CN113109482A - Method for establishing fingerprint of astragalus membranaceus medicinal material, extract and single preparation - Google Patents

Method for establishing fingerprint of astragalus membranaceus medicinal material, extract and single preparation Download PDF

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CN113109482A
CN113109482A CN202110476535.6A CN202110476535A CN113109482A CN 113109482 A CN113109482 A CN 113109482A CN 202110476535 A CN202110476535 A CN 202110476535A CN 113109482 A CN113109482 A CN 113109482A
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peak
relative
retention time
peak area
astragalus
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孙欣光
姚静
杨晓宁
朱平
李昆
王红宇
王玉龙
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Shanxi Zhendong Daodi Medicinal Material Development Co ltd
Beijing Zhendong Guangming Pharmaceutical Research Institute Co ltd
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Shanxi Zhendong Daodi Medicinal Material Development Co ltd
Beijing Zhendong Guangming Pharmaceutical Research Institute Co ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8624—Detection of slopes or peaks; baseline correction
    • G01N30/8631—Peaks
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
    • G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components

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Abstract

The invention relates to the technical field of traditional Chinese medicine analysis, in particular to a method for establishing a fingerprint of a radix astragali medicinal material, an extract and a single preparation. The method for establishing the fingerprint of the astragalus medicinal material, the extract and the single preparation comprises the following steps: detecting the sample solution by adopting HPLC-CAD to obtain a fingerprint containing 31 common characteristic peaks; the detection conditions of HPLC-CAD include: acetonitrile is used as a mobile phase A, and water containing formic acid is used as a mobile phase B, and gradient elution is carried out. The fingerprint spectrum of the radix astragali medicinal material established by the invention has 31 common characteristic peaks, can comprehensively represent the physicochemical properties of the radix astragali medicinal material, can comprehensively represent the saponins, flavonoids and other components in the radix astragali medicinal material, the extract and the single preparation, has strong specificity, good durability and good repeatability and accuracy, and can better realize the overall quality control of the radix astragali medicinal material and the preparation thereof.

Description

Method for establishing fingerprint of astragalus membranaceus medicinal material, extract and single preparation
Technical Field
The invention relates to the technical field of traditional Chinese medicine analysis, in particular to a method for establishing a fingerprint of a radix astragali medicinal material, an extract and a single preparation.
Background
The traditional Chinese medicine has the characteristics of complex chemical components, various structures and multi-component and multi-target action, the traditional Chinese medicine quality can be integrally and macroscopically controlled by the traditional Chinese medicine fingerprint spectrum through the description of the overall characteristics of the chemical components of the traditional Chinese medicine, the types and the quantity of the chemical components contained in the traditional Chinese medicine can be comprehensively reflected, and the traditional Chinese medicine quality control method is a modern traditional Chinese medicine quality control method starting from the 'integrity' perspective and has the more comprehensive characteristic of quality control of non-single medicines.
At present, the part of '2020 edition of Chinese pharmacopoeia' records a method for measuring calycosin glucoside in astragalus root by HPLC-UV and a method for measuring astragaloside content in the astragalus root by HPLC-ELSD, and does not record a fingerprint detection method. In the research related to the astragalus fingerprint, the currently established astragalus fingerprint tends to mark a single component, and the technical methods such as HPLC-DAD, HPLC-ELSD, LC-MS and the like are mainly adopted. Wherein HPLC-DAD can detect flavonoid components, and can not well detect saponin components with weak ultraviolet absorption or no ultraviolet absorption; although HPLC-ELSD is used as a general detector, flavonoid components are weakly absorbed under the ELSD detector, basically have no absorption, and are limited in application by factors such as low sensitivity, poor reproducibility and the like; although LC-MS has high sensitivity, it is expensive, which greatly limits its popularity and use. In a word, the overall quality of the astragalus medicinal material cannot be comprehensively evaluated due to the complex diversity of chemical components contained in the astragalus and the limitation of a detection method.
