CN109085257B

CN109085257B - Method for simultaneously and quantitatively detecting astragaloside IV and cycloastragenol in mouse plasma

Info

Publication number: CN109085257B
Application number: CN201810730008.1A
Authority: CN
Inventors: 王中华; 何秉淑; 陈路路; 再帕尔·阿不力孜
Original assignee: Minzu University of China
Current assignee: Minzu University of China
Priority date: 2018-07-05
Filing date: 2018-07-05
Publication date: 2021-04-06
Anticipated expiration: 2038-07-05
Also published as: CN109085257A

Abstract

The invention establishes a UPLC-HRMS-based method for simultaneously quantifying in mouse plasma aiming at astragaloside IV and a main metabolite thereof, namely cycloastragenol. The method has the advantages of rapid speed, high sensitivity and strong specificity, and takes digoxin as an internal standard, and only 20 mu L of mouse plasma is needed. After protein precipitation, the sample is filtered by using a phospholipid removing plate, so that the matrix effect of the phospholipid endogenous metabolites in the plasma on the substances to be detected is effectively reduced. An ultra-high performance C18 chromatographic column is used as an analytical column, and two substances to be detected and an internal standard are detected in an electrospray ion source positive ion selective ion monitoring mode. The linear range of the two substances to be detected is 1-200 ng/mL; the precision in and during the day is less than or equal to 8.6 percent, and the precision is less than or equal to 8.8 percent, which shows that the method has good precision and accuracy. The method is successfully applied to the research of the pharmacokinetics of the astragaloside IV mice.

Description

Method for simultaneously and quantitatively detecting astragaloside IV and cycloastragenol in mouse plasma

Technical Field

The invention belongs to the field of medicines, and particularly relates to establishment of a method for simultaneously quantifying astragaloside IV and cycloastragenol in mouse plasma based on UPLC-HRMS and application of pharmacokinetics.

Background

Astragaloside IV (AST) is used as the main active component of astragalus root, and has several pharmacological actions including antiphlogistic, antihypertensive, cardioprotective, antioxidant and anti-apoptosis. AST is also reported to be a potential therapeutic agent for various metabolic syndromes. The major bioactive metabolite of AST is Cycloastragenol (CST), a small molecule telomerase activator and a potential adipogenesis inhibitor. In addition, recent studies have shown that AST and CST have equal efficacy in inhibiting Reactive Oxygen Species (ROS) -related endoplasmic reticulum stress and inflammatory factors, etc., and they can stimulate phosphorylation of extracellular signal-regulated protein kinase to improve immunity and promote wound healing in vitro and in vivo. Although there are a large number of pharmacodynamic action studies on AST and CST, their pharmacological action relationship is not clear yet. Therefore, we still need to utilize quantitative methods of both compounds to study their pharmacokinetics and pharmacodynamics and further evaluate the potential of AST as a clinical therapeutic.

The existing methods for separately quantifying the AST or the CST based on liquid chromatography-mass spectrometry (LC-MS or LC-MS/MS) still have some defects, such as complex sample pretreatment process, long detection time, large required sample amount or low sensitivity. In order to measure two substances simultaneously, Zeng et al established a quantitative method with high sensitivity for the analysis of AST and CST within 10 minutes, but this method requires positive and negative ion mode switching during the analysis. Another method requires a long data acquisition time (17 minutes). In addition, the sample size of both methods is 50 μ L plasma, and is not suitable for quantification in mouse plasma. In addition, the bioavailability in animals is not high due to the higher relative molecular mass and lower solubility of AST. In view of the above, it is necessary to establish a more sensitive method for simultaneous quantification of AST and CST.

Disclosure of Invention

The invention aims to provide a method for detecting astragaloside IV and cycloastragenol in mouse plasma.

The method for detecting astragaloside IV and cycloastragenol in mouse plasma provided by the invention comprises the steps of (1) sample preparation and (2) UPLC-HRMS detection.

The preparation of the sample in the step (1) adopts a method comprising the following steps:

a) adding an internal standard working solution and methanol into a plasma sample to be detected to obtain plasma to be detected containing the internal standard and the methanol;

b) adding acetonitrile precipitated protein into the to-be-detected plasma containing the internal standard and methanol, uniformly mixing, centrifuging, and collecting supernatant;

c) and adding a formic acid solution into the supernatant, filtering by using a phospholipid removing plate, and collecting filtrate to obtain a sample to be detected.

