CN109085257A - A kind of method that simultaneous quantitative detects Astragaloside IV-IV, cycloastragenol in mice plasma - Google Patents
A kind of method that simultaneous quantitative detects Astragaloside IV-IV, cycloastragenol in mice plasma Download PDFInfo
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Abstract
The present invention establishes a kind of simultaneous quantitative method in the mice plasma based on UPLC-HRMS for Astragaloside IV-IV and its main metabolites cycloastragenol.This method amount of time is 3min, using digoxin as internal standard, it is only necessary to which 20 μ L mice plasmas have the advantages that quick, high sensitivity, specificity are strong.Sample, using going phosphatide plate to filter, effectively reduces in blood plasma phospholipid endogenous metabolism object for the matrix effect of determinand after protein precipitation.It uses ultra high efficiency C18 chromatographic column for analytical column, two kinds of determinands and internal standard is detected under electric spray ion source cation Salbutamol Selected Ion Monitoring mode.The range of linearity of two kinds of determinands is 1-200ng/mL；In a few days and day to day precision≤8.6%, precision≤8.8%, show that this method precision, accuracy are good.This method is successfully applied to Astragaloside IV-IV mouse Pharmacokinetics research.
Description
Technical field
The invention belongs to field of medicaments, and in particular to Astragaloside IV-IV in a kind of mice plasma based on UPLC-HRMS,
The foundation and pharmacokinetics application of cycloastragenol simultaneous quantitative method.
Background technique
Main active of the Astragaloside IV-IV (Astragaloside IV, AST) as Chinese herb astragalus, it has
Multiple pharmacological effect, including anti-inflammatory, anti-hypertension, Cardioprotective, anti-oxidant and anti-apoptotic.It is reported that AST is also various
The potential treatment drug of metabolic syndrome.The main biologically active metabolin of AST is cycloastragenol
(Cycloastragenol, CST), it is a kind of small molecule telomerase activating agent and potential Adipogenesis inhibitor.In addition,
Recent studies have shown that AST and CST is answered in the relevant endoplasmic reticulum of inhibitory activity oxygen (Reactive oxygen species, ROS)
Swash and inhibit inflammatory factor etc. has equivalent efficacy, they can stimulate the phosphorylation of extracellular signal-regulated kinase
To improve immunity, promote the wound healing of in vitro and in vivo.Although thering is a large amount of drug action to study AST and CST, he
Pharmacological action relationship it is not yet clear.Therefore, we there is still a need for the quantitative approach using two kinds of compounds to study theirs
Pharmacokinetics and pharmacodynamics, and further assess a possibility that AST is as clinical treatment drug.
It is more existing that the method for AST or CST is individually quantified still based on liquid chromatography-mass spectrography (LC-MS or LC-MS/MS)
So there are some disadvantages, such as sample pretreatment process complexity, detection time is long, and required sample size is big or sensitivity is low.In order to same
When measure two kinds of substances, Zeng et al. is established a kind of analyzes quantifying with higher sensitivity for AST and CST in 10 minutes
Method, but negative ions pattern switching is needed in this method analytic process.The data acquisition time that another method needs to grow very much
(17 minutes).In addition, the sample size of above two method is 50 μ L blood plasma, it is not appropriate for for quantifying in mice plasma.
In addition, bioavilability in animal body is not high since AST is with higher relative molecular mass and compared with low solubility.It is comprehensive
Upper described, we come simultaneous quantitative AST and CST it is necessary to establish the higher method of sensitivity.
Summary of the invention
The object of the present invention is to provide a kind of methods of Astragaloside IV-IV, cycloastragenol in detection mice plasma.
The method of Astragaloside IV-IV, cycloastragenol in detection mice plasma provided by the present invention, including step (1) sample
The preparation of product and step (2) are detected using UPLC-HRMS.
The preparation of step (1) sample uses method comprising the following steps:
A) internal standard working solution and methanol are added into test plasma sample, obtains the blood to be measured containing internal standard and methanol
Slurry；
B) acetonitrile precipitation albumen is added into the test plasma containing internal standard and methanol, mixes, supernatant is collected in centrifugation
Liquid；
C) formic acid solution is added into the supernatant, spends the filtering of phosphatide plate, collects filtrate, obtain sample to be tested.
It is interior described in the internal standard working solution to be designated as digoxin in step a).
The internal standard working solution is prepared especially by the method included the following steps: it is suitable that precision weighs digoxin standard items
Amount, the standard solution stock solution of 1mg/mL is dissolved into methanol；With methanol dilution at concentration be 100ng/mL internal standard work it is molten
Liquid.
The volume ratio of the test plasma sample and internal standard working solution and methanol successively may be used are as follows: 2:1:1.
In step b), the volume ratio of acetonitrile and the test plasma sample can be 15:2.