In summary, the prior art has many defects and shortcomings in the fingerprint spectrum determination method for the astragalus root medicinal material.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for establishing a fingerprint of a radix astragali medicinal material, an extract and a single preparation, so as to solve the technical problem that the overall quality of the radix astragali medicinal material and the like cannot be comprehensively evaluated in the prior art.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the method for establishing the fingerprint of the astragalus medicinal material, the extract and the single preparation comprises the following steps:
detecting the sample solution by adopting HPLC-CAD to obtain a fingerprint containing 31 common characteristic peaks;
the detection conditions of HPLC-CAD include:
taking acetonitrile as a mobile phase A, and taking 0.05-0.1 vol.% formic acid aqueous solution as a mobile phase B, and carrying out gradient elution; the gradient elution mode comprises the following steps: the volume fraction of the mobile phase A is 14-16% within 0-3 min; changing the volume fraction of the mobile phase A from 14-16% to 16.5-18% within 3-5 min; within 5-8 min, the volume fraction of the mobile phase A is changed from 16.5-18% to 19-21%; changing the volume fraction of the mobile phase A from 19-21% to 42-44% within 8-25 min; changing the volume fraction of the mobile phase A from 42-44% to 59-61% within 25-35 min; changing the volume fraction of the mobile phase A from 59-61% to 97-99% within 35-45 min; the volume fraction of the mobile phase A is 97-99% within 45-55 min.
The fingerprint spectrum of the astragalus mongholicus medicinal material, the extract and the single preparation, which are established by the invention, has 31 common characteristic peaks, and can comprehensively represent the physicochemical properties of the astragalus mongholicus medicinal material, the extract and the single preparation. The fingerprint spectrum established by the method can comprehensively represent the components such as saponins and flavonoids in the astragalus mongholicus medicinal material, the extract and the single preparation, and the method has the advantages of strong specificity, good durability, good repeatability and accuracy, and can better realize the overall quality control of the astragalus mongholicus medicinal material and the preparation thereof.
In a specific embodiment of the invention, the gradient elution mode comprises: the volume fraction of the mobile phase A is 15% within 0-3 min; changing the volume fraction of the mobile phase A from 15% to 17% within 3-5 min; changing the volume fraction of the mobile phase A from 17% to 20% within 5-8 min; changing the volume fraction of the mobile phase A from 20% to 43% within 8-25 min; changing the volume fraction of the mobile phase A from 43% to 60% within 25-35 min; changing the volume fraction of the mobile phase A from 60% to 98% within 35-45 min; and within 45-55 min, the volume fraction of the mobile phase A is 98%.
In a specific embodiment of the invention, the mobile phase B is 0.1 vol.% formic acid in water.
In a specific embodiment of the invention, the HPLC-CAD uses a C18 column, preferably an Agilent ZORBAX SB C18 column. The Agilent ZORBAX SB C18 column has the specification of 150 x 4.6mm and 3.5 mu m, shortens the sampling time and can further detect and obtain more chromatographic peaks.
In a specific embodiment of the present invention, the detection conditions further include:
the temperature of the chromatographic column is 30-45 ℃;
the flow rate of the mobile phase is 1-2 mL/min;
the sample injection amount is 5-20 mu L;
the CAD parameters include: the temperature of the drift tube is 35 ℃ or 50 ℃, and the gas flow rate is 2.2-2.6L/min.
As in the different embodiments, the column temperature of the chromatography column may be 30 ℃, 35 ℃, 40 ℃, 45 ℃ etc., preferably 40 ℃.
As in the various embodiments, the flow rate can be 1mL/min, 1.2mL/min, 1.4mL/min, 1.5mL/min, 1.6mL/min, 1.8mL/min, 2mL/min, and the like, preferably 1.5 mL/min.
As in various embodiments, the sample size can be 5. mu.L, 10. mu.L, 15. mu.L, 20. mu.L, etc., such as 10. mu.L.
As in various embodiments, the CAD parameters may include a gas flow rate of 2.2L/min, 2.3L/min, 2.4L/min, 2.5L/min, 2.6L/min, etc., such as 2.4L/min.
In a specific embodiment of the present invention, the detection conditions are:
the chromatographic column is an Agilent ZORBAX SB C18 column;
the temperature of the chromatographic column is 40 ℃;
the flow rate is 1.5 mL/min;
the sample injection amount is 10 mu L;
the drift tube temperature was 50 ℃.
In a specific embodiment of the present invention, when the substance to be tested is an astragalus root, the method for preparing the test solution comprises:
(a) performing ultrasonic extraction on a substance to be detected (the astragalus root medicinal material) by taking liquid containing methanol as an extraction solvent, collecting the liquid after the ultrasonic extraction, and concentrating to dry to obtain residues;
(b) redissolving the residue with water, passing through SP825 chromatographic column, eluting with water and methanol respectively, collecting methanol elution part, removing solvent to obtain solid, and dissolving the solid with methanol water solution to obtain solution.