In the step a), the internal standard in the internal standard working solution is digoxin.

The internal standard working solution is prepared by a method comprising the following steps: precisely weighing a proper amount of digoxin standard substance, and dissolving the digoxin standard substance into 1mg/mL standard solution stock solution by using methanol; diluted with methanol to an internal standard working solution with a concentration of 100 ng/mL.

The volume ratio of the plasma sample to be detected to the internal standard working solution and the methanol can be sequentially as follows: 2:1:1.

In step b), the volume ratio of acetonitrile to the plasma sample to be tested may be 15: 2.

The blending condition can be as follows: vortex at 2000rpm for 5 min.

The conditions of the centrifugation may be: centrifuge at 4000rpm for 5 min.

In step c), the formic acid solution is 10% by volume.

The volume ratio of the formic acid solution to the plasma sample to be tested in step a) may be: 1:1.

The conditions of the ultra-high performance liquid chromatography used in the step (2) are as follows:

the chromatographic column is as follows: ZORBAX extended-C18 RRHD;

the mobile phase adopts water containing 0.1% formic acid as phase A, and a mixed solution of methanol and acetonitrile containing 0.1% formic acid (the volume ratio of the methanol to the acetonitrile is 50: 50) as phase B; (0.1% formic acid is the volume ratio of formic acid to the mixture of water/methanol acetonitrile);

the elution mode is gradient elution;

the procedure for the gradient elution was as follows:

0-0.2 min: the phase B accounts for 25% of the total volume of the mobile phase;

0.2-1.0 min: the volume fraction of phase B increased from 25% to 85%;

1.0-2.0 min: the volume fraction of the phase B is kept constant at 85 percent;

2.0min-2.1 min: the volume fraction of phase B increased from 85% to 95%;

2.1min-2.5 min: the volume fraction of the phase B is kept constant at 95%;

2.5min-2.51 min: the volume fraction of the B phase is reduced from 95% to 25%;

2.51min-3 min: the volume fraction of phase B remained constant at 25%.

The mass spectrum conditions used in the detection in the step (2) are as follows: a Q-OT-qIT hybrid mass spectrometer is adopted, an ESI source and an APCI source are arranged, an ESI detection mode of a positive ion mode is adopted, and a scanning mode is Selected Ion Monitoring (SIM).

In the condition of the ultra-high performance liquid chromatography, the chromatographic column is ZORBAX extended-C18 RRHD (2.1 × 50mm,1.8 μm; Agilent corporation, USA).

The flow rate of the mobile phase is 0.5ml/min, the total time of gradient elution is 3min, the sample amount is 10 mu L, and the column temperature is 40 ℃.

In the mass spectrum conditions, other mass spectrum parameters are as follows: spraying voltage: 3800V; sheath gas: 25; auxiliary gas: 15; back blowing 0; ion transfer tube temperature: 350 ℃; ion source temperature: at 450 ℃; mass spectrum resolution: 120000; isolating the window: 1 Da; maximum injection time: 150 ms.

The monitored ion mass-to-charge ratios (m/z) are as follows: the main ion molecules of astragaloside IV, cycloastragenol and digoxin are [ M + Na]⁺The mass-to-charge ratios were m/z807.4501(AST), m/z 513.3550(CST), and m/z803.4197 (digoxin), respectively.

Retention times for AST, CST, and internal standard were 1.93min, 2.13min, 1.66min, respectively.

The invention also comprises a method for simultaneously detecting the content of astragaloside IV and cycloastragenol in the plasma of a mouse, which comprises the following steps:

1) preparation of a standard curve: adding mixed standard solutions of astragaloside IV and cycloastragenol with a series of concentrations into a blank plasma sample, preparing according to the sample preparation method, detecting the obtained supernatant according to the UPLC-HRMS method, and respectively recording the peak areas corresponding to the astragaloside IV and the cycloastragenol with each concentration; taking the peak area ratio Y of the astragaloside IV to the internal standard as a vertical coordinate, and taking the concentration X of the astragaloside IV as a horizontal coordinate to prepare a linear regression equation of the astragaloside IV; taking the peak area ratio Y of the cycloastragenol to the internal standard as a vertical coordinate, and taking the concentration X of the cycloastragenol as a horizontal coordinate, and preparing a linear regression equation of the cycloastragenol;

2) determining the content of astragaloside IV and cycloastragenol in the blood plasma sample to be detected: preparing a plasma sample to be detected according to the sample preparation method, detecting the obtained supernatant according to the liquid chromatography tandem mass spectrometry, and respectively recording peak areas corresponding to astragaloside IV and cycloastragenol; calculating the peak area ratio Y of the astragaloside IV to the internal standard, substituting the value of Y into the linear regression equation of the astragaloside IV, and calculating to obtain the concentration of the astragaloside IV in the plasma sample to be detected; and calculating the peak area ratio Y of the cycloastragenol to the internal standard, substituting the value of Y into the linear regression equation of the cycloastragenol, and calculating to obtain the concentration of the cycloastragenol in the plasma sample to be detected.

