower and saline cistanche, extraction efficiency is higher than existing
There is the ultrasonic extraction of technology --- the chemical composition of extraction is more, and the content of each composition is higher.Cause
This, The inventive method achieves to the medicinal plant including Chinese medicine is quick, trace detection.Compare
Prior art, the organic solvent and sample size of consuming are greatly decreased, and testing cost is also accordingly reduced.
Brief description of the drawings
Hereinafter, embodiment of the present invention is described in detail with reference to accompanying drawing, wherein：
Fig. 1 is illustrated that the liquid chromatogram of the Flos Carthami extract of embodiment 3, wherein No. 1 chromatographic peak is
The absworption peak of hydroxyl radical carthamin yellow carthamus A.
Fig. 2 is illustrated that the micro- extraction of the pressurization of embodiment 5 and online detection instrument structural representation, its
Middle Fig. 2A is illustrated that device connection when extracting and being enriched with, and Fig. 2 B are illustrated that analysis yes
Device connection.In figure, 1 is high pressure pump, and 2 is extraction column, and 3 is analytical column, and 4 is detection
Device, 5 is six-way valve.
Fig. 3 is illustrated that the chromatogram of the extract solution that the safflower Different Extraction Method of embodiment 5 is obtained；Its
Middle A is the chromatogram that the extract solution on-line checking that the pressurized micro- extraction of safflower is obtained is obtained, and B is safflower warp
The chromatogram of the extract solution that warm extraction is obtained.
Fig. 4 is illustrated that the safflower of embodiment 5 is pressurized and micro- extract the extract solution that obtains and taken through temperature extraction
The chromatographic peak peak area ratio of the extract solution for obtaining relatively is schemed, and wherein Fig. 4 A show the color of C1-C21 compounds
Spectral peak Area comparison result, Fig. 4 B show the chromatographic peak area comparative result of C22-C43 compounds.Figure
Middle ■ represents the micro- extraction of pressurization, and represents temperature extraction and takes.
Fig. 5 is illustrated that the micro- extraction of the pressurization of embodiment 6 and online detection instrument structural representation, wherein
Fig. 5 A are illustrated that device connection when extracting and being enriched with, and Fig. 5 B are illustrated that dress during analysis
Put connection.In figure, 1 is high pressure pump, and 2 is extraction column, and 3 is solid phase extraction column (SPE
Post), 4 is analytical column, and 5 is detector, and 6 is six-way valve.
Fig. 6 is illustrated that embodiment 6 is molten using the reference substance that pressurize micro- extraction and online test method are obtained
The chromatogram of liquid, Desert Herba Cistanches and Cistanche tubulosa, wherein A are the chromatogram of reference substance solution, B
It is the chromatogram of Desert Herba Cistanches, C is the chromatogram of Cistanche tubulosa.No. 1 peak is boschnaloside in figure
The absworption peak of E, No. 2 peaks are the absworption peaks of echinacoside, and No. 3 peaks are the absworption peak of acteoside, 4
Number peak is the absworption peak of different acteoside, and No. 5 peaks are the absworption peaks of boschnaloside C, and No. 6 peaks are different
The absworption peak of boschnaloside C, No. 7 peaks are the absworption peaks of 2 '-acetyl group poliumoside, and No. 8 peaks are pipe flowers
The absworption peak of glycosides B.
Specific embodiment
The present invention is illustrated referring to specific embodiment.It will be appreciated by those skilled in the art that these
Embodiment is merely to illustrate the present invention, and it limits the scope of the present invention never in any form.
Experimental technique in following embodiments, unless otherwise specified, is conventional method.Following implementations
Medicinal raw material, reagent material used etc., unless otherwise specified, are commercially available purchase product in example.
Wherein,Laboratory apparatus：
Chromatograph：1) Shimadzu high performance liquid chromatograph (pump：LC-20ADx2, automatic sampler SIL-20A,
Column oven CTO-20A), ultrapure water purification system, Millipore companies；
2) Waters ACQUITY UPLC H-Class systems, equipped with quaternary solvent manager
(QSM), sample manager (SM-FTN) and photodiode array detector (PDA), U.S.'s water generation
Science and Technology Ltd..
Mass spectrograph：The triple compound tandem mass spectrums of level Four bar linear ion hydrazine of ABSciex QTrap4500 types
Instrument (is furnished with ionspray ion gun, Analysis 1.6.2 data handling systems, American AB Sciex companies).
Chromatographic column：1) UPLC HSS T3 chromatographic columns (100mm × 2.1mm, 1.8 μm), 2) CSH
Chromatographic column (150mm × 4.6mm, 3.5 μm), 3) ACE UltraCore super C18 chromatographic columns (150
mm×3.0mm,2.5μm)。
Pre- column sleeve Phenomenex Security GuardTM, post core Phenomenex cartridge (3.0
Mm × 4mm i.d.) and removal RP-C18Filler.
Experiment medicinal material：
Flos carthami dries flower for feverfew safflower Carthamus tinctorius L.'s.Saline cistanche medicinal material
It is Orobanchaceae plant Desert Herba Cistanches Cistanche deserticola Y.C.Ma or Cistanche tubulosa
The fleshy stem of the dry zone scale leaf of Cistanche tubulosa (Schrenk) Wight.
