CN112630339A - Method for simultaneously and quantitatively measuring 4 blood-entering components in agilawood alcohol extract - Google Patents

Abstract

The invention discloses a method for simultaneously and quantitatively measuring 4 blood-entering components in an agilawood alcohol extract. The 4 blood components include linalool, isolinalool, 4' -methoxy linalool and 8-chloro-2 (2-phenylethyl) -5,6, 7-trihydroxy-5, 6,7, 8-tetrahydrochromone. The method provided by the invention has the advantages of high sensitivity, stability and accuracy, can accurately, reliably and simultaneously quantitatively determine the concentration of 4 components in blood plasma obtained at different time points after the agilawood alcohol extract is administrated by gavage of rats, and can provide technical support for the pharmacokinetics research of the agilawood alcohol extract.

Description

Method for simultaneously and quantitatively measuring 4 blood-entering components in agilawood alcohol extract

Technical Field

The invention belongs to the technical field of pharmacokinetic analysis of traditional Chinese medicines, and particularly relates to a method for simultaneously and quantitatively determining 4 blood-entering components in an agilawood alcohol extract.

Background

The lignum Aquilariae Resinatum is resin-containing wood of Aquilaria sinensis (Lour.) Gilg of Thymelaeaceae, and is mainly distributed in southeast Asia and Guangdong and Hainan provinces in China, with pungent, bitter, and mild-warm taste, and enters spleen, stomach, and kidney meridians; has effects of activating qi-flowing, relieving pain, warming middle energizer, relieving vomit, receiving qi, and relieving asthma, and can be used for treating abdominal distention, pain, stomach cold, emesis, singultus, and asthma due to deficiency of the kidney. Modern pharmacological research finds that the agilawood has the effects of relieving pain, calming, resisting inflammation, resisting tumors, depression, insomnia, bacteria, ulcer, spasmolysis, acetylcholinesterase inhibition and the like. Researches show that the agilawood component mainly comprises sesquiterpenes and 2- (2-phenethyl) chromone components.

The absorption of the traditional Chinese medicine components into blood is the prerequisite basis for the efficacy of the traditional Chinese medicine components, and the pharmacokinetic behavior of the traditional Chinese medicine components in the body has important significance on the efficacy activity, the administration dosage and the administration time. However, after the traditional Chinese medicine is taken, the exposure level of the effective components in blood plasma is low, and the integral evaluation of the pharmacokinetic behavior of the traditional Chinese medicine is limited, so that the establishment of a sensitive and accurate method for measuring the blood-entering components of the traditional Chinese medicine is the basis of the pharmacokinetic research of the traditional Chinese medicine.

In the present stage, chemical and pharmacological researches on agilawood are more, but pharmacokinetics researches on agilawood are not reported yet, and the wide application of agilawood in pharmacological research and clinic is severely restricted. Meanwhile, the research laboratory finds that the agaroterol, the isoagaroterol, the 4 ' -methoxy agaroterol and the 8-chloro-2 (2-phenylethyl) -5,6, 7-trihydroxy-5, 6,7, 8-tetrahydrochromone can be detected in plasma collected after the agaroterol extract is administrated by intragastric lavage to rats, and the agaroterol and the 8-chloro-2 (2-phenylethyl) -5,6, 7-trihydroxy-5, 6,7, 8-tetrahydrochromone are quality control index components of a control characteristic map of ' Chinese pharmacopoeia ' of 2015 edition. Therefore, the method for simultaneously and quantitatively measuring the 4 blood components of the agilawood is established, the pharmacokinetic characteristics of the agilawood are further researched, and the method has important significance for deep research and development of the medicinal resources of the agilawood.

Disclosure of Invention

The invention aims to find 4 blood-entering components in the agilawood alcohol extract, establish and provide a UHPLC-MS/MS (ultra high performance liquid chromatography-mass spectrometry) determination method for the 4 blood-entering components, and provide technical support for further researching the pharmacokinetic characteristics of the agilawood alcohol extract.