The method can be successfully used for the pharmacokinetic research of the astragaloside IV.

The invention establishes a rapid, sensitive and high-specificity method based on Ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS), and AST and CST can be quantified simultaneously within 3 minutes. Furthermore, the method of the invention requires only 20 μ L of plasma for sample preparation. After a simple protein precipitation process, the sample was further filtered through a phospholipid plate. Detection was performed on an Orbitrap Fushion Lumos hybrid mass spectrometer (Quadrupole-Orbitrap-Quadrupole ion trap, Q-OT-qIT) in positive ion Electrospray ionization (ESI) and Selective Ion Monitoring (SIM) modes, and this method was successfully applied to quantify AST and CST levels in the plasma of mice administered with 30mg/kg/d AST.

Drawings

FIG. 1 is an ion flow chromatogram of an astragaloside IV extract injected after filtration using (a) and without a dephosphatation plate (b).

FIG. 2 shows the extraction effects of astragaloside IV (AST, 100ng/mL), cycloastragenol (CST, 100ng/mL) and digoxin (100 ng/mL) using different amounts of acetonitrile as extraction solvents, (a) the extraction results of 1-fold acetonitrile, (b) the extraction results of 2-fold acetonitrile, (c) the extraction results of 3-fold acetonitrile, (d) the extraction results of 5-fold acetonitrile, (e) the extraction results of 7.5-fold acetonitrile.

FIG. 3 is the ion flow chromatogram of two internal standards and astragaloside IV, AST is astragaloside IV, Rg1 is ginsenoside Rg1, digoxin is digoxin.

FIG. 4 is the mass spectrum of astragaloside IV (a), cycloastragenol (b), digoxin (c) under positive ion full scan mode.

FIG. 5 shows the mass spectra of Astragaloside IV (A), cycloastragenol (B), and digoxin (C) at the resolution of 15000(I),30000(II),60000(III),120000(IV), and 240000 (V); wherein, the concentration of astragaloside IV and cycloastragenol in blood plasma is 1.5ng/mL, and the concentration of digoxin is 50 ng/mL.

FIG. 6 shows the response intensity of astragaloside IV (1.5ng/mL), cycloastragenol (1.5ng/mL) and digoxin (50ng/mL) at different resolutions.

FIG. 7 is the ion flow chromatogram of the extracts of astragaloside IV (I), cycloastragenol II, digoxin III in blank plasma A, plasma B with 1ng/mL standard solution and plasma C after administration of astragaloside IV 30 mg/kg/d.

FIG. 8 is a plasma concentration-time curve of astragaloside IV (AST) and Cycloastragenol (CST).

Detailed Description

The present invention will be described below with reference to specific examples, but the present invention is not limited thereto.

The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, biomaterials, etc. used in the following examples are commercially available unless otherwise specified.

Examples

1.1 Experimental reagents

Astragaloside IV (Astragaloside IV, AST) with molecular formula C₄₁H₆₈O₁₄Relative molecular mass 784.4609, purchased from china pharmaceutical biologicals institute; cycloastragenol (CST), molecular formula C₃₀H₅₀O₅Relative molecular mass 490.3658, internal standard Digoxin (Digoxin), molecular formula C₄₁H₆₄O₁₄Relative molecular mass 780.4296, available from WUDUCHAN Biotech GmbH, purity HPLC>98 percent, sealed and protected from light and stored at 2-8 ℃. Acetonitrile, methanol, (chromatographically pure) from Merck, germany, formic acid (chromatographically pure) from Roe, usa. The water for the experiment is the Wahaha purified water.

