CN107907619A - A kind of blood concentration quantitative analysis method of radix astragali broken wall medicine materical crude slice active ingredient - Google Patents
A kind of blood concentration quantitative analysis method of radix astragali broken wall medicine materical crude slice active ingredient Download PDFInfo
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- CN107907619A CN107907619A CN201711010087.0A CN201711010087A CN107907619A CN 107907619 A CN107907619 A CN 107907619A CN 201711010087 A CN201711010087 A CN 201711010087A CN 107907619 A CN107907619 A CN 107907619A
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- G—PHYSICS
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Abstract
The present invention relates to a kind of blood concentration quantitative analysis method of radix astragali broken wall medicine materical crude slice active ingredient.Described method includes following steps：S1：Linear gradient solution, quality-control product solution and inner mark solution are prepared, establishes standard curve；S2：Plasma sample processing；S3：It is measured using HPLC MS；Wherein, mobile phase A is aqueous formic acid, and Mobile phase B is formic acid acetonitrile solution；Condition of gradient elution is：0.0~5.0 min：A mobile phases：85%→50%；5.0~9.0 min：A mobile phases：50%→30%；9.0~9.5 min：A mobile phases：30%→10%；9.5~12.5 min：A mobile phases：10%；12.5~13 min：A mobile phases：10% → 85%, and control mass spectrometry parameters.Quantitative analysis method provided by the invention passes through the selection to specific HPLC MS, the blood concentration that a variety of active ingredients can be achieved at the same time quantitatively detects, detection limit is in the range of 0.30~0.33ng/ml, high sensitivity, it is convenient and efficient, it can be applicable in the pharmacokinetic of the Absorption Characteristics of radix astragali broken wall medicine materical crude slice a variety of active ingredients in vivo.
Description
Technical field
The present invention relates to the technical field of radix astragali broken wall medicine materical crude slice active component detection, more particularly to a kind of radix astragali broken wall medicine materical crude slice
The blood concentration quantitative analysis method of active ingredient.
Background technology
Radix astragali is legume astragalus mongolicus Astragalus membranaceus (Fisch.)
Bge.var.mongholicus (Bge.) Hsiao or Astragalus membranacus Astragalus membranaceus (Fisch.) Bge.'s
Dry root.Radix astragali is sweet in flavor, slightly warm in nature, return lung, the spleen channel, has the work(such as tonifying Qi and lifting yang, invigorating qi for consolidating superficies, promoting pus discharge and tissue regeneration and profit water detumescence
Effect；It is clinically used for the spleen-lung Qi deficiency, sinking of qi of middle-jiao, exterior deficiency spontaneous perspiration, insufficiency of vital energy and blood, ulcer is difficult to burst, burst for a long time and does not holds back, blood stagnancy due to deficiency of QI, floating
The diseases such as swollen oliguria, hemiplegia and numbness pain numbness.
Chemical composition contained by radix astragali is broadly divided into three classes, i.e. astragaloside, flavones ingredient and astragalus polyose, additionally
There are amino acid, trace element etc., wherein astragaloside and flavones ingredient is active ingredient mostly important in radix astragali.
Chinese medicine wall cell disruption medicine materical crude slice is will to meet statutory standards to require and have cyto-architectural plant medicine medicine materical crude slice, through the modern times
Crushing technology is machined to the powder of 45 μm of D90 ﹤ (more than 300 mesh), by inducing the adhesiveness of Self substances, is made 30~100
The evenly drying graininess medicine materical crude slice of the full component of purpose original medicine materical crude slice.Compared to traditional medicine materical crude slice, Chinese medicine wall cell disruption medicine materical crude slice is homogeneous with quality, medicine
Thing utilization rate height, stability are good and using conveniently advantage.But since broken wall medicine materical crude slice diameter of particle is small, its active ingredient
In vitro dissolution rate and stripping quantity and vivo biodistribution availability also will be above traditional medicine materical crude slice, the pharmacokinetics of active ingredient
PK parameters and behavior compare also that there will be difference with traditional medicine materical crude slice.