The condition of the mixing can are as follows: 2000rpm, which is vortexed, mixes 5min.
The condition of the centrifugation can are as follows: 4000rpm is centrifuged 5min.
In step c), the formic acid solution is the formic acid solution of volumetric concentration 10%.
The volume ratio of the formic acid solution and the test plasma sample in step a) can are as follows: 1:1.
It is as follows that step (2) detects ultra performance liquid chromatography condition used:
Chromatographic column are as follows: ZORBAX Extend-C18RRHD；
Mobile phase uses the water containing 0.1% formic acid for A phase, the mixed liquor (first of methanol and acetonitrile containing 0.1% formic acid
The volume ratio of alcohol and acetonitrile is 50:50) it is B phase；(0.1% formic acid is the volume that formic acid is equivalent to water/methanol acetonitrile mixed liquor
Than)；
Type of elution is gradient elution；
The program of the gradient elution is as follows:
0-0.2min:B phase accounts for the 25% of mobile phase total volume；
The volume fraction of 0.2-1.0min:B phase increases to 85% by 25%；
The volume fraction maintenance 85% of 1.0-2.0min:B phase is constant；
The volume fraction of 2.0min-2.1min:B phase increases to 95% by 85%；
2.1min-2.5min:B the volume fraction maintenance 95% of phase is constant；
2.5min-2.51min:B the volume fraction of phase is reduced by 95% to 25%；
The volume fraction maintenance 25% of 2.51min-3min:B phase is constant.
It is as follows that step (2) detects Mass Spectrometry Conditions used: Q-OT-qIT heterozygous mass spectrograph is used, is furnished with ESI and the source APCI,
Using the ESI detection mode of positive ion mode, scanning mode be Salbutamol Selected Ion Monitoring (selected ion monitoring,
SIM)。
In the ultra performance liquid chromatography condition, chromatographic column be specially ZORBAX Extend-C18RRHD (2.1 × 50mm,
1.8μm；AgilentCorporation, USA).
Flow rate of mobile phase is 0.5ml/min, and gradient elution total time is 3min, and sample volume is 10 μ L, and column temperature is 40 DEG C.
In the Mass Spectrometry Conditions, other mass spectrometry parameters are as follows: spray voltage: 3800V；Sheath gas: 25；Auxiliary gas: 15；Blowback
Gas 0；Ion transfer tube temperature: 350 DEG C；Ion source temperature: 450 DEG C；Mass resolution: 120000；Window: 1Da is isolated；It is maximum
Injection length: 150ms.
The monitoring ion mass-to-charge ratio (m/z) is as follows: Astragaloside IV-IV, cycloastragenol and digoxin leading ion molecule
For [M+Na]+, mass-to-charge ratio is respectively m/z 807.4501 (AST), m/z 513.3550 (CST), m/z 803.4197 (it is high
It is pungent).
AST, CST and interior target retention time are respectively 1.93min, 2.13min, 1.66min.
The invention also includes a kind of method for detecting the content of Astragaloside IV-IV, cycloastragenol in mice plasma simultaneously, tools
Body includes the following steps:
1) preparation of standard curve: the hybrid standard product solution of the Astragaloside IV-IV, cycloastragenol that take a series of concentration add
Enter in blank plasma samples, prepared according to above-mentioned sample preparation methods, then to obtained supernatant according to above-mentioned
UPLC-HRMS method is detected, and records the corresponding peak area of Astragaloside IV-IV, cycloastragenol of each concentration respectively；With Huang
Stilbene first glycosides-IV and internal standard peak area ratio Y prepares Astragaloside IV-IV using Astragaloside IV-IV concentration X as abscissa for ordinate
Equation of linear regression；Using cycloastragenol and internal standard peak area ratio Y as ordinate, using cycloastragenol concentration X as abscissa, system
The equation of linear regression of standby cycloastragenol；
2) in test plasma sample Astragaloside IV-IV, cycloastragenol assay: by test plasma sample according to above-mentioned
Sample preparation methods prepared, then obtained supernatant is examined according to above-mentioned liquid chromatography tandem mass spectrometry
It surveys, and records Astragaloside IV-IV, the corresponding peak area of cycloastragenol respectively；Calculate Astragaloside IV-IV and internal standard peak area ratio
Y substitutes into Y value in the equation of linear regression of the Astragaloside IV-IV, and Radix Astragali first in the test plasma sample is calculated
The concentration of glycosides-IV；Cycloastragenol and internal standard peak area ratio Y are calculated, Y value is substituted into the equation of linear regression of the cycloastragenol
In, the concentration of cycloastragenol in the test plasma sample is calculated.
The method of the invention can be used successfully to the pharmacokinetic of Astragaloside IV-IV.