2.2 plasma sample Collection

24 male KM mice (8 weeks old, 35-40g in body weight) were purchased from experimental animal technology ltd, viton, beijing. All animals were acclimatized for one week under 12h light-dark cycle, constant temperature (23 + -2 deg.C), constant humidity (60%), free food and water. No water is forbidden 4h before blood is taken from inner canthus every time. Animal experimental procedures followed the welfare and ethical requirements of the laboratory animals of the central ethnic university.

2.3 sample preparation

Preparing a standard solution: accurately weighing proper amount of astragaloside IV, cycloastragenol and digoxin standard substance, and dissolving with methanol to obtain 1mg/mL standard solution stock solution respectively. Taking a proper amount of astragaloside IV and a proper amount of cycloastragenol standard solution stock solution, and diluting the stock solution with methanol into standard series solutions with the concentrations of 2,3,10,30,100,200 and 400 ng/mL. And taking a proper amount of digoxin standard solution stock solution, and diluting the digoxin standard solution stock solution into an internal standard working solution with the concentration of 100ng/mL by using methanol. In addition, accurately weighing one part of each of astragaloside IV and cycloastragenol standard substance, dissolving with methanol, diluting into 1mg/mL standard solution stock solution, and diluting into Quality Control (QC) working solution with concentration of 3,30,300 ng/mL.

Preparation of dosing solution: at room temperature, an appropriate amount of sodium carboxymethylcellulose was precisely weighed, dissolved with water and stirred overnight on a magnetic stirrer. Weighing 75mg of astragaloside IV, dissolving in 50mL of 0.5% sodium carboxymethylcellulose solution, and mixing thoroughly for 3h with magnetic stirrer to make the final concentration of astragaloside IV be 1.5 mg/mL.

Plasma sample pretreatment: before experiment, plasma samples are unfrozen at room temperature, after the plasma samples are fully and uniformly mixed, 20 mu L of blank plasma is taken, 10 mu L of internal standard working solution and 10 mu L of methanol (or 10 mu L of standard solution or quality control solution containing astragaloside IV and cycloastragenol) are added, 150 mu L of acetonitrile is added for precipitating protein, the mixture is evenly mixed by vortex at 2000rpm for 5min, after the mixture is centrifuged at 4000rpm for 5min, 20 mu L of 10% formic acid solution is added into supernate, and then the mixture is filtered by a phospholipid removing plate. 150 μ L of the filtered sample was taken for testing.

2.4 Experimental instruments and conditions

2.4.1 Experimental instruments

Pipettors (Thermo corporation, usa); centrifuge and mixer (Eppendorf, germany); waters Ostro^TM96-Well Plate 25mg 1/Pkg phospholipid-free Plate (Waters, USA). Q-OT-qIT hybrid mass spectrometer (Orbitrap Fusion Lumos, Thermo Co., USA).

2.4.2LC-MS analysis

(1) Chromatographic conditions

Chromatograph: ultra high performance liquid chromatography (Ultramate 3000, Thermo Co., USA)

A chromatographic column: ZORBAX extended-C18 RRHD (2.1X 50mm,1.8 μm; Agilent Corporation, USA).

Mobile phase: water (0.1% formic acid) B methanol: acetonitrile (50: 50, v/v) is added with 0.1% formic acid, the flow rate is 0.5ml/min, the total time of gradient elution is 3min, the sample amount is 10 mu L, and the column temperature is 40 ℃.

(2) Conditions of Mass Spectrometry

Mass spectrometry: Q-OT-qIT hybrid mass spectrometer (Orbitrap Fusion Lumos, Thermo Co., USA) was equipped with ESI and APCI sources.

The ESI detection mode in positive ion mode, scanning mode was Selected Ion Monitoring (SIM), and monitored ion mass-to-charge ratio (m/z) is shown in the following table:

the gas used in the experiment was nitrogen. The data acquisition and processing software adopts an Xcaliibur 2.2 data processing system. Other mass spectral parameters were as follows:

2.5 methodological validation

The method has the characteristics that: taking 20 mu L of blank mouse plasma sample, and preparing a blank sample according to the item of 'plasma sample pretreatment'; adding a certain concentration of astragaloside IV, a cycloastragenol standard solution and an internal standard digoxin solution into blank plasma, and preparing a blank labeled sample according to the same method. And respectively performing LC-MS analysis on the blank sample and the blank labeled sample, and recording corresponding chromatograms for evaluating the specificity of the method.