The preparation method of the sample solution of the fingerprint spectrum of the solid or semisolid formulation of the astragalus extract and the single astragalus preparation is the same as that of the astragalus medicinal material. The method specifically comprises the following steps: carrying out ultrasonic extraction on the astragalus medicinal material by taking liquid containing methanol as an extraction solvent, collecting the liquid after the ultrasonic extraction, and concentrating to dry to obtain residue; redissolving the residue with water, passing through SP825 chromatographic column, eluting with water and methanol respectively, collecting methanol elution part, removing solvent to obtain solid, and dissolving the solid with methanol water solution to obtain solution.
For the liquid formulation of the astragalus extract and the single preparation of the astragalus, the liquid extract or the liquid preparation can be directly taken as a test solution for analysis.
In a specific embodiment of the present invention, the single astragalus formulation comprises any one of an astragalus injection, an astragalus essence oral liquid, an astragalus granule, an astragalus tablet, an astragalus essence granule and an astragalus tablet. Wherein the radix astragali granule, radix astragali essence granule, radix astragali tablet, and radix astragali tablet are solid preparations, and the radix astragali injection, radix astragali essence oral liquid, and radix astragali essence are liquid preparations.
The preparation conditions of the test solution are described below by taking astragalus root as an example:
in a specific embodiment of the invention, the ultrasonic extraction time is 45-120 min; the power of the ultrasonic wave is 400-500W, and the frequency of the ultrasonic wave is 38-40 kHz.
In a specific embodiment of the invention, the time for ultrasonic extraction is 45-60 min.
In a specific embodiment of the invention, the extraction solvent is a methanol aqueous solution with a volume fraction of 70% to 90%.
As in various embodiments, the volume fraction of methanol in the extraction solvent can be 70%, 75%, 80%, 85%, 90%, etc., preferably 75% to 85%, such as 80%.
In a specific embodiment of the invention, the ratio of the astragalus root crude drug to the extraction solvent is 1 g: 20-30 mL.
In various embodiments, the amount of the extraction solvent may be 20mL, 22mL, 24mL, 25mL, 26mL, 28mL, 30mL, etc., preferably 24-26 mL, such as 25mL, based on 1g of the astragalus root.
In practical operation, in step (a), the liquid after ultrasonic extraction is collected by filtration, and the filter residue is washed by methanol.
In a specific embodiment of the invention, the amount of water used for reconstitution is 2-3 mL based on 1g of the astragalus root.
In practical operation, the specification of the SP825 chromatographic column can be 1cm in inner diameter and 10cm in length, and the SP825 chromatographic column is filled with the column in a conventional mode.
In a specific embodiment of the invention, the volume ratio of water used for elution to water used for reconstitution is (10-15): 1; the volume ratio of methanol used for elution to water used for redissolution is (10-15): 1. Further, the volume ratio of water used for elution to water used for redissolution is (11.5-12.5): 1, such as 12: 1; the volume ratio of methanol used for elution to water used for redissolution is (11.5-12.5): 1, such as 12: 1.
In practice, after loading the sample on the column, the sample is eluted with water and then methanol.
In a specific embodiment of the present invention, in the step (b), the volume fraction of methanol in the methanol aqueous solution is 70% to 90%.
As in the different embodiments, the volume fraction of methanol in the aqueous methanol solution may be 70%, 75%, 80%, 85%, 90% or the like, preferably 75% to 85%, such as 80%.
In a specific embodiment of the invention, the amount of the methanol aqueous solution in the step (b) is 2-3 mL based on 1g of the astragalus root.
In a specific embodiment of the present invention, the preparing of the test solution further comprises: filtering the obtained solution by a microporous filter membrane, and taking a subsequent filtrate as the test solution.
In actual operation, the ultrasonic extraction is carried out by adopting a powdery astragalus root medicinal material. Further, the powdered astragalus root is sieved by a No. 4 sieve.