The present invention establishes a kind of based on ultra performance liquid chromatography-high resolution mass spectrum (Ultra-high-performance
Liquid chromatography-high-resolution mass spectrometry, UPLC-HRMS) it is quick, spirit
It is quick, the method for high-specificity, can in 3 minutes simultaneous quantitative AST and CST.In addition, the method for the present invention only needs 20 μ L blood plasma
Carry out sample preparation.After simple protein precipitation process, further by going phosphatide plate filtered sample.In positive ion electrospray
Spraying ionization (Electrospray ionization, ESI) and Salbutamol Selected Ion Monitoring (Selected ion monitoring,
SIM) under mode, in Orbitrap Fushion Lumos heterozygous mass spectrograph (Quadrupole-Orbitrap-
Quadrupole ion trap, Q-OT-qIT) on detected, and this method is applied successfully and carrys out dosed administration 30mg/kg/d
AST and CST content in the mice plasma of AST.
Detailed description of the invention
Fig. 1 be use (a) and without using go phosphatide plate (b) filter after sample introduction Astragaloside IV-IV extract ion flow chromatography
Figure.
Fig. 2 different multiples acetonitrile as Extraction solvent to Astragaloside IV-IV (AST, 100ng/mL), cycloastragenol (CST,
100ng/mL) and the extraction effect of internal standard digoxin (digoxin, 100ng/mL), (a) 1 times of acetonitrile extract result (b) 2 times of second
Nitrile extracts result (c) 3 times of acetonitriles and extracts 5 times of result (d) 7.5 times of acetonitriles extraction result (e) acetonitriles extraction results.
Fig. 3 is the extraction ion stream chromatogram of two kinds of internal standards and Astragaloside IV, AST: Astragaloside IV-IV, Rg1: ginseng soap
Glycosides Rg1, digoxin: digoxin.
Fig. 4 is the mass spectrum of Astragaloside IV-IV (a), cycloastragenol (b), digoxin (c) under cation full scan mode
Figure.
Fig. 5 be Astragaloside IV-IV (A), cycloastragenol (B), digoxin (C) resolution ratio be 15000 (I), 30000
(II), 60000 (III), 120000 (IV), mass spectrogram when 240000 (V)；Wherein, Astragaloside IV-IV, cycloastragenol is in blood
Concentration in slurry is 1.5ng/mL, digoxin concentration 50ng/mL.
Fig. 6 is Astragaloside IV-IV (1.5ng/mL), cycloastragenol (1.5ng/mL) and digoxin under different resolution
The response intensity of (50ng/mL).
Fig. 7 is Astragaloside IV-IV (I), and cycloastragenol (II), digoxin (III) is in blank plasma (A), addition 1ng/mL
Extraction ion stream chromatogram after the blood plasma (B) and administration 30mg/kg/d Astragaloside IV-IV of standard solution in blood plasma (C).
Fig. 8 is the blood concentration-time curve of Astragaloside IV-IV (AST) and cycloastragenol (CST).
Specific embodiment
The present invention will be described below by way of specific embodiments, but the present invention is not limited thereto.
Experimental method used in following embodiments is conventional method unless otherwise specified；Institute in following embodiments
Reagent, biomaterial etc., are commercially available unless otherwise specified.
Embodiment
1.1 experiment reagent
Astragaloside IV-IV (Astragaloside IV, AST), molecular formula C41H68O14, relative molecular mass 784.4609,
It is purchased from Nat'l Pharmaceutical & Biological Products Control Institute；Cycloastragenol (Cycloastragenol, CST), molecular formula C30H50O5, opposite point
Protonatomic mass 490.3658, internal standard digoxin (Digoxin), molecular formula C41H64O14, relative molecular mass 780.4296 is purchased from
Chengdu Kang Bang Biotechnology Co., Ltd, purity HPLC > 98%, sealing are kept in dark place at 2-8 DEG C.Acetonitrile, methanol, (chromatographically pure)
Purchased from German Merck company, formic acid (chromatographically pure) is purchased from U.S. Roe company.Experimental water is Wahaha Pure Water.
The acquisition of 2.2 plasma samples
24 male KM mouse (8 week old, weight 35-40g) are purchased from Beijing Vital River Experimental Animals Technology Co., Ltd..
All animals are in 12h fight-darkness cycle, constant temperature (23 ± 2 DEG C), constant humidity (60%), adaptability under ad lib conditions of water drinking
Raising one week.Each endocanthion takes 4h before blood to be deprived of food but not water.Zoopery operation follows Central University for Nationalities's Laboratory Animal Welfare
And Ethical Demand.