Linear range and quantitative lower limit: taking 20 mu L of blank mouse plasma sample, adding 10 mu L of each standard series solution of astragaloside IV and cycloastragenol to prepare the plasma sample with the concentration of 1, 1.5, 5,15, 50, 100 and 200ng/ml of astragaloside IV and cycloastragenol, and preparing a working curve according to the operation under the item of 'plasma sample pretreatment'. Taking the concentration of the substance to be detected in the plasma sample as an abscissa and the peak area ratio of the substance to be detected to the internal standard as an ordinate, and weighting (W is 1/x)²) And performing regression operation by using a least square method, wherein the obtained linear regression equation is the working curve. The Lower Limit of quantitation (LLOQ) of the analytical method is defined as the lowest concentration with accuracy and precision in the range of. + -. 20%. The concentration of the analyte at a signal-to-noise ratio of 3 is defined as the lowest Limit of Detection (LOD) of the method.

Accuracy and precision: taking 20 mu L of blank mouse plasma sample, adding 10 mu L of QC working solution respectively, preparing quality control samples with low, medium and high concentrations (respectively 3,30 and 300ng/mL) of astragaloside IV and cycloastragenol according to the item of 'plasma sample pretreatment', continuously measuring for 3 days for each concentration of 6 samples, and obtaining the measured concentration of the QC sample according to the working curve of the day. The Relative Deviation (RE) and Relative Standard Deviation (RSD) of the QC samples were calculated and used to evaluate the accuracy and precision of the methods, respectively. Wherein the accuracy RE should be within 15% and the daily and diurnal precision RSD should be < 15%.

Extraction recovery rate: taking 20 mu L of blank mouse plasma sample, preparing samples with low, medium and high concentrations (the concentrations of astragaloside IV and cycloastragenol are respectively 3,30 and 300ng/mL) according to the operation under the item of 'plasma sample pretreatment', and repeating 6 times per concentration. And taking 20 mu L of blank mouse plasma sample, operating according to the item of 'plasma sample pretreatment', adding standard solutions with low, medium and high concentrations into the obtained sample, carrying out vortex mixing at 2000rpm, and carrying out LC-MS analysis to obtain corresponding chromatographic peak areas (average values of three parallel samples). And calculating the extraction recovery rate of the two to-be-detected substances according to the ratio of the chromatographic peak areas obtained by the two treatment modes at each concentration.

Matrix effect: respectively taking 20 mu L of blank mouse plasma, preparing samples with low, medium and high concentrations (the concentrations of astragaloside IV and cycloastragenol are respectively 3,30 and 300ng/mL) according to the operation under the item of 'plasma sample pretreatment', and repeating 6 times per concentration. In addition, equivalent water is taken to replace the plasma of the mouse, samples with low, medium and high concentrations (the concentrations of the astragaloside IV and the cycloastragaloside are respectively 3,30 and 300ng/mL) are prepared according to the operation under the item of 'plasma sample pretreatment', and 6 times of the samples are repeated for each concentration. And calculating the matrix effect of the two to-be-detected substances according to the ratio of the chromatographic peak areas of the two treatment methods at each concentration.

And (3) stability investigation: QC samples with low, medium and high concentrations (the concentrations of astragaloside IV and cycloastragenol are respectively 3,30 and 300ng/mL) are prepared according to the item of 'plasma sample pretreatment', the QC samples are respectively inspected to be stored for one month at minus 80 ℃, stored for 8h at room temperature, repeatedly frozen and thawed for 3 times, the to-be-detected object and the internal standard stock solution are stored for 2 weeks at 2-8 ℃, and the stability of the to-be-detected object and the internal standard stock solution after sample treatment is stored for 2h in a sample injection vial at room temperature. The effect of dilution was explored by analyzing 6 replicate samples of mouse plasma to which 10. mu.g/mL of test substance was added and diluting them to 1.5,15 and 150ng/mL with blank mouse plasma.

2.6 pharmacokinetics

AST 30mg/kg was administered to all mice, and approximately 100. mu.L of angular blood was collected at 20min, 40min, 1h, 1.5h, 2h, 3h, 4h, 6h, 8h, 10h, 12h, 16h, 20h and 24h post-administration. AST 30mg/kg was gavaged daily for the next six days to assess whether repeated dosing would have a drug accumulating effect. Blood was collected daily before dosing. Blood was collected in a centrifuge tube coated with heparin sodium, centrifuged at 4,000rpm for 10min, and plasma was stored at-80 ℃ until use.