At present, the pharmacokinetic for the radix astragali active ingredient being related in the prior art, focuses mostly on single in radix astragali
Component drugs are in terms of dynamic analysis method；Internal pharmacokinetics to reflect a variety of main actives of radix astragali at the same time comprehensively are advised
Rule, carries out the pharmacokinetic of the Absorption Characteristics of radix astragali broken wall medicine materical crude slice a variety of active ingredients in vivo, it is necessary to realizes yellow
Detected while stilbene broken wall medicine materical crude slice a variety of active ingredients, at present on rarely having report to the method for a variety of active ingredients detection at the same time
Road.
Therefore, a kind of high sensitivity is developed, fast and easily radix astragali broken wall medicine materical crude slice a variety of active ingredients detection method has
Important research significance and application value.
The content of the invention
It is a variety of it is an object of the invention to overcome detection method of the prior art to detect radix astragali broken wall medicine materical crude slice at the same time
A kind of the defects of content of active ingredient, there is provided blood concentration quantitative analysis method of radix astragali broken wall medicine materical crude slice active ingredient.This hair
The quantitative analysis method of bright offer can be achieved at the same time five kinds of active ingredient blood concentration quantitative analyses, detection limit 0.30~
In the range of 0.33ng/ml, high sensitivity is convenient and efficient.
For achieving the above object, the present invention adopts the following technical scheme that：
A kind of blood concentration quantitative analysis method of radix astragali broken wall medicine materical crude slice active ingredient, described method includes following steps：
S1：Linear gradient solution, quality-control product solution and inner mark solution are prepared, establishes standard curve；
S2：Plasma sample processing；
S3：It is measured using HPLC-MS；Wherein, mobile phase A is aqueous formic acid, and Mobile phase B is molten for formic acid acetonitrile
Liquid；Condition of gradient elution is：0.0~5.0min：A mobile phases：85% → 50%；5.0~9.0min：A mobile phases：50% →
30%；9.0~9.5min：A mobile phases：30% → 10%；9.5~12.5min：A mobile phases：10%；12.5~13min：A flows
Dynamic phase：10% → 85%.
The present invention by carrying out test of many times discovery to mobile phase and Parameters of gradient elution, using above-mentioned flowing phase composition and
During condition of gradient elution, the blood concentration that a variety of radix astragali broken wall medicine materical crude slice active ingredients can be achieved at the same time quantitatively detects, high sensitivity,
Operate quick and convenient.
Preferably, the active ingredient is Astragaloside IV, calycosin glucoside, ononin, calycosin
Or the one or several kinds in onocerin.
Preferably, the volume fraction of formic acid is 0.1% in the mobile phase A and Mobile phase B.
Preferably, Astragaloside IV, calycosin glucoside, ononin, Mao Ruiyi in the linear gradient solution
The concentration of flavones and onocerin is followed successively by 0.91~468.00ng/mL, 0.98~504.00ng/mL, 0.94~482.90ng/
At least five various concentrations in the range of mL, 0.95~489.00ng/mL and 0.97~499.80ng/mL.
It is further preferable that in the linear gradient solution Astragaloside IV concentration for 468.00ng/mL, 234.00ng/mL,
117.00ng/mL, 58.50ng/mL, 29.25ng/mL, 14.63ng/mL, 7.31ng/mL, 3.66ng/mL, 1.82ng/mL and
0.91ng/mL。
It is further preferable that in the linear gradient solution calycosin glucoside concentration for 504.00ng/mL,
252.00ng/mL、126.00ng/mL、63.00ng/mL、31.50ng/mL、15.75ng/mL、7.88ng/mL、3.94ng/mL、
1.96ng/mL and 0.98ng/mL.