According to the invention, pharmacokinetics research shows that 4 components of the linalool, the isolinalool, the 4' -methoxy linalool and the 8-chloro-2 (2-phenylethyl) -5,6, 7-trihydroxy-5, 6,7, 8-tetrahydrochromone can be detected in plasma of rats after the rats are gavaged with the linalool extract, so that the 4 components are determined to be blood-entering components of the linalool extract. A method for simultaneously and quantitatively determining the 4 blood components in the agilawood alcohol extract in the plasma of the rat by adopting a UHPLC-MS/MS technology is established, so that the aim of the invention is realized.

The method for simultaneously and quantitatively measuring 4 blood-entering components in the agilawood alcohol extract comprises the following steps of:

(1) preparation of stock solution: precisely weighing reference substances respectively, and preparing single reference substance stock solutions of 0.540mg/mL of agarotetrol, 0.803mg/mL of isoagaroterol, 0.346mg/mL of 4' -methoxy agarotetrol, 8-chloro-2 (2-phenylethyl) -5,6, 7-trihydroxy-5, 6,7, 8-tetrahydrochromone and 0.151mg/mL of wrinkled gianthyssop flavonol respectively by using HPLC-grade methanol as a solvent;

(2) preparation of working solution: respectively and precisely measuring a single reference substance stock solution of linalool, isolinalool, 4 '-methoxy-linalool and 8-chloro-2 (2-phenylethyl) -5,6, 7-trihydroxy-5, 6,7, 8-tetrahydrochromone, diluting the single reference substance stock solution by using HPLC-grade methanol by 10 times, and respectively and precisely measuring diluent to prepare a mixed reference substance solution, wherein the concentration of 4' -methoxy-linalool is 1000ng/mL, the concentration of linalool is 2000ng/mL, the concentration of isolinalool is 2000ng/mL, and the concentration of 8-chloro-2 (2-phenylethyl) -5,6, 7-trihydroxy-5, 6,7, 8-tetrahydrochromone is 2000 ng/mL; precisely measuring the mixed reference substance solution, adding HPLC-grade methanol to gradually dilute to obtain a series of standard working solutions with concentration, precisely measuring the stock solution of the single reference substance of Agastache Rugosa flavonol, and adding HPLC-grade methanol to dilute to obtain an internal standard working solution with concentration of 500 ng/mL;

(3) establishment of a standard curve: precisely absorbing 100 mu L of blank plasma, adding 20 mu L of internal standard working solution, respectively adding 20 mu L of standard working solution, vortexing for 30s, then adding 260 mu L of acetonitrile (v: v ═ 1:100) containing 1% formic acid, vortexing for 60s, centrifuging at 14000rpm and 4 ℃ for 10min, taking supernate, passing through a 0.22 mu m microporous filter membrane, and performing UHPLC-ESI-MS/MS analysis; taking the ratio of the peak area of the component to be detected to the peak area of the internal standard as a vertical coordinate (y), taking the concentration of a reference substance of the component to be detected added into plasma as a horizontal coordinate (x), and performing linear regression by adopting a 1/x weighted least square method to establish a curve;

(4) and (3) detection of the sample: precisely sucking 100 mu L of rat drug-containing plasma, adding 20 mu L of internal standard working solution, vortexing for 30s, then adding 280 mu L of acetonitrile containing 1% formic acid (v: v ═ 1:100), vortexing for 60s, centrifuging at 14000rpm and 4 ℃ for 10min, taking supernate, filtering the supernate with a 0.22 mu m microporous filter membrane, carrying out UHPLC-MS/MS analysis, and calculating the concentration of 4 components in a plasma sample according to a following standard curve;

4 blood-entering components in the agarol extract are agarotetraol, isoagarotetraol, 4' -methoxy agarotetraol and 8-chloro-2 (2-phenethyl) -5,6, 7-trihydroxy-5, 6,7, 8-tetrahydrochromone respectively.