2.3 sample preparation
The preparation of standard solution: precision weighs Astragaloside IV-IV, and cycloastragenol, digoxin standard items are appropriate, uses respectively
Methanol is dissolved into the standard solution stock solution of 1mg/mL.It takes Astragaloside IV-IV and cycloastragenol standard solution stock solution appropriate, uses
The standard serial solution that methanol dilution is 2,3,10,30,100,200,400ng/mL at concentration.Take digoxin standard solution deposit
Appropriate liquid, the internal standard working solution for being 100ng/mL at concentration with methanol dilution.In addition precision weighs Astragaloside IV-IV and ring is yellow
Each portion of stilbene alcohol standard items, dissolves with methanol and is diluted to the standard solution stock solution of 1mg/mL, and being diluted to concentration is 3,30,
The quality of 300ng/mL controls (QC) working solution.
To the preparation of drug solns: at room temperature, it is appropriate that precision weighs sodium carboxymethylcellulose, is dissolved with water and in magnetic
It is stirred overnight on power blender.75mg Astragaloside IV-IV is weighed, is dissolved in 0.5% carboxymethylcellulose sodium solution of 50mL,
3h is mixed well using magnetic stirring apparatus, makes the final concentration of 1.5mg/ml of Astragaloside IV-IV.
Plasma sample pre-treatment: by plasma sample thaw at RT before experiment, after mixing well, 20 μ L of blank plasma is taken, is added
Enter 10 μ L of internal standard working solution, 10 μ L of methanol (or 10 μ L-IV containing Astragaloside IV, the standard solution of cycloastragenol or Quality Control are molten
Liquid), 150 μ L acetonitrile precipitation albumen are added, 2000rpm, which is vortexed, mixes 5min, after 4000rpm is centrifuged 5min, adds in supernatant
The formic acid solution for entering 20 μ L10% then spends the filtering of phosphatide plate.Take the 150 filtered samples of μ L to be measured.
2.4 laboratory apparatus and condition
2.4.1 laboratory apparatus
Pipettor (Thermo company of the U.S.)；Centrifuge and blending instrument (German Eppendorf company)；Waters
OstroTM96-Well Plate 25mg 1/Pkg goes to phosphatide plate (Waters, US).Q-OT-qIT heterozygous mass spectrograph
(Orbitrap Fusion Lumos, Thermo company of the U.S.).
2.4.2LC-MS analysis
(1) chromatographic condition
Chromatograph: ultra performance liquid chromatography (Ultramate 3000, Thermo company of the U.S.)
Chromatographic column: ZORBAX Extend-C18RRHD (2.1 × 50mm, 1.8 μm；Agilent Corporation,
USA)。
Mobile phase: A: water (0.1% formic acid) B: methanol: acetonitrile (50:50, v/v) plus 0.1% formic acid, flow velocity 0.5ml/
Min, gradient elution total time 3min, sample volume 10 μ L, 40 DEG C of column temperature.
(2) Mass Spectrometry Conditions
Mass spectrograph: Q-OT-qIT heterozygous mass spectrograph (Orbitrap Fusion Lumos, Thermo company of the U.S.) is matched
There are ESI and the source APCI.
Using the ESI detection mode of positive ion mode, scanning mode is Salbutamol Selected Ion Monitoring (selected ion
Monitoring, SIM), monitoring ion mass-to-charge ratio (m/z) is as shown in the table:
Gas used in experiment is nitrogen.Data acquisition and processing software use 2.2 data processing of Xcalibur
System.Other mass spectrometry parameters are as follows:
2.5 methodology validation
Method specificity: taking 20 μ L of blank mice plasma sample, by operating under " plasma sample pre-treatment " item, prepares blank
Sample；Certain density Astragaloside IV-IV, cycloastragenol standard solution and internal standard digoxin solution are added in blank plasma,
It is operated according to same method, prepares blank mark-on sample.Blank sample and blank mark-on sample are subjected to LC-MS analysis respectively, record phase
The chromatogram answered, the specificity for evaluation method.
The range of linearity and lower limit of quantitation: taking 20 μ L of blank mice plasma sample, and Astragaloside IV-IV, cycloastragenol mark is added
Quasi- each 10 μ L of serial solution is configured to be equivalent to Astragaloside IV-IV, cycloastragenol concentration to be 1,1.5,5,15,50,100,
The plasma sample of 200ng/ml is pressed and is operated under " plasma sample pre-treatment " item, preparation work curve.With determinand in plasma sample
Concentration is abscissa, and the peak area ratio of determinand and internal standard compound is ordinate, with weighting (W=1/x2) least square method progress
Regressing calculation, the linear regression equation acquired are working curve.Lower limit of quantitation (the Lower Limit of of analysis method
Quantification, LLOQ) it is defined as the minimum concentration of accuracy and precision in ± 20% range.When signal-to-noise ratio is 3
Testing concentration be defined as the minimum detectability (Limit of Detection, LOD) of method.