It is further preferable that in the linear gradient solution ononin concentration for 482.80ng/mL, 241.40ng/mL,
120.70ng/mL, 60.35ng/mL, 30.18ng/mL, 15.09ng/mL, 7.54ng/mL, 3.77ng/mL, 1.88ng/mL and
0.94ng/mL。
It is further preferable that the concentration of calycosin is 489.00ng/mL, 244.50ng/ in the linear gradient solution
mL、122.25ng/mL、61.13ng/mL、30.56ng/mL、15.28ng/mL、7.64ng/mL、3.82ng/mL、1.90ng/mL
And 0.95ng/mL.
It is further preferable that in the linear gradient solution onocerin concentration for 499.80ng/mL, 249.90ng/mL,
124.95ng/mL, 62.48ng/mL, 31.24ng/mL, 15.62ng/mL, 7.81ng/mL, 3.90ng/mL, 1.94ng/mL and
0.97ng/mL。
Preferably, Astragaloside IV, calycosin glucoside, ononin, Mao Ruiyi are yellow in the quality-control product solution
Ketone, the concentration of onocerin be followed successively by 4.68~468.00ng/mL, 5.04~504ng/mL, 4.83~482.80ng/mL,
At least three difference in the range of 4.89~489.00ng/mL, 4.89~489.00ng/mL and 5.00~499.80ng/mL is dense
Degree.
It is further preferable that in the quality-control product solution concentration of Astragaloside IV for 468.00ng/mL, 46.80ng/mL and
4.68ng/mL。
It is further preferable that in the quality-control product solution calycosin glucoside concentration for 504.00ng/mL,
50.40ng/mL、5.04ng/mL。
It is further preferable that in the quality-control product solution ononin concentration for 482.8ng/mL, 48.280ng/mL,
4.83ng/mL。
It is further preferable that in the quality-control product solution calycosin concentration for 489.00ng/mL, 48.90ng/mL,
4.89ng/mL。
It is further preferable that in the quality-control product solution onocerin concentration for 499.80ng/mL, 49.98ng/mL,
5.00ng/mL。
Preferably, the inner mark solution is naringenin methanol solution, and the concentration of naringenin is in the inner mark solution
294ng/mL。
Preferably, the plasma sample preparation process is as follows：
S1：Water is added into radix astragali broken wall medicine materical crude slice, grinding is uniform must be for reagent liquid；
S2：Will be for reagent liquid gavage in animal body, eye socket takes blood, and separated plasma, obtains for examination blood plasma, freezing
It is spare；
S3：Inner mark solution is placed in centrifuge tube, and blood plasma, protein precipitant after defrosting are added after drying, mix, is ultrasonic,
Take supernatant to dry up after centrifugation, redissolve, gained supernatant is the plasma sample after mixing, ultrasound, centrifugation.
Preferably, it is 0.14~0.21g/ml that S1, which supplies the mass concentration of radix astragali broken wall medicine materical crude slice in reagent liquid,.
Preferably, animal described in S2 is rat, 12h fasting, free water before the gavage, described for medicine test solution
Measure as 2.5ml/100g, gavage after 5min, 15min, 30min, 1h, 2h, 3h, 4h, 6h, 8h, 12h, 24h, 36h, 48h or
72h eye sockets take blood 0.5ml, and the freezen protective condition is -20 DEG C.
Preferably, mixing, ultrasound and centrifugal condition are in S3：Vortex mixed 2min, ultrasonic 4min, 13000rpm centrifugation
5min；The protein precipitant is the formic acid solution that volume fraction is 0.1%.
Preferably, chromatographic condition is：Flow velocity is 0.2mL/min；Sampling volume is 2 μ L；Injector temperature is 20 DEG C；Chromatographic column
For Agilent XDB-C18；Column oven is 30 DEG C；Run time is 13min；It is 50% methanol to wash pin liquid.
Preferably, mass spectrometer system is the triple level Four bar mass spectrometer systems of U.S.'s Agilent.
Compared with prior art, the present invention has the advantages that：
The blood concentration quantitative analysis side of radix astragali broken wall medicine materical crude slice active ingredient provided by the invention passes through to specific flowing
Mutually with the selection of gradient concentration condition, the blood concentration that a variety of active ingredients can be achieved at the same time quantitatively detects, and detection limit is 0.30
In the range of~0.33ng/ml, high sensitivity is convenient and efficient, can be applicable to radix astragali broken wall medicine materical crude slice a variety of active ingredients in vivo
In the pharmacokinetic of Absorption Characteristics.