Chromatographic conditions of the UHPLC-MS/MS analysis are as follows: the column was an ACQUITY UPLC BEH C18 column, 100mm X2.1 mm, 1.8 μm, mobile phase A: b is acetonitrile: 0.2% formic acid water, gradient elution program is 0-1.0 min, 15% A; 1.00-4.00 min, 15 → 40% A; 4.00-5.00 min, 40 → 70% A; 5.00-5.50 min, 70 → 80% A; 5.50-7.50 min, 80% A; 7.50-7.51 min, 80 → 95% A; 7.51-9.00 min, 95% A; 9.00-9.01 min, 95 → 15% A; 9.01-11.00 min, 15% A; the flow rate is 0.3 mL/min; the column temperature is 30 ℃; the sample injection amount is 2 mu L;

mass spectrum conditions: electrospray (ESI) ion source, positive ion detection mode; capillary voltage 4000V; spraying gas (N2) pressure 45psi, drying gas (N2) flow rate 10L/min, drying gas (N2) temperature 325 ℃; the flow rate of the sheath gas (N2) is 11L/min, and the temperature of the sheath gas (N2) is 350 ℃; nozzle voltage 500V; the scanning mode is Multiple Reaction Monitoring (MRM); the mass spectral parameters are given in the following table:

retention time and mass spectral parameters for quantitative analysis of target compounds

Quantitative ions

The invention has the beneficial effects that:

the invention discovers 4 blood-entering components in the agilawood alcohol extract: the method comprises the steps of taking 4 blood-entering components as detection indexes and using Agastache rugosa flavonol as an internal standard, and establishing an analysis method for determining 4 components in the agilawood alcohol extract in rat plasma by adopting an ultra high liquid chromatography tandem mass spectrometry (UHPLC-MS/MS), wherein the method can accurately and reliably simultaneously determine the concentrations of the 4 components in the agilawood alcohol extract in the rat plasma obtained at different time points after the agilawood alcohol extract is administrated by rat gavage, and can provide technical support for researching the pharmacokinetic characteristics of the agilawood alcohol extract.

Drawings

FIG. 1 is a graph of Multiple Reaction Monitoring (MRM) of 4 bleeding components and an internal standard, in which: a is blank plasma, B is blank plasma, standard working solution of reference substance and internal standard working solution, and C is plasma containing medicine; 1 IS linalool, 2 IS isolinalool, 3 IS 4' -methoxy linalool, 4 IS 8-chloro-2 (2-phenylethyl) -5,6, 7-trihydroxy-5, 6,7, 8-tetrahydrochromone, and IS IS interior standard Agastache flavonol.

FIG. 2 is the mean drug concentration-time curve of 4 blood-entering components after three doses of the linalol extract are gavaged in rats.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1:

1. apparatus and materials

1.1 instruments

Agilent1290 HPLC-6470Triple Quard MS (Agilent Inc., USA); sidoris TP-114 electronic balance (Sartorious, USA), H1850R centrifuge (Hunan instruments laboratory, Inc., Hunan), XW-80A rapid mixer (Kalima Kaisha).

1.2 materials

Aquilaria tetrol (batch number: RFS-C07411812016, purity: not less than 98%), isoagaroterol (batch number: RFS-Y22701904002, purity: not less than 98%), 4' -methoxyagaroterol (batch number: RFS-DFZY-200407, purity: not less than 90%) and 8-chloro-2 (2-phenylethyl) -5,6, 7-trihydroxy-5, 6,7, 8-tetrahydrochromone (batch number: RFS-DFZY-200407, purity: not less than 98%) were purchased from Chengdu Elfin scientific and Tech Biotech limited, and patchouli flavonol (batch number: P18A11S111514, purity: not less than 98%) were purchased from Shanghai leaf Biotech limited. Methanol, ethanol and acetonitrile are used as chromatographic pure reagents (Merck company in Germany) and distilled water (Drech company in hong Kong); formic acid (Fisher scientific, USA); ammonium acetate (Sigma-Aldrich, USA).

SPF grade SD rat, male, body weight 210 + -20 g, provided by Guangdong provincial animal center for medical experiment, rat qualification number SYXK (Guangdong) 2018-.