Experiment reagent and medicine：
Hydroxyl radical carthamin yellow carthamus A (HSYA, lot number：MUST-14101810, purity HPLC are examined
Survey,>98%), it is purchased from Chengdu Man Site bio tech ltd；
Boschnaloside E (Cistanoside E), echinacoside (Echinacoside), acteoside
(Acteoside), different acteoside (Isoacteoside), boschnaloside C (Cistanoside C), 2 '-
Acetyl group poliumoside (2'-Aceylpoliumoside), different boschnaloside C (Isocistanoside C), pipe
Flower glycosides B (Tubuloside B) is extracted from Desert Herba Cistanches by Peking University's Chinese medicine modern study center and divided
From purity is more than 95%.
The pure acetonitrile of liquid matter, methyl alcohol, formic acid, are purchased from silent winged generation that (China) Science and Technology Ltd. of match；Diatom
Soil, chemistry is pure, is purchased from Xilong Chemical Co., Ltd；Purification on normal-phase silica gel, 100-200 mesh, is purchased from green grass or young crops
Island marine chemical industry Co., Ltd.
Embodiment 1Safflower pressurization is micro- to extract research and condition optimizing
1. primary condition
Index components：Hydroxymethyl-Safflor yellow A (HSYA)
Chromatograph：Waters ACQUITY UPLC H-Class systems
Chromatographic column：UPLC HSS T3 chromatographic columns (100mm × 2.1mm, 1.8 μm)
Extraction column：Flos carthami powder (crossing 60 mesh sieves) 1.0mg is loaded in pre-column core and uses inertia material
Material diatomite is filled, and obtains extraction column, is fitted into afterwards in pre- column sleeve and is placed in column oven.
It is prepared by reference substance solution：Weigh appropriate HSYA standard items, plus concentration expressed in percentage by volume 25% methyl alcohol
The aqueous solution (hereinafter referred to as " 25% methyl alcohol ") dissolving is configured to the stock solution that concentration is 6.925mg/mL.
Take above-mentioned standard product storing solution to mix in right amount, it is 35 to be diluted to compound concentration with 25% methanol solution
The titer of μ g/mL.
Detection mode：The assay of HSYA is carried out using UPLC-PDA systems
Chromatographic condition：UPLC HSS T3 chromatographic columns (100mm × 2.1mm, 1.8 μm)；Column temperature：
35℃；Mobile phase：The formic acid acetonitrile (B) of 0.01% formic acid water (A) -0.01%, flow velocity：0.4mL/min,
Gradient elution：0-5min, 0%-23%B, 5-8min, 23%-100%B；Detection wavelength 403nm.
2. safflower pressurizes micro- extraction and optimization for extracting condition
Extraction column is connected with chromatographic high-pressure pump, Extraction solvent is delivered to extraction column through high-pressure pump, received
Collection efflux, takes appropriate (5 μ L) injection UPLC-PDA systems and is separated and detected, records HSYA
Chromatographic peak area, and carry out cubage.
2.1 Extraction solvents
In order to have comparativity with ultrasonic extraction, micro- extraction of pressurizeing is also adopted by concentration of volume percent 25%
Methanol aqueous solution is Extraction solvent.
2.2 investigations for extracting flow velocity
The pressure during flow rate effect the speed and extraction process extracted is extracted, is influence extraction efficiency
Key factor.Therefore different extraction flow velocitys is investigated.With 25% methyl alcohol, 60 DEG C of (column ovens
Temperature) it is primary condition, 3mL/min (pressure has been investigated respectively：2400psi) extract 3min and 5
ML/min (pressure：The extraction efficiency of 1.8min 3600psi) is extracted, 1 is the results are shown in Table.
The different flow velocitys that extract of table 1 investigate result (n=3)
Flow velocity 5mL/min, the content of HSYA is high in extract solution, illustrates under the flow velocity to HSYA's
Extraction efficiency is more preferable, it is therefore preferable that it is 5mL/min to extract flow velocity.
The investigation of 2.3 extraction times
Extraction time is too short, and sample can not be extracted completely, causes extraction efficiency low；Extraction time is long,
Solvent consumption is big, and sample is easily degraded at high operating temperatures, again results in extraction efficiency low.In flow velocity 5
ML/min, under the conditions of temperature 60 C, has investigated different extraction times：27 seconds (s), 54 seconds (s)
Extraction efficiency with 108 seconds (s), the results are shown in Table 2.
The extraction time of table 2 investigates result (n=3)
a：Extraction efficiency is calculated by equation below
Medicinal material amount × 100% (W/W%) that the HSYA of extraction efficiency=measure is measured/weighed
Result shows that 27~54 seconds extraction times, extraction efficiency is basically unchanged, but enters one over time
Step extension, extraction efficiency is reduced on the contrary.Therefore, micro- extraction of pressurizeing need to only be extracted 27~54 seconds and can just reached
To the purpose extracted completely；And extraction time is shorter, required reagent is fewer；Therefore it is preferred that 27 seconds.
The investigation of 2.4 Extracting temperatures
Temperature is too low, and chemical composition dissolution efficiency is low；Temperature is too high, and HSYA easily degrades, and also causes
Extraction efficiency is low.It is 5mL/min flow velocity is extracted, it is right under extraction time is for the primary condition of 27s
50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C are investigated with 70 DEG C of five different Extracting temperatures, as a result
It is shown in Table 3.
The Extracting temperature of table 3 investigates result (n=3)
The data display of table 3, safflower is extracted at 55~65 DEG C, the concentration of HSYA in extract solution
It is all higher, wherein highest at 55 DEG C.Therefore, Extracting temperature is preferably 55~65 DEG C, most preferably 55 DEG C.