Accuracy and precision: taking 20 μ L of blank mice plasma sample, and each 10 μ L of QC working solution is added, and presses " blood plasma sample
It is operated under product pre-treatment " item, prepares basic, normal, high three concentration (respectively 3,30,300ng/ of Astragaloside IV-IV, cycloastragenol
ML Quality control samples), 6 samples of every concentration METHOD FOR CONTINUOUS DETERMINATION 3 days, according to the working curve on the same day, obtain the survey of QC sample
Obtain concentration.Calculate the relative deviation (Relative Error, RE) and relative standard deviation (Relative of QC sample
Standard Deviation, RSD), it is respectively used to the accuracy and precision of evaluation method.Wherein accuracy RE should ±
In the range of 15%, < 15% in a few days is answered with day to day precision RSD.
Extraction recovery: taking 20 μ L of blank mice plasma sample, by operating under " plasma sample pre-treatment " item, prepare it is low,
The sample of middle and high three concentration (Astragaloside IV-IV, cycloastragenol concentration are respectively 3,30,300ng/mL), 6 weights of every concentration
It is multiple.Separately take 20 μ L of blank mice plasma sample, by operating under " plasma sample pre-treatment " item, be added in the sample of acquisition it is low,
The standard solution of middle and high three concentration, 2000rpm vortex mixed carry out LC-MS analysis, obtain corresponding chromatographic peak area (three
The average value of a parallel samples).With two kinds of the ratio calculation of two kinds of processing mode chromatographic peak areas obtained of each concentration to
Survey the extraction recovery of object.
Matrix effect: taking 20 μ L of blank mice plasma respectively, by operating under " plasma sample pre-treatment " item, prepare it is low, in,
The sample of high three concentration (Astragaloside IV-IV, cycloastragenol concentration are respectively 3,30,300ng/mL), 6 repetitions of every concentration.
In addition it takes the water of equivalent to substitute mice plasma, by operating under " plasma sample pre-treatment " item, it is (yellow to prepare basic, normal, high three concentration
Stilbene first glycosides-IV, cycloastragenol concentration are respectively 3,30,300ng/mL) sample, 6 repetitions of every concentration.With two kinds of each concentration
The matrix effect of two kinds of determinands of ratio calculation of the chromatographic peak area of processing method.
Study on the stability: according to the basic, normal, high three concentration (Astragaloside IVs-of operation preparation under " plasma sample pre-treatment " item
IV, cycloastragenol concentration are respectively 3,30,300ng/mL) QC sample, investigate respectively its -80 DEG C save one month, room temperature
Save 8h, multigelation 3 times, determinand and internal standard stock solution saved 2 weeks at 2 DEG C -8 DEG C and sample treatment after exist at room temperature
The stability of 2h is saved in sample introduction bottle.By the mice plasma repeated sample of 6 10 μ g/mL determinands of addition of analysis, it is used in combination
Blank mice plasma is diluted to 1.5,15 and 150ng/mL to probe into dilution effect.
2.6 pharmacokinetics
AST 30mg/kg is administered in all mouse, upon administration 20min, 40min, 1h, 1.5h, 2h, 3h, 4h, 6h, 8h,
10h, 12h, 16h, 20h and for 24 hours endocanthion take about 100 μ L of blood.AST 30mg/kg is given in daily stomach-filling in next six days,
To assess whether repeat administration has drug accumulation effect.It takes a blood sample before administration daily.It is collected with the centrifuge tube for being coated with heparin sodium
Blood plasma is stored in -80 DEG C for use by blood, 4,000rpm centrifugation 10min.
3 results and discussion
The optimization of 3.1 sample pre-treatments conditions
According to the design feature and chemical property of determinand, in conjunction with document report, this research is for precipitation of protein, liquid
The method of liquid extraction, use acetonitrile, n-butanol, ether: methylene chloride (3:2, v/v), ethyl acetate organic solvent etc. is to extraction
Method is optimized, and since the polarity difference of two kinds of determinands is larger, the resulting extraction recovery of liquid-liquid extraction is below
50%, so finally being extracted using acetonitrile precipitation method.In addition, in order to reduce in blood plasma endogenous metabolism object as far as possible for be measured
Sample is spent the filtering of phosphatide plate before sample introduction by the interference of object, is inhibited with eliminating phosphatide micromolecular for the ion of determinand
Effect (Fig. 1).In addition, in order to obtain optimal extraction effect, it is respectively adopted 1 times of Plasma volumes used, 2 times, 3 times, 5 times, 7.5
Acetonitrile again extracts determinand, the results showed that extraction effect is best (Fig. 2) when using 7.5 times of acetonitriles.
In the measurement of biological sample, select suitable internal standard that the accuracy and precision of mass spectrometry method can be improved.Reason
The analyte in internal standard and sample thought should have similar physicochemical property and response characteristic.This investigation and comparison ginsenoside
Rg1 and digoxin are as interior target effect (Fig. 3), wherein digoxin and Astragaloside IV-IV have more similar retention time and
Response characteristic can be used as the internal standard in quantitative approach to improve the accuracy and precision of this method.