2. Method of producing a composite material

2.1 chromatographic conditions

A chromatographic column: waters BEH C18, 2.1 mm. times.100 mm, 1.7 μm, guard column: waters Van Guard BEH C18, 2.1 mm. times.5 mm, 1.7 μm, mobile phase A: b is acetonitrile: 0.2% formic acid water, gradient elution program is 0-1.0 min, 15% A; 1.00-4.00 min, 15 → 40% A; 4.00-5.00 min, 40 → 70% A; 5.00-5.50 min, 70 → 80% A; 5.50-7.50 min, 80% A; 7.50-7.51 min, 80 → 95% A; 7.51-9.00 min, 95% A; 9.00-9.01 min, 95 → 15% A; 9.01-11.00 min, 15% A; the flow rate is 0.3 mL/min; the column temperature is 30 ℃; the sample injection amount is 2 mu L;

2.2 Mass Spectrometry conditions

Electrospray (ESI) ion source, positive ion detection mode; capillary voltage 4000V; spraying gas (N2) pressure 45psi, drying gas (N2) flow rate 10L/min, drying gas (N2) temperature 325 ℃; the flow rate of the sheath gas (N2) is 11L/min, and the temperature of the sheath gas (N2) is 350 ℃; nozzle voltage 500V; the scanning mode is Multiple Reaction Monitoring (MRM); the mass spectral parameters are shown in table 1:

TABLE 1 Retention time and Mass Spectrometry parameters for quantitative analysis of target Compounds

Quantitative ions

2.3 preparation of solution

2.3.1 preparation of stock solutions: precisely weighing reference substances, respectively placing the reference substances in 10mL volumetric flasks, adding HPLC-grade methanol to fully dissolve the reference substances and fixing the volume, respectively preparing a single reference substance stock solution of 0.540mg/mL of agarotetraol, 0.803mg/mL of isoagarotetraol, 0.346mg/mL of 4' -methoxy agarotetraol, 8-chloro-2 (2-phenylethyl) -5,6, 7-trihydroxy-5, 6,7, 8-tetrahydrochromone and 0.151mg/mL of agastache flavonol, respectively transferring the single reference substance stock solution into a liquid storage bottle, and placing the liquid storage bottle in a refrigerator at the temperature of-20 ℃ for later use;

2.3.2 preparation of working solution: precisely measuring single reference substance stock solutions of agarotetraol, isoagarotetraol, 4 '-methoxy agarotetraol and 8-chloro-2 (2-phenylethyl) -5,6, 7-trihydroxy-5, 6,7, 8-tetrahydrochromone respectively, diluting the single reference substance stock solutions by 10 times by using methanol, and precisely measuring diluent solutions respectively to prepare mixed reference substance solutions, wherein the 4' -methoxy agarotetraol is 1000ng/mL, the agarotetraol is 2000ng/mL, the isoagarotetraol is 2000ng/mL, and the 8-chloro-2 (2-phenylethyl) -5,6, 7-trihydroxy-5, 6,7, 8-tetrahydrochromone is 2000 ng/mL; precisely measuring the mixed reference substance solution, adding methanol, diluting gradually to obtain a series of standard working solutions with concentration, precisely measuring Agastache Rugosa flavonol stock solution, diluting with methanol to obtain an internal standard working solution with concentration of 500ng/mL, and storing in refrigerator at-20 deg.C for use.

2.4 plasma sample processing method

Rat blank plasma 100 μ L was placed in a 1.5mL EP tube, 20 μ L of internal standard working solution was added, vortexed for 30s, then 280 μ L of 1% formic acid acetonitrile (v: v ═ 1:100), vortexed for 60s, 14000rpm, centrifuged at 4 ℃ for 10min, the supernatant was passed through a 0.22 μm microfiltration membrane and subjected to UHPLC-MS/MS analysis (see 2.1 and 2.2).

2.5 analysis methodology investigation

2.5.1 specialization examination

The blank plasma, the blank plasma and the standard working solution of 6 rats with different sources are taken, and the plasma containing the medicine is processed and analyzed by the sample injection according to the item of 2.4. Comparing the chromatograms of the three, and observing whether the component to be detected and the internal standard have interference. As shown in FIG. 1, the retention time of the component to be measured and the internal standard are respectively 3.57min for linalool, 3.83min for isolinalool, 3.62min for 4' -methoxy linalool, 5.20min for 8-chloro-2 (2-phenylethyl) -5,6, 7-trihydroxy-5, 6,7, 8-tetrahydrochromone, and 6.50min for Agastache flavonol. And as a result, the area of the hetero peak at the retention time of the substance to be detected is less than 20% of the area of the lower limit of quantitation (LLOQ) peak, and the area of the hetero peak at the retention time of the internal standard is less than 5% of the area of the internal standard peak, which shows that the method has good specificity, and endogenous substances in plasma do not interfere with the determination of the component to be detected and the internal standard.