The investigation of 2.5 diatomite loadings
The effect of inert material is mainly dispersed sample particulate and prevents meticulous sample particles holography from carrying
Take the outlet of pipe.Therefore the different amounts of diatomaceous extraction result of filling has been investigated.The results are shown in Table 4.
The diatomite loadings of table 4 investigate result (n=3)
The result of table 4 shows that display diatomite 4mg or 6mg is little on extraction efficiency influence.But filling
Amount is more, more consolidation, and sample dispersion degree is bigger, and stability is better between batch, therefore it is preferred that diatomaceous dress
The amount of filling out is 6.0mg.
2.6 extract influence of the matrix to extraction efficiency investigates
Prepare blank extraction column：Pre-column core does not load safflower, only loads 6.0mg diatomite.
By concentration for the μ L of reference substance storing solution 2 (equivalent to 13.85 μ g HSYA) of 6.925mg/mL enter
Sample detects HSYA peak areas by the blank extraction column, is designated as " sample peak area ".Simultaneously will
Concentration for 6.925mg/mL the μ L direct injecteds of reference substance storing solution 2 to UPLC HSS T3 chromatographic columns,
The peak area of HSYA is detected, is designated as " reference substance peak area ".The results are shown in Table 5.
Table 5 extracts influence of the matrix to extraction efficiency and investigates result (n=6)
a：Matrix effect is calculated by equation below：
Matrix effect=sample peak area/reference substance peak area × 100%
The result of table 5 shows that average matrix effect is that 96.50%, RSD values are 4.50%；Show diatom
Soil does not interfere with the efficiency of the micro- extraction of online pressurized solvent.
The micro- extraction optimum condition of 2.7 safflowers pressurization
By the studies above, the preferred process conditions of the safflower micro- extraction of pressurization are established：
Flos carthami is crushed, and crosses 60~80 mesh sieves, is taken 1.0mg and is loaded in the pre-column core for eliminating filler
And be filled with 4.0~6.0mg of inert material diatomite, it is then charged into being placed in liquid phase color in pre- column sleeve
In spectrometer column oven；Column oven temperature is 55~65 DEG C, and the methanol aqueous solution with volumetric concentration 25% is
Extraction solvent, 3~5mL/min of flow velocity, 20~60 seconds extraction times；Collect efflux.
Safflower pressurization is micro- to extract most preferred process conditions and is：
Flos carthami is crushed, and crosses 60 mesh sieves, is taken 1.0mg and is loaded on and eliminates in the pre-column core of filler simultaneously
It is filled with inert material diatomite 6.0mg, is then charged into being placed in liquid chromatograph post in pre- column sleeve
In incubator；Column oven temperature be 55 DEG C, the methanol aqueous solution with volumetric concentration 25% as Extraction solvent,
Flow velocity 5mL/min, 27 seconds extraction times；Collect efflux.
Embodiment 2The micro- extracting method of safflower pressurization is investigated
The most preferred safflower micro- extraction conditions of pressurization set up with embodiment 1, and described in embodiment 1
Under chromatographic condition, methodological study is carried out.
1. the range of linearity is investigated
The μ L of sample introduction 0.1,0.2,0.5,1,2,5 are distinguished with the reference substance titer that embodiment 1 is equipped with,
Record corresponding chromatographic peak area.Linear regression is carried out to sample size X (μ g) with reference substance peak area Y.
Regression equation is：Y=5196429.6X-3599.9 (R2=1), as a result show sample size in 3.5-175ng
Linear relationship is good.
2. precision is investigated
Flos carthami powder about 1.0mg is taken, it is accurately weighed, loaded in pre-column core and using diatomite 6.0mg
After being filled, it is fitted into pre- column sleeve, obtains extraction column.Extraction column is placed in column oven, by implementation
After the condition of the most preferred flos carthami micro- extraction of pressurization that example 1 is set up is extracted, extract solution sample introduction 5
μ L, continuous sample introduction 6 times records chromatographic peak area.The results are shown in Table 6.
The precision of table 6 investigates result (n=6)
The as shown by data instrument precision of table 6 is good, and RSD values are 0.2%, meets wanting for analysis method
Ask.
3. repeatability is investigated
Flos carthami powder about 1.0mg is taken, it is accurately weighed, loaded in pre-column core and using diatomite 6.0mg
After being filled, it is fitted into pre- column sleeve, obtains extraction column.6 samples of parallel preparation.Extraction column is put
In column oven, the condition of the most preferred flos carthami micro- extraction of pressurization set up by embodiment 1 is carried
After taking, the μ L of sample introduction 5 calculate the content of HSYA.The results are shown in Table 7.
The repeatability of table 7 investigates result (n=6)
The result of table 7 shows RSD<5%, show repeated good, meet the requirement of analysis method.
4. average recovery is investigated
Flos carthami powder about 1.0mg is taken, it is accurately weighed, loaded in pre-column core and using diatomite 6.0mg
It is filled, is fitted into pre- column sleeve, parallel 6 samples.Extraction column is placed in column oven, by concentration
For 6.925mg/mL the μ L of reference substance storing solution 2 (equivalent to 13.85 μ g HSYA) sample injections in
In extraction column, the condition of the most preferred flos carthami micro- extraction of pressurization then set up by embodiment 1 is carried out
After extraction, extract solution is collected, the μ L of sample introduction 5 determine and record peak area, and the sample-adding for calculating HSYA is returned
Yield, the results are shown in Table 8.