The optimization of 3.2LC-MS condition
The mass spectrum of Astragaloside IV-IV, cycloastragenol and its internal standard digoxin are examined first by the way of peristaltic pump sample introduction
ESI is respectively adopted in survey condition and the negative ions mode of two kinds of ion sources of APCI is investigated and optimized.As a result, it has been found that be measured
The Ionization Efficiency of object is better than APCI under the conditions of ESI, and positive ion mode responds height compared with negative ion mode.In positive ion mode
Under, determinand and interior target major molecular ion are [M+Na]+, mass-to-charge ratio is respectively m/z807.4501 (AST), m/z
513.3550 (CST), m/z 803.4197 (digoxin).Then to sheath gas, auxiliary gas, the mass spectrometry parameters such as blowback air carried out into
One-step optimization, to obtain the maximum mass spectrum response intensity of determinand quota ion.
SIM is two kinds in Orbitrap matter with parallel reaction monitoring (parallel reaction monitoring, PRM)
Most common quantitative approach in spectrum.The ion monitoring method that PRM and SIM is respectively adopted in this research is quantitative to determinand, discovery two
The kind determinand quantitative sensitivity of SIM method and linear PRM method, the especially CST of being better than are because having saponin(e structure and not having
Branch, it is difficult to ionize, and as the strong lipophilicity of CST and with relative molecular mass similar in phospholipid molecule, detecting
It is easy the interference by matrix effect in journey, therefore uses SIM method quantitative to two kinds of determinands, monitors the mass-to-charge ratio of ion as Huang
Stilbene first glycosides-IV m/z 807.4501, cycloastragenol m/z 513.3550, digoxin m/z803.4197.
In addition, mass resolution has biggish shadow for the sensitivity of method and specificity in the detection of SIM method
It rings.As mass resolution increases to 120,000 from 15,000, failing can be by by the total effluent of liquid chromatogram post separation
(Fig. 5) is gradually efficiently separated by mass spectrum, significantly improves specificity.This phenomenon is especially apparent (Fig. 5 B) in the detection of CST.
But further increasing with MS resolution ratio, scanning of the mass spectrum speed will affect detection spirit in trend (Fig. 6) is gradually decreased instead
Sensitivity.When mass resolution is 240,000, it can be seen that the signal strength of AST and CST significantly reduces, in 500,000 point
The signal of two kinds of determinands is nearly no detectable under resolution.Therefore, final choice 120,000 be m/z 200 when resolution ratio.Most
Afterwards, by spray voltage, evaporator temperature, sheath gas assists gas, and other mass spectrometry parameters such as scavenging and isolation window width also carry out
Optimization, to improve the response intensity of determinand to the maximum extent.
In order to which in the shortest possible time two kinds of determinands are quantified and are obtained with preferable separating effect and peak type,
Chromatographic condition is optimized.It is selected firstly for different mobile phases, the methanol/water including different proportion, acetonitrile/
Water and methanol/acetonitrile/water, as a result, it has been found that AST and CST is equal when using with methanol/acetonitrile (1:1, v/v) and water as mobile phase
There is better response.In addition, 0.1% formic acid is added in mobile phase can increase the symmetry of chromatographic peak, facilitate AST and
The ionizing efficiency of CST.Then test a series of chromatographic column in different lengths and aperture.Have finally chosen ZORBAX Extend-
C18RRHD (50 × 2.1mm, 1.8 μm) chromatographic column, can make analysis time most short and obtain optimal separation using the chromatographic column
Effect.After condition optimizing, the total time of gradient elution is 3min, and flow velocity 0.5mL/min, AST, CST and interior target are protected
Staying the time is respectively 1.93min, 2.13min, 1.66min.
3.3 methodology validation
The plasma sample of the specificity of method treated blank plasma samples, blank mark-on sample and administration animal mentions
Ion stream chromatogram is taken to see Fig. 7.The result shows that endogenous material in mice plasma is for Astragaloside IV-IV and cycloastragenol
Measurement nothing significantly interferes with.
The range of linearity and lower limit of quantitation linearly investigates the result shows that, the model of the method for this research institute foundation in 1-200ng/mL
Enclose that interior linear relationship is good, the equation of linear regression of AST and CST are respectively y=0.0129x-0.0114 (R2=0.9976) and y
=0.0375x+0.0467 (R2=0.9983).Astragaloside IV-IV lower limit of quantitation (LLOQ) 1ng/mL and detection limit (LOD) are
0.005ng/mL, the lower limit of quantitation of cycloastragenol are 1ng/mL, and detection is limited to 0.016ng/mL.