2.5.2 Linear Range and lower quantitative limits:

a series of standard working solutions with a series of concentrations, 20 μ L, were sequentially added to rat blank plasma of 100 μ L, and a standard curve was established by performing the procedure under "2.4". The concentration of the component to be measured is taken as a horizontal coordinate, the peak area ratio of the component to be measured and an internal standard is taken as a vertical coordinate, weighted least square regression calculation is adopted, the weight coefficient is 1/X, the results of a standard curve, a correlation coefficient, a linear range and a quantitative Lower Limit (LLOQ) are shown in a table 2, and the results show that the component to be measured has a good linear relation in each concentration range.

TABLE 24 Standard Curve, correlation coefficient, Linear Range and lower quantitative bound for the ingredients

2.5.3 precision and accuracy test

Quality control samples (QC) with three concentrations of quantitative lower limit, low concentration, medium concentration and high concentration of 4 component rat plasma are prepared respectively according to the item of '2.4' (prepared by adding standard working solution to blank rat plasma), each concentration is analyzed by 6 samples, continuous sample injection is carried out in the day, and a follow-up standard curve is prepared. Preparing 6 parts of each series of concentration, continuously measuring for 3d, substituting into the following standard curve, respectively calculating the concentration of each component, and calculating the precision and accuracy within and during the day. The results of the precision and accuracy of the 4 components are shown in table 3, the data show that the precision and accuracy are within the standard range, and the results show that the method has good precision and accuracy and can be used for analyzing biological samples.

Precision and accuracy within and between days for the 34 components of Table

2.5.4 matrix Effect and extraction recovery test

Taking 100 mu L of rat blank plasma, preparing quality control samples (QC) with three concentrations of 4 components, namely low, medium and high rat plasma (prepared by adding standard working solution into the blank rat plasma) under the item of '2.4', analyzing 6 samples at each concentration, and operating the rest under the item of 2.4 (A sample); separately, 100. mu.L of rat blank plasma was taken, and standard solutions (each concentration was analyzed for 6 samples) (sample B) having the concentration corresponding to the above were added to the obtained supernatant by the procedure under "2.4" except that the mixed standard solution was not added; separately taking 100 μ L of pure water, preparing quality control samples (QC) with low, medium and high water concentrations, analyzing 6 samples at each concentration, and performing the other operations under the item of '2.4' (C sample). The extraction recovery rate calculation method is the ratio of the chromatographic peak areas of the components to be detected of the sample A and the sample B, and the matrix effect calculation method comprises the following steps: and respectively calculating the peak area ratio of the chromatographic peak areas of the component to be measured and the internal standard of the sample B and the sample C to obtain the matrix factor of the component to be measured and the internal standard, and further dividing the matrix factor of the component to be measured by the matrix factor of the internal standard to calculate the normalized matrix factor, namely the matrix effect. The results of the matrix effect and the extraction recovery rate of the 4 components are shown in table 4, and the data show that the matrix effect and the extraction recovery rate of the components to be detected are stable and can be used for analyzing biological samples.

TABLE 44 matrix Effect and extraction recovery of the components

2.5.5 stability test

And (3) analyzing 5 samples of low and high QC samples (prepared by blank rat plasma and standard working solution) with 2 concentrations, wherein each concentration is subjected to stability inspection, and the stability inspection comprises short-term stability of the analyzed samples after being placed at 25 ℃ for 4 hours, long-term stability of the analyzed samples after being placed at 20 ℃ for 30 days, stability after 3 freeze-thaw cycles (-20 ℃), and stability of the samples after being processed and placed in an automatic sample injector for 24 hours. Substituting the following standard curve to calculate the precision and accuracy. The stability of 4 components to be tested was investigated using 2 concentrations of QC samples under different conditions (samples placed at 25 ℃ for 4h, -20 ℃ for 30 days, 3 freeze-thaw cycles, placed in an autosampler for 24h) and the results are given in Table 5. The results show that all analytes are stable throughout the analysis.