The average recovery of table 8 investigates result (n=6)
The average recovery of the data display HSYA of table 8 is that 95.71%, RSD values are 3.64%, is met
The requirement of analysis method.
The result of the present embodiment shows, the safflower that the present invention sets up pressurize micro- extracting method stability, reappear
Property is good.
Embodiment 3A kind of micro- extracting method of safflower pressurization
Flos carthami is crushed, and crosses 60 mesh sieves, is taken 1.0mg and is loaded on and eliminates in the pre-column core of filler simultaneously
It is filled with inert material diatomite 6.0mg, is then charged into being placed in liquid chromatograph post in pre- column sleeve
In incubator；Column oven temperature be 55 DEG C, the methanol aqueous solution with volumetric concentration 25% as Extraction solvent,
Flow velocity 5mL/min, 27 seconds extraction times；Collect efflux, as Flos Carthami extract.
Using the detection mode and chromatographic condition of embodiment 1, the liquid chromatogram of the extract solution is obtained,
See Fig. 1.
Embodiment 4The comparing of the micro- extracting method of pressurization of the invention and traditional extraction process
1. the preparation of need testing solution
A. the micro- extraction of the online pressurized solvent of flos carthami：Flos carthami powder 1.0mg is loaded on pre-column core
In and be filled with inert material diatomite 6.0mg, be fitted into afterwards in pre- column sleeve, extraction column is placed in
It is Extraction solvent, 55 DEG C of Extracting temperature, flow velocity 5mL/min (pressure with 25% methyl alcohol in column oven：
3600psi), pressurised extraction 27 seconds.Extract solution is collected, analysis, record in 5 μ L injections UPLC is taken
Corresponding chromatographic peak area simultaneously calculates extraction efficiency.
B. flos carthami ultrasonic extraction：According to《Chinese Pharmacopoeia》HSYA in flos carthami in (2010 editions)
The method of assay is extracted, that is, take flos carthami powder (crossing 60 mesh sieves) about 0.1g, and precision claims
In fixed rearmounted conical flask with cover, precision adds 25% methyl alcohol 12.5mL, weighed weight, ultrasonically treated (work(
Rate 300W, frequency 5kHz) 40 minutes, let cool, then weighed weight, supply less loss with 25% methyl alcohol
Weight, shake up, filter, take the μ L of subsequent filtrate 0.5 injection UPLC in analyze, record corresponding chromatogram
Peak area simultaneously calculates extraction efficiency.
2. chromatographiccondition：
With the chromatographic condition of embodiment 1.
3. measurement result：Be shown in Table 9, at the same by two kinds of samples and solvent load of extracting method, extract when
Between etc. be also found in table 9, to compare.
The micro- extraction of the online pressurized solvent of table 9 and the comparing (n=3) of ultrasonic extraction
The data display of table 9, the micro- extracting method of pressurization of the invention compared with ultrasonic extraction, recovery rate
Improve, and extraction time can be significantly shorter, the consumption of sample and solvent is reduced, with significant excellent
Gesture.
Embodiment 5A kind of micro- extraction of safflower pressurization and the method for on-line checking
1. pressurize micro- extraction and online detection instrument
According to the micro- extraction of foundation pressurization shown in Fig. 2 and online detection instrument, wherein 1 is high pressure pump,
2 is extraction column, and 3 is analytical column, and 4 is detector, and 5 is six-way valve.
2. testing conditions：
2.1 chromatographic conditions
Analytical column：Waters CSH chromatographic columns (150mm × 4.6mm, 3.5 μm, article No.：
186005270)；
Column temperature：Room temperature；
Mobile phase：The formic acid acetonitrile (B) of 0.01% formic acid water (A) -0.01%, room temperature, flow velocity：1mL/min；
Gradient elution program is shown in Table 10.
The online pressurized solvent micro-extraction of table 10 is extracted and elution flow phase program to micro constitutent in safflower
2.2 detectors：
The triple compound tandem mass spectrometers of level Four bar linear ion hydrazine of ABSciex QTrap4500 types, use
Many reaction detection patterns (MRM) of Qtrap 4500 detect to the micro constitutent in safflower, accordingly
Mass Spectrometry Conditions are as follows：Ion gun is electrospray ion sources；Negative ion mode detects that ion injection electric is
-4500V；Temperature is 650 DEG C；Gas 1 and gas 2 in source：Nitrogen pressure is to be all 65psi；Gas
Curtain gas：Nitrogen pressure is 40.0psi；Scan mode is detected for multiple reaction.
3. extract and detect
Flos carthami powder (crossing 60 mesh sieves) 1.0mg is loaded in pre-column core and uses inert material diatomite
6.0mg is filled, and populated pre-column core is fitted into protection column sleeve and is placed in column oven, and six are led to
Position shown in valve regulation to Fig. 2A, makes extraction element be connected with analytical column.Extraction solvent is 0.01%
Formic acid water, flow velocity is 1mL/min, and temperature when column oven is extracted is 55 DEG C.Sample by high temperature,
Pressurised extraction, and in analysis on-column enrichment.After the completion of extraction, six-way valve is adjusted to shown in Fig. 2 B
Position, makes mobile phase be directly over analytical column, and the composition to being retained in analytical column front end carries out wash-out separation,
And inject in mass spectrum and be analyzed.Chromatogram is shown in Fig. 3, records corresponding peak area, as a result see Fig. 4 A and
Fig. 4 B.