In a specific embodiment of the present invention, with peak number 21 as a reference peak, the relative retention time and the relative peak area of the 31 common characteristic peaks are respectively as follows:
peak No. 1: the relative retention time is 0.17-0.19, and the relative peak area is 0.21-0.94;
peak No. 2: the relative retention time is 0.28-0.30, and the relative peak area is 0.01-0.19;
peak No. 3: the relative retention time is 0.33-0.35, and the relative peak area is 0.14-1.86;
peak No. 4: the relative retention time is 0.40-0.42, and the relative peak area is 0.07-0.47;
peak No. 5: the relative retention time is 0.44-0.46, and the relative peak area is 0.01-0.13;
peak No. 6: the relative retention time is 0.45-0.47, and the relative peak area is 0.01-0.09;
peak No. 7: the relative retention time is 0.47-0.49, and the relative peak area is 0.12-0.86;
peak No. 8: the relative retention time is 0.50-0.52, and the relative peak area is 0.11-1.17;
peak No. 9: the relative retention time is 0.52-0.54, and the relative peak area is 0.02-0.17;
peak No. 10: the relative retention time is 0.52-0.54, and the relative peak area is 0.05-0.82;
peak No. 11: the relative retention time is 0.54-0.56, and the relative peak area is 0.01-0.12;
peak No. 12: the relative retention time is 0.56-0.58, and the relative peak area is 0.01-1.97;
peak No. 13: the relative retention time is 0.58-0.60, and the relative peak area is 0.01-0.79;
peak No. 14: the relative retention time is 0.70-0.72, and the relative peak area is 0.04-0.30;
peak No. 15: the relative retention time is 0.74-0.76, and the relative peak area is 0.01-0.25;
peak 16: the relative retention time is 0.75-0.77, and the relative peak area is 0.06-0.19;
peak No. 17: the relative retention time is 0.80-0.82, and the relative peak area is 0.13-0.63;
peak No. 18: the relative retention time is 0.83-0.85, and the relative peak area is 0.26-0.50;
peak No. 19: the relative retention time is 0.85-0.87, and the relative peak area is 0.09-0.40;
peak No. 20: the relative retention time is 0.88-0.90, and the relative peak area is 0.01-0.12;
peak No. 21: the relative retention time is 1.00, and the relative peak area is 1.00;
peak No. 22: the relative retention time is 1.04-1.06, and the relative peak area is 0.06-0.18;
peak No. 23: the relative retention time is 1.17-1.19, and the relative peak area is 0.01-0.20;
peak No. 24: the relative retention time is 1.19-1.21, and the relative peak area is 0.01-0.90;
peak No. 25: the relative retention time is 1.30-1.32, and the relative peak area is 0.16-0.85;
peak number 26: the relative retention time is 1.45-1.47, and the relative peak area is 0.01-0.10;
peak No. 27: the relative retention time is 1.52-1.55, and the relative peak area is 0.03-0.20;
peak number 28: the relative retention time is 1.53-1.56, and the relative peak area is 0.10-1.43;
peak No. 29: the relative retention time is 1.59-1.61, and the relative peak area is 0.22-2.73;
peak No. 30: the relative retention time is 1.64-1.66, and the relative peak area is 0.08-0.57;
peak No. 31: the relative retention time is 1.65-1.67, and the relative peak area is 0.04-0.56.
In a specific embodiment of the invention, in the fingerprint, the peak 1 is calycosin glucoside, the peak 4 is formononetin, the peak 7 is pterocarpan glycoside, the peak 8 is astragaloside isoflavan glycoside, the peak 15 is astragaloside IV, the peak 16 is formononetin, the peak 17 is 7,2' -dihydroxy-3 ',4' -dimethoxy isoflavan, the peak 18 is astragaloside II, the peak 20 is isoastragaloside II, the peak 21 is astragaloside I, and the peak 22 is isoastragaloside I.
The invention identifies chemical components corresponding to 11 common characteristic peaks in the fingerprint, wherein the saponin components identify 5 and the flavonoid components identify 6.
Compared with the prior art, the invention has the beneficial effects that:
(1) the fingerprint spectrum of the radix astragali medicinal material established by the invention has 31 common characteristic peaks, and can comprehensively represent saponins, flavonoids and other components in the radix astragali medicinal material;
(2) the method has the advantages of strong specificity, good durability, good repeatability and accuracy, and can better realize the overall quality control of the astragalus mongholicus medicinal material and the preparation thereof.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a fingerprint of Astragalus membranaceus as a medicinal material measured in example 1 of the present invention;
FIG. 2 shows the measured fingerprints and reference spectra of different Astragalus membranaceus medicinal materials in example 2 of the present invention;
FIG. 3 is a chromatogram obtained by different extraction methods according to the present invention;
FIG. 4 is a chromatogram obtained for different extraction solvents according to the present invention; wherein the A-extraction solvent is 60 vol.% aqueous methanol solution, the B-extraction solvent is 80 vol.% aqueous methanol solution, and the C-extraction solvent is pure methanol;
FIG. 5 is a chromatogram obtained corresponding to different extraction times according to the present invention; wherein, the A-extraction time is 45min, the B-extraction time is 60min, and the C-extraction time is 120 min;
FIG. 6 is chromatograms obtained from a sample not treated with the SP825 column chromatography and a sample treated with the SP825 column chromatography; wherein, A-is not processed by SP825 chromatographic column, B-is processed by SP825 chromatographic column;
FIG. 7 is a chromatogram of example 7 of the present invention under different elution conditions;
FIG. 8 shows chromatograms obtained in comparative example 1 and comparative example 2.