TABLE 54 stability of ingredients

2.5.5 dilution effect test

Preparing a plasma sample with the plasma drug concentration of the linalool, the isolinalool and the 8-chloro-2 (2-phenylethyl) -5,6, 7-trihydroxy-5, 6,7, 8-tetrahydrochromone being 1000ng/mL and the plasma drug concentration of the 4 ' -methoxylinalool being 500ng/mL, diluting the plasma sample by 10 times (dilution factor) with blank rat plasma to obtain plasma samples with theoretical concentrations of 100ng/mL and 50ng/mL respectively, analyzing the 6 samples, treating according to a treatment method under ' 2.4 ', then carrying out sample injection and determination, calculating the concentration of each sample according to a follow-up standard, comparing the concentration with the theoretical concentration, respectively calculating the accuracy and precision, researching the dilution effect of the components to be tested, and the result is shown in Table 6 and shows that the components to be tested do not have the dilution effect.

TABLE 64 dilution Effect of the ingredients

2.6 blood Collection and concentration determination at different time points

SPF grade SD rats 18, male, weighing 210 + -20 g. Rats were randomly divided into 3 groups of 6 rats each, and three groups of low (70mg/kg), medium (140mg/kg) and high (280mg/kg) eaglewood alcohol extract doses were assigned. Fasting is not prohibited for 12h before administration, blood is taken for 0.3mL before administration, blood is taken for 0.167, 0.333, 0.5, 0.75, 1, 2, 3, 4, 8, 12 and 24h after intragastric administration, 0.3mL is taken from orbital venous plexus of rats, the blood is placed in a 1.5mL tube containing 0.1% heparin sodium anticoagulant, the tube is stood for 1h and then centrifuged (4000rpm, 4 ℃, 10min), blood plasma is separated and stored at-20 ℃ for testing.

Taking rat plasma, unfreezing at room temperature, whirling for 30s, absorbing 100uL, processing samples according to the item of '2.4', preparing a random standard curve, low, medium and high QC samples (prepared by blank rat plasma and standard working solution), wherein two parts of QC samples with each concentration are parallel, the quantity of quality control samples is not less than 5% of the total quantity of the samples, simultaneously determining the concentration of a component to be detected in the rat plasma by adopting an established UPLC-MS/MS method, and calculating the concentration of the component to be detected in the rat plasma sample according to the random standard curve corrected by quality control.

3 results

The concentration of the component to be detected in the plasma of the rat is determined by adopting the established UPLC-MS/MS method, the concentration and the corresponding time point are input into DAS 3.0 software, a non-atrioventricular model is adopted to carry out data processing on each dose group, the pharmacokinetic parameters of 4 blood-entering components are respectively shown in tables 7,8, 9 and 10, and meanwhile, the average drug concentration-time curve of the 4 blood-entering components after the rat orally takes the linalol extract is drawn, which is shown in figure 2.

TABLE 7 pharmacokinetic parameters of agarotetrol

TABLE 8 pharmacokinetic parameters of isoagarotetraol

TABLE 94 pharmacokinetic parameters of methoxyagarotetraol

TABLE 108 chloro-2 (2-phenylethyl) -5,6, 7-trihydroxy-5, 6,7, 8-tetrahydrochromone

4. Discussion of the related Art

The UHPLC-MS/MS method for measuring 4 blood-entering components of the agarotetraol, the isoagarotetraol, the 4' -methoxy agarotetraol and the 8-chloro-2 (2-phenethyl) -5,6, 7-trihydroxy-5, 6,7, 8-tetrahydrochromone in the agarol extract, which is established by the invention, has the advantages of sensitivity, stability and reliability, can accurately and quantitatively measure 4 components in the agarol extract at the same time, is applied to the pharmacokinetics research of the agarol extract, and has guiding significance for the pharmacology and clinical application of medicinal resources of agarol.

The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.