The accuracy of veracity and precision this method and precision data are shown in Table 1, wherein in a few days, day to day precision RSD
It is below 8.6%, accuracy relative deviation RE shows that this method accuracy is high within 8.8%, highly reliable.
1. Astragaloside IV-IV (AST) of table, the veracity and precision of cycloastragenol (CST)
A: actual average concentration ± standard deviation (SD)
B:SD/ actual average concentration × 100
C:(actual measurement-theoretical concentration)/theoretical concentration × 100
Two kinds of determinands of extraction recovery and matrix effect and interior target extraction recovery and matrix effect are as shown in table 2.
The extraction recovery of Astragaloside IV-IV, cycloastragenol and internal standard digoxin under 1.5,15,150ng/mL tri- concentration is above
75%, matrix effect RSD value are < 15%.Show that this method is reproducible.
All be under stability determinand and the interior condition of storage for being marked on all assessments it is stable, determinand measured concentration exists
In ± the 11.5% of theoretical concentration.The plasma sample that standard solution is added is diluted to 1.5,15 or 150ng/mL from 10 μ g/mL
When, it is found that the RE of the measured concentration of three concentration is below ± 15%, shows when testing concentration exceeds this method in sample
Method accuracy will not be significantly affected when the range of linearity.
2. Astragaloside IV-IV (AST) of table, the extraction of cycloastragenol (CST) and digoxin (digoxin) in mice plasma
The rate of recovery and matrix effect (n=18)
3.4 pharmacokinetics
By the UPLC-HRMS method of foundation applied to AST in mice plasma sample after administration 30mg/kg AST and its mainly
Metabolin CST's quantifies.The blood concentration-time curve of AST and CST is as shown in Figure 8.After stomach-filling 2 hours, AST is in blood plasma
Concentration start to increase, blood concentration maximum value (C is reached after 4 hoursmax).(every mouse is about with initial given low
It 1.2mg) compares, the C of ASTmax(59.2ng/mL) shows that its bioavilability in Mice Body is low.3 hours CST blood medicines after administration
Concentration starts to increase, TmaxFor 8h, CmaxFor 474.9ng/mL.Drug eliminated half life (the t of AST and CST1/2) it is respectively 1.85
And 0.32h.Significant drug accumulation is not observed after successive administration 7 days.Area under the curve (the 2299.9hng/ of CST
ML it) is much larger than AST (289.8hng/mL), shows that AST is likely to be and plays its drug action by being hydrolyzed to CST in vivo,
Specific mechanism of drug action is up for further studying.
4 conclusions
This research establishes the quick, sensitive, exclusive of in mice plasma simultaneous quantitative Astragaloside IV-IV and cycloastragenol
The strong HPLC-HRMS method of property.The quantitative approach only needs 20 μ L plasma samples, and lower limit of quantitation can reach 1ng/mL, when analyzing total
Between be 3min；Using digoxin as internal standard, the accuracy and precision of analysis method are improved；Acetonitrile is taken in extraction process
The extracting method that the precipitation method are combined with solid phase extractions, processing method is easy, improves extraction efficiency；Chromatographic isolation uses C18
Chromatographic column improves chromatographic isolation efficiency, shortens analysis time, it can be achieved that high-throughput isolation is analyzed；Ion monitoring mode is adopted
With Salbutamol Selected Ion Monitoring method, the sensitivity of method is improved.Methodology validation of this method Jing Guo system, accuracy is high, in a few days
Day to day precision is good, and linear relationship is good in the range of 1-200ng/mL, and can be applied in the measurement of actual sample.
Therefore, the LC-MS quantitative approach that this research is established Astragaloside IV-IV and quantifies while cycloastragenol suitable for mice plasma
Analysis.
Claims (9)
1. the method for Astragaloside IV-IV, cycloastragenol, preparation and step including step (1) sample in a kind of detection mice plasma
Suddenly (2) are detected using UPLC-HRMS；
The preparation of step (1) sample is using the method included the following steps:
A) internal standard working solution and methanol are added into test plasma sample, obtains the test plasma containing internal standard and methanol；
B) acetonitrile precipitation albumen is added into the test plasma containing internal standard and methanol, mixes, supernatant is collected in centrifugation；
C) formic acid solution is added into the supernatant, spends the filtering of phosphatide plate, collects filtrate, obtain sample to be tested.
2. according to the method described in claim 1, it is characterized by: in step a), internal standard described in the internal standard working solution
For digoxin；
The internal standard working solution passes through the method that includes the following steps and prepares: it is appropriate that precision weighs digoxin standard items, uses first
Alcohol is dissolved into the standard solution stock solution of 1mg/mL；The internal standard working solution for being 100ng/mL at concentration with methanol dilution；
The volume ratio of the test plasma sample and internal standard working solution and methanol is successively are as follows: 2:1:1；
In step b), the volume ratio of acetonitrile and the test plasma sample is 15:2；
The condition of the mixing are as follows: 2000rpm, which is vortexed, mixes 5min；
The condition of the centrifugation are as follows: 4000rpm is centrifuged 5min；
In step c), the formic acid solution is the formic acid solution of volumetric concentration 10%；
The volume ratio of the formic acid solution and the test plasma sample in step a) are as follows: 1:1.