Brief description of the drawings
Fig. 1 is blank plasma total ion current figure result；
Fig. 2~5 are blank plasma+reference substance ion flow graph；
Fig. 6~9 are plasma sample ion flow graph；
Wherein, A is Astragaloside IV, and B is calycosin glucoside, and C is ononin, and D is calycosin, and E is
Onocerin, F are naringenin.
Embodiment
With reference to embodiment, the present invention is further explained.These embodiments are merely to illustrate the present invention rather than limitation
The scope of the present invention.The experimental method of actual conditions is not specified in lower example embodiment, usually according to this area normal condition or presses
The condition suggested according to manufacturer；Used raw material, reagent etc., unless otherwise specified, being can be from the business such as conventional market
The raw materials and reagents that approach obtains.The change for any unsubstantiality that those skilled in the art is done on the basis of the present invention
And replace and belong to scope of the present invention.
A kind of blood concentration quantitative analysis method of radix astragali broken wall medicine materical crude slice active ingredient of embodiment 1
The blood concentration quantitative analysis method of radix astragali broken wall medicine materical crude slice active ingredient provided in this embodiment includes the following steps：
The blood concentration quantitative analysis method of 5 kinds of active ingredients of radix astragali broken wall medicine materical crude slice, comprises the following steps：
(1) prepared by linear gradient solution, quality-control product solution and inner mark solution, the foundation of standard curve；
(2) plasma sample is handled；
(3) it is measured using HPLC-MS；
It is prepared by step (1) linear gradient solution, quality-control product solution and inner mark solution and the foundation of standard curve include with
Lower step and method：
The preparation of reference substance storing solution：Precision weighs Astragaloside IV, calycosin glucoside, ononin, Mao Rui
Isoflavones, onocerin in 50mL volumetric flasks, add methanol to dissolve and are diluted to scale, shake up in right amount, are stored up up to each reference substance
Standby liquid, corresponding concentration is respectively 23.4 μ gmL-1、33.6μg·mL-1、28.4μg·mL-1、32.6μg·mL-1、29.4μg·
mL-1Reference substance storing solution.
The preparation of inner mark solution：Take naringenin appropriate, it is accurately weighed, into 50mL volumetric flasks, add methanol to dissolve and dilute
To scale, shake up, it is 29.4 μ gmL to obtain concentration-1Internal standard storing solution, then it is accurate draw internal standard storing solution 1.00mL in
In 100mL volumetric flasks, add methanol constant volume, be 294ngmL up to concentration-1Inner mark solution.
The preparation of linear gradient solution and Quality Control solution：The accurate reference substance storing solution that measures is appropriate respectively, adds methanol dilution
Linear gradient solution and quality-control product solution as shown in table 1 are configured to, is preserved at 4 DEG C of refrigerator, it is spare.
1 linear gradient solution of table and Quality Control solution concentration table (ng/ml)
Above-mentioned gradient reference substance solution is injected into LC-MS instrument, is examined by mass spectrum under step (3) item and chromatographic condition sample introduction
Survey, using reference substance concentration as abscissa (X), peak area ratio is ordinate (Y), draws standard curve.
The preparation of step (2) plasma sample is comprised the following steps and method：
A：Radix astragali broken wall medicine materical crude slice is weighed, is placed in mortar, is slowly added into water, grinding is uniform, makes into 0.14~0.21g/ml.
B：Take healthy rat, 12h fasting before experiment, free water, will configure for reagent liquid 2.5ml/100g gavages to
Medicine, it is interior fasting for solids and liquids after 4h after administration, be administered before collection blank blood sample (blank plasma, can freeze spare), gavage after 5min,
15min, 30min, 1h, 2h, 3h, 4h, 6h, 8h, 12h, 24h, 36h, 48h, 72h eye sockets take blood 0.5ml, put in EDTA pipes,
5000rpm centrifuges 10min, and separated plasma, obtains spare for examination blood plasma, -20 DEG C of freezen protectives.