3 results and discussion

3.1 optimization of sample pretreatment conditions

According to the structural characteristics and chemical properties of the substance to be detected and combined with literature reports, acetonitrile, n-butanol and diethyl ether are used for protein precipitation and liquid-liquid extraction: the extraction method is optimized by dichloromethane (3:2, v/v), ethyl acetate organic solvent and the like, and the extraction recovery rate obtained by liquid-liquid extraction is lower than 50 percent due to the large polarity difference of two substances to be detected, so that the extraction is finally carried out by adopting an acetonitrile precipitation method. In addition, in order to minimize the interference of the endogenous metabolites in plasma with the analyte, the sample was filtered with a phospholipid-removing plate before injection to eliminate the ion-inhibiting effect of phospholipid-like small molecules on the analyte (fig. 1). In addition, in order to obtain the best extraction effect, acetonitrile with the volume of 1 time, 2 times, 3 times, 5 times and 7.5 times of the used plasma is respectively used for extracting the object to be detected, and the result shows that the extraction effect is best when the acetonitrile with the volume of 7.5 times is used (figure 2).

In the determination of biological samples, the accuracy and precision of mass spectrometry methods can be improved by selecting an appropriate internal standard. An ideal internal standard should have similar physicochemical properties and response characteristics as the analyte in the sample. The research compares the effects of ginsenoside Rg1 and digoxin as internal standards (figure 3), wherein digoxin and astragaloside IV have relatively similar retention time and response characteristics, and can be used as the internal standards in a quantitative method to improve the accuracy and precision of the method.

3.2 optimization of LC-MS conditions

Firstly, the mass spectrum detection conditions of astragaloside IV, cycloastragenol and digoxin as internal standard thereof are respectively inspected and optimized by adopting positive and negative ion modes of ESI and APCI ion sources in a peristaltic pump sample injection mode. The result shows that the ionization efficiency of the analyte is better than APCI under the ESI condition, and the positive ion mode has higher response than the negative ion mode. In positive ion mode, the main molecular ion of the object to be detected and the internal standard is [ M + Na ]]⁺The mass-to-charge ratios were m/z807.4501(AST), m/z 513.3550(CST), and m/z803.4197 (digoxin), respectively. And then further optimizing mass spectrum parameters such as sheath gas, auxiliary gas, back-blowing gas and the like to obtain the maximum mass spectrum response intensity of quantitative ions of the object to be detected.

SIM and Parallel Reaction Monitoring (PRM) are the two most commonly used quantification methods on Orbitrap mass spectrometry. The study respectively adopts PRM and SIM ion monitoring methods to quantify the objects to be tested, finds that the sensitivity and linearity of the two objects to be tested quantified by the SIM method are superior to those of the PRM method, particularly CST has saponin structure and no branched chain, is difficult to ionize, and is easily interfered by matrix effect in the detection process due to the strong lipophilicity of CST and the relative molecular mass close to phospholipid molecules, so the SIM method is adopted to quantify the two objects to be tested, and the mass-to-charge ratio of the monitoring ions is astragaloside IV m/z807.4501, cycloastraganol m/z 513.3550 and digoxin m/z 803.4197.

In addition, when the SIM method is used for detection, the mass spectrometry resolution has a large influence on the sensitivity and specificity of the method. As the mass spectral resolution increased from 15,000 to 120,000, co-effluents that failed to be separated by the liquid chromatography column could gradually be effectively separated by mass spectrometry (fig. 5), making the specificity significantly higher. This phenomenon is particularly evident in the detection of CST (fig. 5B). However, as the resolution of the MS is further increased, the scanning speed of the mass spectrometer gradually decreases (fig. 6), which adversely affects the detection sensitivity. When the mass spectrum resolution was 240,000, a significant decrease in AST and CST signal intensity was observed, and almost no signals of both analytes were detected at a resolution of 500,000. Therefore, a resolution of 120,000 at m/z 200 is finally selected. And finally, optimizing other mass spectrum parameters such as spray voltage, evaporator temperature, sheath gas, auxiliary gas, scavenging gas, isolation window width and the like so as to improve the response intensity of the object to be detected to the maximum extent.