3. method according to claim 1 or 2, it is characterised in that: step (2) detects ultra performance liquid chromatography condition used
It is as follows:
Chromatographic column are as follows: ZORBAX Extend-C18 RRHD；
Mobile phase: the water containing 0.1% formic acid is A phase, the mixed liquor of methanol and acetonitrile containing 0.1% formic acid, wherein methanol
Volume ratio with acetonitrile is 50:50, is B phase；
Type of elution is gradient elution；
The program of the gradient elution is as follows:
0-0.2min:B phase accounts for the 25% of mobile phase total volume；
The volume fraction of 0.2-1.0min:B phase increases to 85% by 25%；
The volume fraction maintenance 85% of 1.0-2.0min:B phase is constant；
The volume fraction of 2.0min-2.1min:B phase increases to 95% by 85%；
2.1min-2.5min:B the volume fraction maintenance 95% of phase is constant；
2.5min-2.51min:B the volume fraction of phase is reduced by 95% to 25%；
The volume fraction maintenance 25% of 2.51min-3min:B phase is constant.
4. method according to any one of claim 1-3, it is characterised in that: step (2) detects Mass Spectrometry Conditions used such as
Under: Q-OT-qIT heterozygous mass spectrograph is used, equipped with ESI and the source APCI；
Using the ESI detection mode of positive ion mode, scanning mode is Salbutamol Selected Ion Monitoring.
5. method according to any of claims 1-4, it is characterised in that: in the ultra performance liquid chromatography condition,
Chromatographic column is ZORBAX Extend-C18 RRHD, 2.1 × 50mm, 1.8 μm；Agilent Corporation；
Flow rate of mobile phase is 0.5ml/min, and gradient elution total time is 3min, and sample volume is 10 μ L, and column temperature is 40 DEG C.
6. method according to any one of claims 1-5, it is characterised in that: in the Mass Spectrometry Conditions, other mass spectrums ginseng
Number is as follows: spray voltage: 3800V；Sheath gas: 25；Auxiliary gas: 15；Blowback air 0；Ion transfer tube temperature: 350 DEG C；Ion source temperature
Degree: 450 DEG C；Mass resolution: 120000；Window: 1Da is isolated；Maximum injection length: 150ms.
7. according to the method described in claim 4, it is characterized by: the monitoring ion mass-to-charge ratio (m/z) is as follows: Radix Astragali first
Glycosides-IV, cycloastragenol and digoxin leading ion molecule are [M+Na]+, mass-to-charge ratio is respectively m/z 807.4501, AST；m/
Z 513.3550, CST, m/z 803.4197, digoxin；
AST, CST and interior target retention time are respectively 1.93min, 2.13min, 1.66min.
8. a kind of method for detecting the content of Astragaloside IV-IV, cycloastragenol in mice plasma simultaneously, includes the following steps:
1) preparation of standard curve: the hybrid standard product solution of the Astragaloside IV-IV, cycloastragenol that take a series of concentration are added empty
It in white plasma sample, is prepared according to above-mentioned sample preparation methods, then to obtained supernatant according to above-mentioned UPLC-
HRMS method is detected, and records the corresponding peak area of Astragaloside IV-IV, cycloastragenol of each concentration respectively；With Radix Astragali first
Glycosides-IV and internal standard peak area ratio Y prepares the line of Astragaloside IV-IV using Astragaloside IV-IV concentration X as abscissa for ordinate
Property regression equation；Using cycloastragenol and internal standard peak area ratio Y as ordinate, using cycloastragenol concentration X as abscissa, ring is prepared
The equation of linear regression of Astragenol；
2) in test plasma sample Astragaloside IV-IV, cycloastragenol assay: by test plasma sample according to above-mentioned sample
Prepared by product preparation method, then detect to obtained supernatant according to above-mentioned liquid chromatography tandem mass spectrometry, and
Astragaloside IV-IV, the corresponding peak area of cycloastragenol are recorded respectively；Astragaloside IV-IV and internal standard peak area ratio Y is calculated, by Y
Value substitutes into the equation of linear regression of the Astragaloside IV-IV, and Astragaloside IV-IV in the test plasma sample is calculated
Concentration；Cycloastragenol and internal standard peak area ratio Y are calculated, Y value is substituted into the equation of linear regression of the cycloastragenol, is calculated
Obtain the concentration of cycloastragenol in the test plasma sample.
9. application of the method described in claim 1 or 8 in the pharmacokinetic of Astragaloside IV-IV.
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