Claims (2)

1. A method for simultaneously and quantitatively measuring 4 blood-entering components in an agilawood alcohol extract is characterized by comprising the following specific steps:

(1) preparation of stock solution: precisely weighing reference substances respectively, and preparing single reference substance stock solutions of 0.540mg/mL of agarotetrol, 0.803mg/mL of isoagaroterol, 0.346mg/mL of 4' -methoxy agarotetrol, 8-chloro-2 (2-phenylethyl) -5,6, 7-trihydroxy-5, 6,7, 8-tetrahydrochromone and 0.151mg/mL of wrinkled gianthyssop flavonol respectively by using HPLC-grade methanol as a solvent;

(2) preparation of working solution: respectively and precisely measuring a single reference substance stock solution of linalool, isolinalool, 4 '-methoxy-linalool and 8-chloro-2 (2-phenylethyl) -5,6, 7-trihydroxy-5, 6,7, 8-tetrahydrochromone, diluting the single reference substance stock solution by using HPLC-grade methanol by 10 times, and respectively and precisely measuring diluent to prepare a mixed reference substance solution, wherein the concentration of 4' -methoxy-linalool is 1000ng/mL, the concentration of linalool is 2000ng/mL, the concentration of isolinalool is 2000ng/mL, and the concentration of 8-chloro-2 (2-phenylethyl) -5,6, 7-trihydroxy-5, 6,7, 8-tetrahydrochromone is 2000 ng/mL; precisely measuring the mixed reference substance solution, adding HPLC-grade methanol to gradually dilute to obtain a series of standard working solutions with concentration, precisely measuring agastache flavonol single reference substance stock solution, and adding HPLC-grade methanol to dilute to obtain an internal standard working solution with concentration of 500 ng/mL;

(3) establishment of a standard curve: precisely absorbing 100 mu L of blank plasma, adding 20 mu L of internal standard working solution, respectively adding 20 mu L of standard working solution, vortexing for 30s, then adding 260 mu L of acetonitrile (v: v ═ 1:100) containing 1% formic acid, vortexing for 60s, centrifuging at 14000rpm and 4 ℃ for 10min, taking supernate, passing through a 0.22 mu m microporous filter membrane, and performing UHPLC-ESI-MS/MS analysis; taking the ratio of the peak area of the component to be detected to the peak area of the internal standard as a vertical coordinate (y), taking the concentration of a reference substance of the component to be detected added into plasma as a horizontal coordinate (x), and performing linear regression by adopting a 1/x weighted least square method to establish a curve;

(4) and (3) detection of the sample: precisely sucking 100 mu L of rat medicated plasma, adding 20 mu L of internal standard, vortexing for 30s, then adding 280 mu L of acetonitrile (v: v ═ 1:100) containing 1% formic acid, vortexing for 60s, centrifuging at 14000rpm and 4 ℃ for 10min, taking supernate, passing through a 0.22 mu m microporous filter membrane, carrying out UHPLC-MS/MS analysis, and calculating the concentration of 4 components in a plasma sample according to a following standard curve; 4 blood-entering components in the agarol extract are agarotetraol, isoagarotetraol, 4' -methoxy agarotetraol and 8-chloro-2 (2-phenethyl) -5,6, 7-trihydroxy-5, 6,7, 8-tetrahydrochromone respectively.

2. The method for simultaneously and quantitatively determining 4 blood-entering components in the agilawood alcohol extract according to claim 1, which is characterized in that: chromatographic conditions of the UHPLC-MS/MS analysis are as follows: the column was an ACQUITY UPLC BEH C18 column, 100mm X2.1 mm, 1.8 μm, mobile phase A: b is acetonitrile: 0.2% formic acid water, gradient elution program is 0-1.0 min, 15% A; 1.00-4.00 min, 15 → 40% A; 4.00-5.00 min, 40 → 70% A; 5.00-5.50 min, 70 → 80% A; 5.50-7.50 min, 80% A; 7.50-7.51 min, 80 → 95% A; 7.51-9.00 min, 95% A; 9.00-9.01 min, 95 → 15% A; 9.01-11.00 min, 15% A; the flow rate is 0.3 mL/min; the column temperature is 30 ℃; the sample injection amount is 2 mu L;

mass spectrum conditions: electrospray (ESI) ion source, positive ion detection mode; capillary voltage 4000V; spraying gas (N2) pressure 45psi, drying gas (N2) flow rate 10L/min, drying gas (N2) temperature 325 ℃; the flow rate of the sheath gas (N2) is 11L/min, and the temperature of the sheath gas (N2) is 350 ℃; nozzle voltage 500V; the scanning mode is Multiple Reaction Monitoring (MRM); the mass spectral parameters are given in the following table:

retention time and mass spectral parameters for quantitative analysis of target compounds

Quantification of ions.

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