4. flos carthami temperature extraction takes
Take flos carthami powder (crossing 60 mesh sieves) about 0.5g, in accurately weighed rearmounted conical flask with cover, essence
0.01% aqueous formic acid 5.0mL of close addition, weighed weight, in extracting 60 under 55 DEG C of bath temperatures
Minute, let cool, then weighed weight, the weight of less loss is supplied with 0.01% aqueous formic acid, shake up, filter
Cross, take the μ L of subsequent filtrate 10 (it is 1.0mg to convert into applied sample amount, and the applied sample amount with micro- extraction of pressurizeing is identical)
Injecting chromatograph, wash-out and detection mode with the present embodiment " pressurization micro- extraction and on-line checking ".Color
Spectrogram is shown in Fig. 3, records peak area, as a result sees Fig. 4 A and Fig. 4 B.
5. conclusion：
The extraction for obtaining is followed the example of by the micro- extraction of pressurization relatively more of the invention and on_line detection method and temperature extraction
The peak area at the respective absorption peak of liquid, it can be seen that：Same Extraction solvent (0.01% aqueous formic acid),
Under Extracting temperature (55 DEG C) and testing conditions, in the extract solution that the micro- extracting method of pressurization of the invention is obtained
The absorption peak area of each composition is all higher than the corresponding composition of the extract solution that warm leaching method is obtained.Show of the invention
Micro- extracting method of pressurizeing not only increases extraction efficiency, and has saved sample and solvent, more suitable for red
Spend the extraction and detection of middle micro constitutent.
Embodiment 6A kind of micro- extraction of saline cistanche pressurization and online test method
1. pressurize micro- extraction and online detection instrument
According to the micro- extraction of foundation pressurization shown in Fig. 5 and online detection instrument, wherein 1 is high pressure pump,
2 is extraction column, and 3 is solid phase extraction column (SPE posts), and 4 is analytical column, and 5 is detector, and 6 is six
Port valve.
The micro- extraction of pressurization and the course of work of on-line checking are：During extraction, two positions of six-way valve 6 are as schemed
Shown in 5A, extraction column 2 (now extraction column is placed in column oven) is connected with SPE posts 3, sample warp
High temperature pressurised extraction is crossed, and is enriched with SPE posts 3.After extraction terminates, two six-way valve position switchings
To as shown in Figure 5 B, SPE posts 3 are connected with analytical column 4, and the composition being retained on SPE posts 3 is anti-
It is flushed to analytical column 4 to be separated, and is detected in Injection Detector 5, records chromatogram and carry out phase
The calculating answered；Can now sample replacing be carried out to extraction column 2.
2. prepared by test sample
2.1 extraction columns
1mg is fitted into pre-column core to weigh saline cistanche medicinal material (crossing No. four sieves), and is filled out with purification on normal-phase silica gel 6mg
Fill, pre-column core is fitted into pre- column sleeve, it is stand-by.Extraction column is placed in column oven during extraction.
It is prepared by 2.2 reference substance solutions
Weigh appropriate boschnaloside E, echinacoside, acteoside, different acteoside, saline cistanche
Glycosides C, 2 '-acetyl group poliumoside, different boschnaloside C, pipe flower glycosides B, plus DMSO dissolvings are configured to
Each standard concentration is the stock solution of 4mg/mL.Take above-mentioned standard product storing solution to mix in right amount, use
50% methyl alcohol dissolved dilution is standby into the hybrid standard storing solution that each standard concentration is 400 μ g/mL.
3. extract and testing conditions：
3.1 extraction conditions
Extraction solvent：0.1% aqueous formic acid
Extracting temperature：60℃
Flow velocity：2mL/min
3.2 testing conditions
Analytical column：ACE UltraCore super C18 chromatographic column (specifications：150mm×3.0mm,2.5
μm) (article No.：CORE-25A-1002U)；
Detection wavelength：278nm；
Column temperature：Room temperature；
Mobile phase：0.1% formic acid water (A)-acetonitrile (B), room temperature；Gradient elution program is shown in Table 11.
Micro- extraction-SPE-on-line checking mobile phase the program of the saline cistanche of table 11 pressurization
4. methodological study
4.1 test limits and quantitative limit
Pre-column core only is filled with clean purification on normal-phase silica gel, is placed in pre- column sleeve, obtain blank extraction column.Will
50% methyl alcohol of hybrid standard stock solution constantly dilutes and is splined on blank extraction column, according to micro- carrying of pressurizeing
Take and be measured with the course of work of on-line checking, respectively with signal to noise ratio 3 and 10 for standard determines eachization
The detection line and quantitative limit of compound.
The results are shown in Table 12.
4.2 standard curves
Pre-column core only is filled with clean purification on normal-phase silica gel, is placed in pre- column sleeve, obtain blank extraction column.Take
Hybrid standard storing solution is appropriate, with 50% methyl alcohol dissolved dilution, is made each standard concentration and is respectively 200
μg/mL、100μg/mL、80μg/mL、50μg/mL、25μg/mL、10μg/mL、5μg/mL、
2.5μg/mL、1μg/mL.The course of work according to pressurize micro- extraction and on-line checking is measured, with
Target compound peak area is ordinate, and concentration is abscissa, draws standard curve, obtains linear regression side
Journey.
As a result：It is shown in Table 12.
The linear equation of the standard items of table 12, test limit and quantitative limit measurement result
The data display of table 12, each standard items compound is linear in test limit and quantitative limit concentration range
Relation is good, and the method sensitivity of the present embodiment is high.