Detailed Description
The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and the detailed description, but those skilled in the art will understand that the following described embodiments are some, not all, of the embodiments of the present invention, and are only used for illustrating the present invention, and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In the specific implementation mode, the verification of the items such as precision, repeatability, stability and the like is carried out according to the relevant guiding principle in the appendix of the pharmacopoeia of the people's republic of China in the current edition.
The information of materials, instruments, reagents and the like adopted in the specific embodiment of the invention can be as follows:
materials: astragalus mongholicus medicinal material samples are purchased from different producing areas and identified as dried roots of astragalus mongholicus astraglus brandanaceus (Fisch.) bge.var. mongholicus (Bge.) Hsiao, and the source information of the specific astragalus mongholicus medicinal material samples is shown in the following table 1:
TABLE 1 Astragalus medicinal material sample source information Table
Medicinal material numbering Producing area Medicinal material numbering Producing area
S1 Gansu S13 Shanxi province
S2 Gansu S14 Shanxi province
S3 Gansu S15 Shanxi province
S4 Gansu S16 Shanxi province
S5 Gansu S16 Inner cover
S6 Gansu S18 Inner cover
S7 Gansu S19 Inner cover
S8 Gansu S20 Inner cover
S9 Shanxi province S21 Inner cover
S10 Shanxi province S22 Inner cover
S11 Shanxi province S23 Inner cover
S12 Shanxi province
The instrument comprises the following steps: thermo U3000 high performance liquid chromatograph (conona Veo model CAD electrospray detector), Mettler Toledo XSE205DU electronic analytical balance (Mettler Toledo, switzerland); a numerical control ultrasonic cleaning machine (KQ-500DE type, Kunshan ultrasonic Instrument Co., Ltd.); Milli-Q water purification machines (Millipore, USA).
Reagent: acetonitrile (chromatographically pure, merck, usa); methanol (chromatographically pure, merck, usa); other chemical reagents are analytically pure;
calycosin glucoside (batch No. 111920-201606, purity 97.6%, China food and drug inspection institute), astragaloside (batch No. 110781-201717, purity 96.9%, China food and drug inspection institute), formononetin (batch No. 3523, purity 98.2%, Schdner Biotech limited), 7,2' -dihydroxy-3 ',4' -dimethoxyisoflavan (batch No. Y31D10H107318, purity 98%, Shanghai-sourced leaf Biotech limited), astragaloside I (batch No. 6107, purity 99.0%, Schdner Biotech limited), astragaloside II (batch No. 3258, purity 100.0%, Schdner Biotech limited), santalin (batch No. P21N9F75533, purity 00098%, Shanghai-sourced leaf Biotech limited), formononetin (batch No. PS, purity 98%, Woosi bioscience) Astragalus isoflavanoside (batch No. P31D10F107320, purity: 98%, Shanghai-sourced leaf Biotechnology Co., Ltd.), isoastragaloside I (batch No. PS020461, purity: 98%, Chengpo Biotechnology Co., Ltd.), and isoastragaloside II (batch No. PS001047, purity: 98%, Chengpo Biotechnology Co., Ltd.).
Example 1
The embodiment provides a method for establishing a fingerprint of a radix astragali medicinal material, which comprises the following steps:
(1) preparation of control solutions
Taking a proper amount of each reference substance of calycosin glucoside, astragaloside IV, astragaloside I, astragaloside II, formononetin and 7,2' -dihydroxy-3 ',4' -dimethoxy isoflavan, pterocarpan glycoside, formononetin, astragaloside I and isoastragaloside II, precisely weighing, respectively preparing a reference substance solution containing 0.1mg of each reference substance in each 1mL by using 80 vol.% methanol aqueous solution, shaking up, and filtering by using a 0.22 mu m microfiltration membrane to obtain the product.