In order to quantify two kinds of objects to be measured in as short a time as possible and to obtain a good separation effect and a good peak pattern, the chromatographic conditions are optimized. First, different mobile phases were selected, including different ratios of methanol/water, acetonitrile/water and methanol/acetonitrile/water, and as a result, it was found that both AST and CST responded better when using both methanol/acetonitrile (1: 1, v/v) and water as the mobile phase. In addition, the addition of 0.1% formic acid in the mobile phase increases the symmetry of the chromatographic peaks, contributing to the ionization efficiency of AST and CST. A series of chromatographic columns of different lengths and pore sizes were subsequently tested. Finally, a ZORBAX extended-C18 RRHD (50X 2.1mm, 1.8 μm) column was selected, with which the analysis time could be minimized and the best separation effect could be obtained. After condition optimization, the total time of gradient elution is 3min, the flow rate is 0.5mL/min, and the retention time of AST, CST and internal standard is 1.93min, 2.13min and 1.66min respectively.

3.3 methodological validation

The chromatogram of the extracted ion current of the processed blank plasma sample, the blank labeled sample and the plasma sample of the administration animal is shown in figure 7. The result shows that the endogenous substances in the blood plasma of the mice have no obvious interference on the determination of the astragaloside IV and the cycloastragenol.

The linear range and quantitative lower limit linear investigation result shows that the method established in the research has good linear relation in the range of 1-200ng/mL, and the linear regression equations of AST and CST are respectively that y is 0.0129x-0.0114 (R)²0.9976) and y 0.0375x +0.0467 (R)²0.9983). The lower limit of astragaloside IV quantification (LLOQ) is 1ng/mL, the detection Limit (LOD) is 0.005ng/mL, the lower limit of cycloastragaloside quantification is 1ng/mL, and the detection limit is 0.016 ng/mL.

The accuracy and precision of the method are shown in table 1, wherein the accuracy RSD in day and day is lower than 8.6%, and the relative accuracy deviation RE is within 8.8%, which shows that the method has high accuracy and strong reliability.

TABLE 1 accuracy and precision of astragaloside IV (AST), Cycloastragenol (CST)

a: mean measured concentration. + -. Standard Deviation (SD)

b: SD/average measured concentration X100

c: (actual measurement-theoretical concentration)/theoretical concentration X100

Extraction recovery and matrix effect the extraction recovery and matrix effect of the two analytes and the internal standard are shown in table 2. The extraction recovery rate of the astragaloside IV, the cycloastragenol and the internal standard digoxin is higher than 75% under the three concentrations of 1.5,15 and 150ng/mL, and the RSD value of the matrix effect is less than 15%. The method is proved to have good repeatability.

Stability both the test substance and the internal standard were stable under all assessed storage conditions, with the measured concentration of the test substance being within ± 11.5% of the theoretical concentration. When plasma samples to which the standard solution was added were diluted from 10. mu.g/mL to 1.5,15 or 150ng/mL, the measured concentrations of RE were found to be less than + -15% for all three concentrations, indicating that the accuracy of the method was not significantly affected when the concentration of the test substance in the sample was outside the linear range of the method.

Table 2. extraction recovery and matrix effect of astragaloside iv (ast), Cycloastragenol (CST) and digoxin (digoxin) in mouse plasma (n ═ 18)

3.4 pharmacokinetics

The established UPLC-HRMS method was applied to the quantification of AST and its major metabolite CST in plasma samples of mice administered 30mg/kg AST. The plasma concentration-time curves for AST and CST are shown in fig. 8. After 2 hours of gavage, the AST concentration in the plasma began to rise and reached a maximum plasma concentration (C) after 4 hours_max). C of AST compared to initial gavage dose (about 1.2mg per mouse)_max(59.2ng/mL) indicated that it had low bioavailability in mice. 3 hours after administration, CST blood concentration began to rise, T_maxIs 8h, C_maxIt was 474.9 ng/mL. Drug elimination half-life (t) of AST and CST_1/2) 1.85 and 0.32h respectively. No significant drug accumulation was observed after 7 days of continuous dosing. The area under the curve of CST (2299.9 h.ng/mL) is much larger than AST (289.8 h.ng/mL), which indicates that AST may exert its pharmacodynamic action through in vivo hydrolysis to CST, and the specific mechanism of action of the drug is to be further studied.