C：After thaw at RT sample, precision is drawn 100.0 μ L inner mark solutions and is placed in 5mL centrifuge tubes, N2 dryings, then draws
100 μ L confession examination blood plasma, vortex mixed, 400 μ L protein precipitants (0.1% formic acid) of addition, vortex mixed 2min, ultrasonic 4min,
13000rpm centrifuges 5min, and separation 400 μ L of supernatant are dried up in N2, and residue is redissolved with 150 μ L methanol, vortex mixed 2min, are surpassed
Sound 4min, 13000rpm centrifugation 5min, it is plasma sample to take supernatant.
In addition, thaw at RT blank plasma, precision is drawn 100.0 μ L inner mark solutions and is placed in 5mL centrifuge tubes, N2 dryings,
100 μ L Astragaloside IVs, calycosin glucoside, ononin, calycosin and onocerin are drawn again, and concentration is successively
In 4.68~468.00ng/mL, 5.04~504ng/mL, 4.83~482.80ng/mL, 4.89~489.00ng/mL and 5.00
In the range of~499.80ng/mL, vortex mixed, adds 400 μ L protein precipitants (0.1% formic acid), vortex mixed 2min, ultrasound
4min, 13000rpm centrifuge 5min, and separation 400 μ L of supernatant are dried up in N2, and residue is redissolved with 150 μ L methanol, vortex mixed
2min, ultrasonic 4min, 13000rpm centrifugation 5min, it is reference substance plasma sample to take supernatant.
Step (3) is measured using Liquid Chromatography/Mass Spectrometry, actual conditions such as table 2~4：
2 chromatographic condition of table
Selection uses the triple level Four bar mass spectrometer systems of U.S.'s Agilent (6470Triple Quad).
3 mass spectrum source parameter of table
4 mass spectrometry parameters of table
Quantitative analysis is carried out to the blood concentration of radix astragali broken wall medicine materical crude slice active ingredient with above-mentioned quantitative analysis method.
Measurement result：
(1) specificity is investigated
Pass through blank plasma (testing result such as Fig. 1), the blood plasma containing reference substance being prepared in plasma sample preparation process
(testing result such as Fig. 2~5) and plasma containing drug (testing result such as Fig. 6~9) respectively sampling 2 μ L, injection liquid phase-mass spectrometer into
Row detection.Such as Fig. 1~9 the results show that under the testing conditions middle endogenous material not jamming target compound and internal standard compound
The measure of matter, 5 kinds of components and internal standard substance retention time are respectively 5.566min (Astragaloside IV), 3.142min in plasma containing drug
(calycosin glucoside), 5.137min (calycosin), 4.314min (ononin), 6.756min (rest-harrows
Element), 5.916min (naringenin).
(2) linear relationship and quantitative limit, test limit are investigated
Linear gradient solution shown in table 1 is injected into LC-MS instrument, by quantitative analysis method sample introduction provided in this embodiment
Detection, using the molten concentration of linear gradient as abscissa (X), peak area ratio is ordinate (Y), draws standard curve, line is calculated
Property regression equation (such as table 5)：By the molten constantly dilution sample introduction of mixed linear gradient, provided according to Mass Spectrometer Method, signal-to-noise ratio (S/N)
3 times are test limit, and 10 times of signal-to-noise ratio are quantitative limit.
The result shows that Astragaloside IV, calycosin glucoside, ononin, calycosin, onocerin is dense
Degree successively 0.91~468.00,0.98~504.00,0.94~482.90,0.95~489.00,0.97~499.80ng
mL-1Linear relationship is good, and correlation coefficient r is all higher than being equal to 0.9954.