(2) Preparation of test solution
Precisely weighing 2g of S1 radix astragali medicinal material powder (passing through a No. 4 sieve), placing the powder into a 100mL conical flask with a plug, precisely adding 50mL of 80 vol.% methanol aqueous solution, ultrasonically extracting for 1 hour, filtering, washing filter residues with 20mL of methanol, recovering filtrate, evaporating to dryness, re-dissolving residues with 5mL of pure water, adding the re-dissolved residues onto a treated SP825 column (with the inner diameter of 1cm and the length of 10cm) to elute with 60mL of pure water and methanol respectively, collecting eluents respectively, evaporating to dryness, dissolving the methanol elution part with 80 vol.% methanol aqueous solution, fixing the volume to a 5mL measuring flask, shaking uniformly, filtering with a 0.22 mu m microfiltration membrane, and taking subsequent filtrate to obtain a sample solution.
(3) Detection conditions
A chromatographic column: agilent SB-C18(150 mm. times.4.6 mm, 3.5 μm);
column temperature: 40 ℃;
flow rate: 1.5 mL/min;
mobile phase: acetonitrile (a) -0.1 vol.% formic acid water (B); gradient elution, elution conditions (volume fraction): 0-3 min, 15% A; 3-5 min, 15% → 17% A; 5-8 min, 17% → 20% A; 8-25 min, 20% → 43% A; 25-35 min, 43% → 60% A; 35-45 min, 60% → 98% A; 45-55 min, 98% A;
sample introduction amount: 10 mu L of the solution;
CAD parameters: the drift tube temperature was 50 ℃ and the gas flow rate was 2.4L/min.
(4) Detection step
And (4) precisely measuring 10 mu L of each of the reference solution and the test solution, injecting samples respectively, detecting according to the detection conditions in the step (3), and recording a chromatogram. The fingerprint measured in this example is shown in FIG. 1.
Example 2
In this example, referring to the method of example 1, the rest 22 batches of astragalus mongholicus medicinal materials recorded in table 1 are respectively detected to obtain 22 fingerprint spectrums.
The 22 fingerprints and the fingerprints obtained in the embodiment 1 are introduced into the traditional Chinese medicine fingerprint similarity evaluation software to synthesize a comparison map.
FIG. 2 shows the finger print and the reference R of 23 batches of radix astragali.
Example 3
This example refers to the method of example 1, with the only difference that: the preparation method of the test solution is different from the extraction method.
The test solution of this example was prepared as follows:
precisely weighing 2g of astragalus mongholicus medicinal material powder (passing through a No. 4 sieve), placing the powder into a 100mL conical flask with a plug, precisely adding 50mL of 80 vol.% methanol aqueous solution, heating and refluxing for extraction for 2h, filtering, washing filter residues with 20mL of methanol, recovering filtrate, evaporating to dryness, re-dissolving residues with 5mL of pure water, adding the re-dissolved residues onto a treated SP825 column (with the inner diameter of 1cm and the length of 10cm) to elute with 60mL of pure water and methanol respectively, collecting eluents respectively, evaporating to dryness, dissolving methanol elution parts with 80 vol.% methanol aqueous solution, fixing the volume into a 5mL measuring flask, shaking uniformly, filtering with a 0.22 mu m microfiltration membrane, and taking subsequent filtrate to obtain a sample solution.
The chromatograms of the sample solution of this example and example 1 are shown in fig. 3, respectively.
When ultrasonic extraction and reflux extraction are adopted, the response values and the separation degrees of all peaks in the chromatogram have no obvious difference, and the ultrasonic extraction method can be selected for extraction based on the consideration of simple method and time saving.
Example 4
This example refers to the method of example 1, with the only difference that: the sample solution is prepared by different extraction solvents. The extraction solvent of this example was 60 vol.% aqueous methanol.
The chromatogram corresponding to the sample solution of this example is shown in fig. 4 a.
Example 5
This example refers to the method of example 1, with the only difference that: the sample solution is prepared by different extraction solvents. The extraction solvent of this example was pure methanol.
The chromatogram corresponding to the sample solution of this example is shown in fig. 4C.
Referring to fig. 4, by comparing the number of peaks extracted with different extraction solvents and the degrees of separation of the peaks, 80 vol.% of methanol in water was selected as the extraction solvent because the number of peaks is large and the degree of separation is good.
Example 6
This example refers to the method of example 1, with the only difference that: the preparation method of the test solution has different extraction time. The ultrasound extraction time of this example was 45 min.
The chromatogram corresponding to the sample solution of this example is shown in fig. 5 a.