4 conclusion

The research establishes a rapid, sensitive and highly specific HPLC-HRMS method for simultaneously quantifying astragaloside IV and cycloastragenol in the plasma of a mouse. The quantitative method only needs 20 mu L of plasma sample, the lower limit of the quantitative method can reach 1ng/mL, and the total analysis time is 3 min; digoxin is used as an internal standard, so that the accuracy and precision of the analysis method are improved; in the extraction process, an extraction method combining an acetonitrile precipitation method and solid phase extraction is adopted, the treatment method is simple and convenient, and the extraction efficiency is improved; the chromatographic separation adopts a C18 chromatographic column, so that the chromatographic separation efficiency is improved, the analysis time is shortened, and high-throughput separation analysis can be realized; the ion monitoring mode adopts a selective ion monitoring method, so that the sensitivity of the method is improved. The method is verified by systematic methodology, has high accuracy, good daytime precision and good linear relation within the range of 1-200ng/mL, and can be applied to the determination of actual samples. Therefore, the LC-MS quantitative method established by the research is suitable for simultaneous quantitative analysis of astragaloside IV and cycloastragenol in the plasma of the mouse.

Claims

1. A method for detecting astragaloside IV and cycloastragenol in mouse plasma comprises the steps of (1) sample preparation and (2) UPLC-HRMS detection;

c) adding a formic acid solution into the supernatant, filtering by using a phospholipid removing plate, and collecting filtrate to obtain a sample to be detected;

in the step a), the internal standard in the internal standard working solution is digoxin;

the internal standard working solution is prepared by a method comprising the following steps: precisely weighing a proper amount of digoxin standard substance, and dissolving the digoxin standard substance into 1mg/mL standard solution stock solution by using methanol; diluting the solution into internal standard working solution with the concentration of 100ng/mL by using methanol;

the volume ratio of the plasma sample to be detected to the internal standard working solution and the methanol is as follows in sequence: 2:1: 1;

in the step b), the volume ratio of acetonitrile to the plasma sample to be detected is 15: 2;

the blending condition is as follows: vortex at 2000rpm and mix for 5 min;

the centrifugation conditions were: centrifuging at 4000rpm for 5 min;

in the step c), the formic acid solution is 10% by volume;

the volume ratio of the formic acid solution to the plasma sample to be detected in the step a) is as follows: 1:1.

2. The method of claim 1, wherein: the conditions of the ultra-high performance liquid chromatography used in the step (2) are as follows:

the chromatographic column is as follows: ZORBAX extended-C18 RRHD;

mobile phase: taking water containing 0.1% formic acid as phase A, and a mixture of methanol and acetonitrile containing 0.1% formic acid, wherein the volume ratio of methanol to acetonitrile is 50: 50, is phase B;

the elution mode is gradient elution;

the procedure for the gradient elution was as follows:

0.2-1.0 min: the volume fraction of phase B increased from 25% to 85%;

1.0-2.0 min: the volume fraction of the phase B is kept constant at 85 percent;

2.0min-2.1 min: the volume fraction of phase B increased from 85% to 95%;

2.1min-2.5 min: the volume fraction of the phase B is kept constant at 95%;

2.5min-2.51 min: the volume fraction of the B phase is reduced from 95% to 25%;

2.51min-3 min: the volume fraction of phase B remained constant at 25%.

3. The method of claim 1, wherein: the mass spectrum conditions used in the detection in the step (2) are as follows: adopting a Q-OT-qIT hybrid mass spectrometer which is provided with an ESI source and an APCI source;

and adopting an ESI detection mode of a positive ion mode, wherein the scanning mode is selected ion monitoring.

4. The method of claim 1, wherein: in the condition of the ultra-high performance liquid chromatography, a chromatographic column is ZORBAX extended-C18 RRHD, 2.1 multiplied by 50mm,1.8 mu m;

the flow rate of the mobile phase is 0.5ml/min, the total time of gradient elution is 3min, the sample injection amount is 10 mu L, and the column temperature is 40 ℃.

5. The method of claim 1, wherein: in the mass spectrum conditions, other mass spectrum parameters are as follows: spraying voltage: 3800V; sheath gas: 25; auxiliary gas: 15; back blowing 0; ion transfer tube temperature: 350 ℃; ion source temperature: 450 ℃; mass spectrum resolution: 120000; isolating the window: 1 Da; maximum injection time: 150 ms.

6. The method of claim 3, wherein: the monitored ion mass-to-charge ratios (m/z) are as follows: astragaloside IV and cyclovirobuxineThe main ionic molecules of the stilbenol and the digoxin are [ M + Na]⁺Mass to charge ratios of m/z807.4501, AST; m/z 513.3550, CST, m/z803.4197, digoxin;

7. Use of the method of claim 1 in the pharmacokinetic study of astragaloside IV.