Astragaloside IV, calycosin glucoside, ononin, calycosin, onocerin quantitative limit are followed successively by
0.91ng·mL-1、0.98ng·mL-1、0.94ng·mL-1、0.95ng·mL-1、0.97ng·mL-1；Test limit is followed successively by
0.30ng·mL-1、0.33ng·mL-1、0.31ng·mL-1、0.32ng·mL-1、0.32ng·mL-1。
5 five kinds of component linear regression equations of table and quantitative limit, test limit form (n=3)
(3) matrix effect is investigated with the rate of recovery
Take high, medium and low three concentration quality-control sample solution and each 100 μ l of internal standard in 5ml centrifuge tubes, N2It is straight after drying
Connect plus 150 μ l methanol redissolve, by the mass spectrum in quantitative analysis method provided in this embodiment and chromatographic condition sample detection, measure
Response A.Each concentration is 6 parts parallel.
100 μ l blank plasmas are taken to be placed in 5mL centrifuge tubes, N2Drying, then 100 μ L are drawn for examination blood plasma, vortex mixed, adds
Enter 400 μ L protein precipitants (0.1% formic acid), vortex mixed 2min, ultrasonic 4min, 13000rpm centrifugation 5min, separates supernatant
400 μ L of liquid are dried up in N2, and residue adds quality-control sample solution and each 100 μ l nitrogen drying of internal standard of high, medium and low three concentration,
Residue adds 150 μ l methanol to redissolve, and by the mass spectrum in quantitative analysis method provided in this embodiment and chromatographic condition sample detection, surveys
Obtain response B.Each concentration is 6 parts parallel.
The quality-control sample solution and each 100 μ l of internal standard for taking high, medium and low three concentration are placed in 5ml centrifuge tubes, N2After drying
Add 100 μ l blank plasmas to be placed in 5mL centrifuge tubes, N2Drying, then 100 μ L are drawn for examination blood plasma, vortex mixed, adds 400 μ
L protein precipitants (0.1% formic acid), vortex mixed 2min, ultrasonic 4min, 13000rpm centrifugation 5min, 400 μ L of separation supernatant
Dried up in N2, residue adds 150 μ l methanol to redissolve, by the mass spectrum in quantitative analysis method provided in this embodiment and chromatographic condition into
Sample detects, and measures response C.Each concentration is 6 parts parallel.
Matrix effect=B/A；The rate of recovery=C/B.
From data in table, the matrix effect and rate of recovery value of five kinds of components are between 80%~120%, RSD
Respectively less than 15%, meet the requirement of biological sample quantitative analysis and guidance principle.
6 matrix effect of table and the rate of recovery (n=6)
(4) precision and accuracy test
Quality-control product (QC) sample of basic, normal, high three concentration is taken, each concentration carries out 6 sample analyses, METHOD FOR CONTINUOUS DETERMINATION three
My god, and concentration is measured with the standard curve calculating QC samples on the same day, the concentration pair with preparation with batch measure with standard curve
According to trying to achieve the preci-sion and accuracy (being shown in Table 7) of method provided in this embodiment.
7 precision of table and accuracy result (n=6)
(5) stability test
Room temperature stability
Each 1 part of the reference substance solution of high, medium and low three concentration is taken, by processing and " chromatography under " test sample treating method " item
And Mass Spectrometry Conditions " condition analysis under item, respectively at 0,1,2,4,8,12,18,24h sample introductions, 2 μ L, as a result obtain RSD every time, knot
Fruit is shown in Table 8.As shown in data in table, response RSD is respectively less than 15% in all the components 24h, meets biology sample detection requirement.
8 short-term stability experimental result (n=8) of table
Freeze-thaw stability
By -20 DEG C of preservations of quality-control sample, then pressed after 3 Frozen-thawed cycleds under item under processing and " Specification Curve of Increasing " item
Each 6 parts of the reference substance solution for high, medium and low three concentration prepared, the results are shown in Table 9.