Example 7
This example refers to the method of example 1, with the only difference that: the preparation method of the test solution has different extraction time. The ultrasound extraction time of this example was 120 min.
The chromatogram corresponding to the sample solution of this example is shown in fig. 5C.
As can be seen from fig. 5, samples extracted at different extraction times have no significant difference in the number of peaks and the degree of separation in the chromatogram, and therefore, ultrasound for 60min can be selected as the extraction time.
Example 8
This example refers to the method of example 1, with the only difference that: the preparation method of the sample solution is different, and the sample is not purified by using the SP825 chromatographic column in the preparation of the sample solution in the embodiment.
In the method for preparing the sample solution of this embodiment, after the residue is obtained in reference example 1, the residue is directly dissolved in 80 vol.% methanol aqueous solution and the volume is determined to be 5mL in a measuring flask, the solution is shaken up and filtered through a 0.22 μm microporous membrane, and a subsequent filtrate is taken, so as to obtain the sample solution.
The chromatogram corresponding to the sample solution of this example is shown in fig. 6 a.
As can be seen from fig. 6, the chromatogram before and after the astragalus root medicinal material passes through the SP825 chromatographic column has no significant change, but the corresponding value of the individual chromatographic peak of the sample after the purification by the chromatographic column is higher, so the SP825 chromatographic column is adopted to purify the sample.
Example 9
This example refers to the method of example 1, with the only difference that: the chromatographic detection conditions are different.
The chromatographic detection conditions of this example are as follows:
a chromatographic column: agilent SB-C18(150 mm. times.4.6 mm, 3.5 μm);
column temperature: 35 ℃;
flow rate: 1.0 mL/min;
mobile phase: 0.05 vol.% formic acid acetonitrile (a) -0.05 vol.% formic acid water (B);
gradient elution conditions (volume fraction): 0-3 min, 20% A; 3-8 min, 20% A → 25% A; 8-18 min, 25% A → 36% A; 18-38 min, 36% A → 43% A;
sample introduction amount: 20 mu L of the solution;
CAD parameters: the drift tube temperature was 50 ℃ and the gas flow rate was 50 psi/min.
The chromatogram corresponding to the sample solution of this example is shown in FIG. 7. The chromatogram of this example has fewer chromatographic peaks than the chromatogram of example 1, and cannot more comprehensively reflect the chemical components in the astragalus root. In fig. 7, peak 1 is calycosin glucoside, peak 2 is astragaloside IV, peak 3 is formononetin, peak 4 is 7,2' -dihydroxy-3 ',4' -dimethoxy isoflavan, peak 5 is astragaloside II, and peak 6 is astragaloside I.
Comparative example 1
Comparative example 1 the sample solution prepared according to example 1 was subjected to HPLC-UV detection and a chromatogram was recorded, as shown in FIG. 8.
Wherein, the conditions of the HPLC-UV method are as follows: ultraviolet detection wavelength 254nm, other chromatographic conditions refer to the method of example 1.
Comparative example 2
Comparative example 2 the sample solution prepared according to example 1 was examined by HPLC-ELSD method and a chromatogram was recorded, as shown in FIG. 8.
Wherein, the conditions of the HPLC-ELSD method are as follows: HPLC-ELSD drift tube temperature 70 deg.C, carrier gas flow 1.5L min-1Other chromatographic conditions refer to the procedure of example 1.
Compared with HPLC-ELSD and HPLC-UV, the method has higher sensitivity and more chromatographic peaks.
Experimental example 1
Methodology investigation
And (3) precision experiment: a sample solution of the astragalus mongholicus medicinal material No. S12 is prepared, sample injection is continuously carried out for 6 times according to the method of example 1, and the relative retention time of 31 common peaks and the RSD value of the relative peak area are calculated.
And (3) repeatability experiment: 6 parts of an S12 Astragalus membranaceus sample was precisely weighed, a test solution was prepared by the method of example 1, and the measurement was carried out under the chromatographic conditions of example 1. RSD values were calculated as relative retention times and relative peak areas of 31 common peaks.
Stability test: taking the astragalus mongholicus No. S12, preparing a test solution according to the method in the embodiment 1, and injecting samples for analysis after 0, 4, 8, 12, 24 and 36 hours respectively. The relative retention times of the 31 common peaks and the RSD values of the relative peak areas were calculated.