4.3 precision
A, withinday precision：Pre-column core only is filled with purification on normal-phase silica gel, is placed in pre- column sleeve, obtain blank
Extraction column.Precision draw high (80 μ g/mL), in (25 μ g/mL), low (5 μ g/mL) three concentration it is mixed
Standardization solution, difference continuous sample introduction six times.The course of work according to pressurize micro- extraction and on-line checking is entered
Row is determined, and records the peak area of each compound, and calculate corresponding RSD values.
B, day to day precision：With purification on normal-phase silica gel packed column core, it is placed in pre- column sleeve.Precision draws high (80
μ g/mL), in (25 μ g/mL), low (5 μ g/mL) three mixed standard solutions of concentration, respectively continuous three
Its sample introduction, daily sample introduction three times.The course of work according to pressurize micro- extraction and on-line checking is measured,
The peak area of each compound is recorded, and calculates corresponding RSD values.
As a result：It is shown in Table 13.
The withinday precision of table 13 (n=6) and day to day precision (n=9) measurement result
The as shown by data of table 13, this method precision is good.
4.4 repeatability
About 1mg Desert Herba Cistanches sample (crossing No. four sieves) is weighed loaded in pre-column core, it is micro- according to pressurizeing
Extract and the course of work of on-line checking is measured, sample introduction simultaneously records retention time and peak area, and count
Calculate the RSD values of retention time and each compound content in the sample.
As a result：It is shown in Table 14.
4.5 average recoveries are tested
Weigh about 0.5mg Desert Herba Cistanches sample (crossing No. four sieves) and loaded in pre-column core, take high (80
μ g/mL), in (25 μ g/mL), low (5 μ g/mL) three hybrid standard product solution of concentration, according to measuring
Amount and addition calculate the rate of recovery of each compound.
As a result：It is shown in Table 14.
14 8 rate of recovery of compound (n=3) of table and repeatability (n=5)
The data display of table 14, this method repeatability is good, and average recovery result is good.Illustrate this
The method degree of accuracy is good.
5. assay
The Desert Herba Cistanches and Cistanche tubulosa 1mg of different sources and batch are taken respectively, are respectively charged into pre-
Post in-core, loads in pre- column sleeve after filling purification on normal-phase silica gel 6mg, obtains extraction column, is placed in column oven,
According to the micro- extraction of foundation pressurization shown in Fig. 6 and on-line measuring device.Extraction solvent is that 0.1% formic acid is water-soluble
Liquid, 60 DEG C of Extracting temperature extracts flow velocity 2mL/min.Testing conditions are as described under 3.2.
During extraction, as shown in Figure 5A, extraction column 2 is connected with SPE posts 3 for two positions of six-way valve 6,
Sample is extracted by high-temperature pressurizing, and is enriched with SPE posts 3.After extraction terminates, two six-way valve positions
Put and switch to as shown in Figure 5 B, SPE posts 3 are connected with analytical column 4, be retained on SPE posts 3 into
Point being sprung back over analytical column 4 is separated, and is detected in Injection Detector 5, and record chromatogram is simultaneously
Calculated accordingly.
The chromatogram of reference substance solution, Desert Herba Cistanches and Cistanche tubulosa is shown in Fig. 6 (A-C).Containing measurement
Surely 15 be the results are shown in Table.
The saline cistanche assay result of table 15 (μ g/mg, n=1)
1-8 compounds are respectively boschnaloside E (1) in table, echinacoside (2), acteoside (3),
Different acteoside (4), boschnaloside C (5), different boschnaloside C (6), 2 '-acetyl group poliumoside (7)
Glycosides B (8) is spent with pipe.
6. the present embodiment method compares with ultrasonic extraction
6.1 saline cistanche ultrasonic extractions
Desert Herba Cistanches (crossing No. four sieves) about 0.5g is taken, it is accurate in accurately weighed rearmounted conical flask with cover
Add 50% methyl alcohol 50mL, weighed weight, 40 points of ultrasonically treated (power 300W, frequency 5kHz)
Clock, is let cool, then weighed weight, and the weight of less loss is supplied with 50% methyl alcohol, is shaken up, filtration, takes continuous filter
Analyzed in the μ L of liquid 10 injections UPLC, record corresponding chromatographic peak area and calculate extraction efficiency.As a result
It is shown in Table 16.
The micro- extraction of the online pressurized solvent of table 16 and the comparing (n=5) of ultrasonic extraction
Compared in terms of Extraction solvent, solvent load, extraction time, medicinal material amount and extraction efficiency etc.
Compared with result show that saline cistanche of the invention micro- extracting method of pressurizeing can realize recovery rate higher, its
Extraction effect of the organic solvent (50% methyl alcohol) to each compound can be reached under conditions of using the aqueous solution
Really, and can significantly shorten extraction time, the consumption of sample and solvent be reduced, no matter in economic benefit
All there is significant advantage with environmental protection aspect.
The present embodiment provide the micro- extraction of pressurization and online test method, saline cistanche can be carried out quickly,
Micro, accurate qualitative and quantitative determination.Sample is enriched with also with online SPE pillars,
In the case of need not being extracted using organic solvent, the compound relatively low to polarity has still reached higher
Extraction efficiency, further illustrate the method more more environmentally friendly than traditional extracting mode.
Specific description of embodiments of the present invention above is not intended to limit the present invention, and those skilled in the art can
Various changes or deformation are made with according to the present invention, without departing from spirit of the invention, this all should be belonged to
Invention scope of the following claims.