9 five kinds of compound freeze-thaw stability experimental results (n=6) of table
Claims (9)
- A kind of 1. blood concentration quantitative analysis method of radix astragali broken wall medicine materical crude slice active ingredient, it is characterised in that the described method includes Following steps：S1：Linear gradient solution, quality-control product solution and inner mark solution are prepared, establishes standard curve；S2：Plasma sample processing；S3：It is measured using HPLC-MS；Wherein, mobile phase A is aqueous formic acid, and Mobile phase B is formic acid acetonitrile solution；Ladder Spending elution requirement is：0.0~5.0 min：A mobile phases： 85%→50%；5.0~9.0 min：A mobile phases： 50%→30%； 9.0~9.5 min：A mobile phases： 30%→10%；9.5~12.5 min：A mobile phases：10%；12.5~13 min：A flows Phase：10%→85%；Mass spectrometry parameters are：Ion gun is：Electric spray ion source（ESI）, detection mode is cation, capillary voltage For 3500V, scan mode MRM, 300 DEG C, nitrogen flow rate 5L/min of nitrogen temperature, 250 DEG C of sheath temperature degree, sheath gas 11L/ Min, spray nozzle voltage 500V.
- 2. the blood concentration quantitative analysis method of radix astragali broken wall medicine materical crude slice active ingredient according to claim 1, it is characterised in that The active ingredient is one in Astragaloside IV, calycosin glucoside, ononin, calycosin or onocerin Kind is several.
- 3. the blood concentration quantitative analysis method of radix astragali broken wall medicine materical crude slice active ingredient according to claim 1, it is characterised in that The volume fraction of formic acid is 0.1% in the mobile phase A and Mobile phase B.
- 4. the blood concentration quantitative analysis method of radix astragali broken wall medicine materical crude slice active ingredient according to claim 2, it is characterised in that Astragaloside IV in the linear gradient solution, calycosin glucoside, ononin, calycosin and onocerin Concentration is followed successively by 0.91 ~ 468.00ng/mL, 0.98 ~ 504.00ng/mL, 0.94 ~ 482.90ng/mL, 0.95 ~ 489.00ng/mL With at least five various concentrations in the range of 0.97 ~ 499.80ng/mL.
- 5. the blood concentration quantitative analysis method of radix astragali broken wall medicine materical crude slice active ingredient according to claim 2, it is characterised in that Astragaloside IV in the quality-control product solution, calycosin glucoside, ononin, calycosin and onocerin it is dense Degree be followed successively by 4.68 ~ 468.00ng/mL, 5.04 ~ 504ng/mL, 4.83 ~ 482.80ng/mL, 4.89 ~ 489.00ng/mL and At least three various concentrations in the range of 5.00 ~ 499.80ng/mL.
- 6. the blood concentration quantitative analysis method of radix astragali broken wall medicine materical crude slice active ingredient according to claim 1, it is characterised in that The inner mark solution is naringenin methanol solution, and the concentration of naringenin is 294ng/mL in the inner mark solution.
- 7. the blood concentration quantitative analysis method of radix astragali broken wall medicine materical crude slice active ingredient according to claim 1, it is characterised in that The plasma sample preparation process is as follows：S1：Water is added into radix astragali broken wall medicine materical crude slice, grinding is uniform must be for reagent liquid；S2：Will be for reagent liquid gavage in animal body, eye socket takes blood, and separated plasma, freezes spare；S3：Inner mark solution is placed in centrifuge tube, and blood plasma, protein precipitant after defrosting, mixing, ultrasound, centrifugation are added after drying After take supernatant to dry up, redissolve, mixing, ultrasound, centrifugation after gained supernatant be the plasma sample.
- 8. the blood concentration quantitative analysis method of radix astragali broken wall medicine materical crude slice active ingredient according to claim 1, it is characterised in that The mass concentration of the plasma sample is 0.14~0.21g/ml.
- 9. the blood concentration quantitative analysis method of radix astragali broken wall medicine materical crude slice active ingredient according to claim 1, it is characterised in that Chromatographic condition is：Flow velocity is 0.2mL/min；Sampling volume is 2 μ L；Injector temperature is 20 DEG C；Chromatographic column is Agilent XDB- C18；Column oven is 30 DEG C；Run time is 13min；It is 50% methanol to wash pin liquid.
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