Claims (10)
1. a kind of micro- extraction equipment of pressurization, including extraction column and pressue device, the extraction column and plus
Pressure device is connected by pipeline；The extraction column is the pre- column sleeve of liquid chromatograph and being filled with for being placed in one
Sample to be measured and the pre-column core of inert filler.
2. the micro- extraction equipment of pressurization according to claim 1, it is characterised in that the inertia is filled out
Material is selected from diatomite, purification on normal-phase silica gel, octadecyl silane, octane bonded silica gel and cellulose
One or more；
Preferably, testing sample is crushed, particle diameter≤0.5mm.
3. the micro- extraction equipment of pressurization according to claim 1 and 2, it is characterised in that it is described plus
Press-fit the high pressure pump for being set to liquid chromatograph；
Preferably, the micro- extraction equipment of pressurization also includes temperature control system；It is described to carry during extraction
Post is taken to be placed in the temperature control system；
It is furthermore preferred that column oven of the temperature control system for liquid chromatograph.
4. the micro- extraction equipment of pressurization any one of claims 1 to 3 is carried in plant chemical ingredient
Take, the application in qualitative detection and/or quantitative analysis, including plant sample to be measured fills with inert filler
In the extraction column, Extraction solvent enters in the extraction column through the pressurized equipment, collects outflow
Liquid, obtains final product plant extraction liquid to be measured, then carries out chemical composition separation, qualitative detection and/or quantitatively divides
Analysis.
5. application according to claim 4, it is characterised in that qualitative detection and/or quantitative point
The detector of analysis is selected from UV-detector, fluorescence detector, electrochemical detector, differential pulse polarograpll
One or more in device, PDAD, EISD and mass detector.
6. a kind of micro- extracting method of the pressurization of safflower, concrete operations are：
I. extraction column is loaded：Flos carthami is crushed, and crosses 60~80 mesh sieves, takes 1.0mg loaded on removal
It is filled in the pre-column core of filler and with 4.0~6.0mg of inert material diatomite, is then charged into pre-
It is placed in column sleeve in liquid chromatograph column oven；
II. pressurize micro- extraction：Column oven temperature is 55~65 DEG C, with the first of concentration expressed in percentage by volume 25%
The aqueous formic acid of alcohol solution or concentration expressed in percentage by volume 0.01% is Extraction solvent, opens phase autoclave
Pump, 3~5mL/min of flow velocity, 20~120 seconds extraction times；Collect efflux；
Preferably, the methanol aqueous solution with volumetric concentration 25% is as Extraction solvent, flow velocity 5mL/min,
27 seconds extraction times.
7. a kind of safflower pressurization it is micro- extraction and offline inspection hydroxyl radical carthamin yellow carthamus A method, specifically
Operate and be：
I. extraction column is loaded：Flos carthami is crushed, and crosses 60~80 mesh sieves, takes 1.0mg loaded on removal
It is filled in the pre-column core of filler and with 4.0~6.0mg of inert material diatomite, is then charged into pre-
It is placed in column sleeve in liquid chromatograph column oven；
II. pressurize micro- extraction：Column oven temperature is 55~65 DEG C, with the first of concentration expressed in percentage by volume 25%
Alcohol solution is Extraction solvent, opens phase autoclave pump, 3~5mL/min of flow velocity, extraction time 20~120
Second；Efflux is collected, is well mixed, that is, obtain Flos Carthami extract；
III. the μ L of Flos Carthami extract 5 that step II is obtained are taken, hydroxyl is detected in injection high performance liquid chromatograph
The content of base carthamus tinctorius yellow colour A；
Preferably, the chromatographic condition in above-mentioned steps III is：
Chromatographic column：UPLC HSS T3,100mm × 2.1mm, 1.8 μm；
Column temperature：35℃；
Mobile phase：0.01% formic acid (concentration of volume percent) water is A phases, concentration of volume percent
0.01% formic acid acetonitrile is B phases, and gradient elution is carried out according to following program：
0-5min, percent by volume 0% → 23%B, balance of A；
5-8min, percent by volume 23% → 100%B, balance of A；
Flow velocity：0.4mL/min；
Detector：Photodiode array detector, Detection wavelength is 403nm；
It is also preferred that above-mentioned steps III also includes for the μ L of reference substance solution 5 injecting high performance liquid chromatography
In instrument, wherein the reference substance solution is prepared via a method which：
Hydroxyl radical carthamin yellow carthamus A standard items are weighed, plus it is 6.925 that the dissolving of 25% methyl alcohol is configured to concentration
The stock solution of mg/mL；Take the storing solution and be diluted to Sydroxy carthamin with 25% methanol solution
A concentration is the titer of 35 μ g/mL, is obtained final product.
8. a kind of method of pressurize micro- extraction and on-line checking, including will be any in claims 1 to 3
The micro- extraction equipment of pressurization and detecting system connection described in, makes Extraction solvent enter through the pressurized equipment
In entering the extraction column, efflux is directly or through entering the detecting system after treatment；
Preferably, the detecting system includes liquid-phase chromatographic analysis post and detector, and the detector is same
Definition in claim 5；
It is also preferred that the micro- extraction equipment of pressurization passes through multi-channel valve and the liquid-phase chromatographic analysis
Post is connected；During extraction, the extraction column is connected with the liquid-phase chromatographic analysis post, open described adding
After pressure device, Extraction solvent flows into the extraction column, and the chemical composition in efflux is in the liquid phase color
Analysis of spectrum post front end is enriched with；After extraction terminates, the pressue device directly connects with the liquid-phase chromatographic column
It is logical, mobile phase is directly entered the liquid-phase chromatographic column, the chemical composition to being enriched with is eluted and divided
From eluent is detected with detector；
It is also preferred that also including SPE between the micro- extraction equipment of the pressurization and the detecting system
Pillar, pressurised extraction liquid enters back into the detecting system after purification by the solid phase extraction column.
9. the method for a kind of micro- extraction of the pressurization of safflower and on-line checking, specific operation is：
I. extraction column is loaded：Flos carthami is crushed, and crosses 60~80 mesh sieves, takes 1.0mg loaded on removal
It is filled in the pre-column core of filler and with 4.0~6.0mg of inert material diatomite, is then charged into pre-
It is placed in column sleeve in liquid chromatograph column oven；
II. the micro- extraction equipment of pressurization and detecting system are connected：The extraction column, phase autoclave pump, point
Analysis chromatographic column is connected on six-way valve, and the analysis chromatographic column and detector are connected；
III. pressurize micro- extraction：Switching six-way valve, makes the phase autoclave pump, extraction column and analysis color
Spectrum post sequential series；Column oven temperature is 50~60 DEG C, with the formic acid water of concentration expressed in percentage by volume 0.01%
Solution is Extraction solvent, opens phase autoclave pump, flow velocity 1mL/min, 3 minutes extraction times；
IV. on-line checking：After extraction terminates, six-way valve is switched immediately to be made the phase autoclave pump and divides
Analysis chromatographic column is directly connected, and is eluted and is detected；
Preferably, in the step I, diatomaceous loading is 6.0mg；
Preferably, in the step II, column oven temperature is 55 DEG C；
Preferably, in the step IV, chromatographic condition is：
Chromatographic column：Waters CSH chromatographic columns, specification 150mm × 4.6mm, 3.5 μm of fixing phase particle diameter,
Column temperature：35 DEG C,
Mobile phase：It is A phases with the aqueous formic acid of concentration expressed in percentage by volume 0.01%, it is dense with volume basis
The formic acid acetonitrile solution of degree 0.01% is B phases；Gradient elution is carried out according to following program：
0-3min, percent by volume 0% → 12%B, balance of A,
3-20min, percent by volume 12% → 30%B, balance of A,
23-26min, percent by volume 30% → 100%B, balance of A,
Flow velocity：1mL/min；
It is also preferred that in the step IV, the detector is mass detector, Mass Spectrometer Method bar
Part is：
Ion gun：Electrospray ion sources,
Detection pattern：Negative ion mode detection,
Ion injection electric：- 4500V,
Temperature：650 DEG C,
Gas 1 and gas 2 in source：All it is nitrogen, pressure is all 65psi,
Curtain gas：Nitrogen, pressure is 40.0psi,
Scan mode：Multiple reaction is detected.
10. the method for a kind of micro- extraction of the pressurization of saline cistanche and on-line checking, specific operation is：
I. extraction column is loaded：Saline cistanche pulverizing medicinal materials, cross 60~80 mesh sieves, take 1.0mg loaded on going
Except being filled in the pre-column core of filler and with 4.0~6.0mg of inert material purification on normal-phase silica gel, then fill
To enter be placed in liquid chromatograph column oven in pre- column sleeve；
II. the micro- extraction equipment of pressurization and detecting system are connected：The extraction column, phase autoclave pump, C18
Chromatographic column and solid phase extraction column are connected on a six-way valve, the C18Chromatographic column, detector and
Solid phase extraction column is connected on another six-way valve；The C18Chromatographic column and detector are connected；
III. pressurize micro- extraction：Two six-way valves of regulation, make the phase autoclave pump, extraction column solid phase
Extraction pillar sequential series；Column oven temperature is 55~65 DEG C, with the formic acid water of volumetric concentration 0.1%
Solution is Extraction solvent, opens phase autoclave pump, 2~4mL/min of flow velocity, extraction time 60~180
Second；
IV. on-line checking：After extraction terminates, two six-way valves of regulation make the phase autoclave pump, institute
Solid phase extraction column and analysis chromatographic column sequential series are stated, is eluted and is detected；
Preferably, in the step I, the loading of purification on normal-phase silica gel is 6.0mg；
Preferably, in the step III, column oven temperature is 60 DEG C；
Preferably, in the step III, the flow velocity of the aqueous formic acid of volumetric concentration 0.1% is 2
ML/min, extraction time is 180 seconds；
Preferably, in the step IV, chromatographic condition is：
Column temperature：Room temperature,
Mobile phase：It is A phases with the aqueous formic acid of concentration expressed in percentage by volume 0.1%, with acetonitrile solution as B
Phase；According to following program gradient elutions：
0-32min, 10% → 30%B, balance of A；
Flow velocity：0.25mL/min,
Detector：Photodiode array detector, Detection wavelength 278nm；
Preferably, in the step IV, also including the μ L of reference substance solution 10 are injected into the height
Effect liquid phase chromatogram instrument；The reference substance solution is prepared via a method which：
Boschnaloside E, echinacoside, acteoside, different acteoside, meat desert are weighed respectively
Rong's glycosides C, 2 '-acetyl group poliumoside, different boschnaloside C, pipe flower glycosides B standard product, plus DMSO
Dissolving is configured to the stock solution that concentration is 4mg/mL；Each standard items storing solution mixing is taken,
With 50% methyl alcohol dissolved dilution into the hybrid standard storing solution that each standard concentration is 400 μ g/mL, i.e.,
.
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