The methodology investigation results were as follows:
and (3) precision experiment: precisely absorbing 10 mu L of test solution, injecting the test solution into a liquid chromatograph, continuously sampling for 6 times, calculating the relative retention time of 31 common peaks and the RSD value of the relative peak area, and indicating that the RSD values of the 31 common peaks are less than 1.0% and the RSD values of the relative peak areas are less than 5.0%, wherein the specific results are shown in tables 2 and 3.
And (3) repeatability experiment: precisely absorbing 10 mu L of test solution, injecting the test solution into a liquid chromatograph, repeating 6 samples, and calculating the relative retention time of 31 common peaks and the RSD value of the relative peak area, wherein the RSD values of the 31 common peaks are less than 1.0% and the RSD values of the relative peak areas are less than 5.0%, which shows that the method has good repeatability, and the specific results are shown in tables 2 and 3.
Stability test: taking an S12 radix astragali medicinal material, preparing a test solution according to the method in the embodiment 1, respectively carrying out sample injection analysis after 0, 4, 8, 12, 24 and 36h, calculating the relative retention time of 31 common peaks and the RSD value of the relative peak area, and indicating that the method has good repeatability, wherein the specific results are shown in tables 2 and 3, and the RSD values of the 31 common peaks are all less than 1.0% and less than 5.0%.
Table 2 methodological validation consensus peak relative retention time results (n ═ 6)
Figure BDA0003047587300000161
Figure BDA0003047587300000171
Table 3 methodological validation consensus peak relative peak area results (n ═ 6)
Figure BDA0003047587300000172
Figure BDA0003047587300000181
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. The method for establishing the fingerprint of the astragalus mongholicus medicinal material, the extract and the single preparation is characterized by comprising the following steps of:
detecting the sample solution by adopting HPLC-CAD to obtain a fingerprint containing 31 common characteristic peaks;
the detection conditions of HPLC-CAD include:
taking acetonitrile as a mobile phase A, and taking 0.05-0.1 vol.% formic acid aqueous solution as a mobile phase B, and carrying out gradient elution; the gradient elution mode comprises the following steps: the volume fraction of the mobile phase A is 14-16% within 0-3 min; changing the volume fraction of the mobile phase A from 14-16% to 16.5-18% within 3-5 min; within 5-8 min, the volume fraction of the mobile phase A is changed from 16.5-18% to 19-21%; changing the volume fraction of the mobile phase A from 19-21% to 42-44% within 8-25 min; changing the volume fraction of the mobile phase A from 42-44% to 59-61% within 25-35 min; changing the volume fraction of the mobile phase A from 59-61% to 97-99% within 35-45 min; the volume fraction of the mobile phase A is 97-99% within 45-55 min.
2. The method of claim 1, wherein the gradient elution profile comprises: the volume fraction of the mobile phase A is 15% within 0-3 min; changing the volume fraction of the mobile phase A from 15% to 17% within 3-5 min; changing the volume fraction of the mobile phase A from 17% to 20% within 5-8 min; changing the volume fraction of the mobile phase A from 20% to 43% within 8-25 min; changing the volume fraction of the mobile phase A from 43% to 60% within 25-35 min; changing the volume fraction of the mobile phase A from 60% to 98% within 35-45 min; within 45-55 min, the volume fraction of the mobile phase A is 98%;
preferably, the mobile phase B is 0.1 vol.% aqueous formic acid.
3. The method of claim 1, wherein the HPLC-CAD uses a C18 column;
preferably, the C18 chromatographic column is an Agilent ZORBAX SB C18 column.
4. The method of claim 1, wherein detecting the condition further comprises:
the temperature of the chromatographic column is 30-45 ℃;
the flow rate of the mobile phase is 1-2 mL/min;
the sample injection amount is 5-20 mu L;
the CAD parameters include: the temperature of the drift tube is 35 ℃ or 50 ℃, and the gas flow rate is 2.2-2.6L/min.
5. The method of claim 1, wherein the detection condition is:
the chromatographic column is an Agilent ZORBAX SB C18 column;
the temperature of the chromatographic column is 40 ℃;
the flow rate is 1.5 mL/min;
the sample injection amount is 10 mu L;
the drift tube temperature was 50 ℃.
6. The method of claim 1, wherein the method for preparing the test solution of the astragalus root, the astragalus extract in solid or semisolid dosage form, and the astragalus single ingredient preparation in solid or semisolid dosage form comprises:
(a) carrying out ultrasonic extraction on the astragalus medicinal material, the astragalus extract or the astragalus single preparation by taking liquid containing methanol as an extraction solvent, collecting the liquid after the ultrasonic extraction, and concentrating to dry to obtain residue;
(b) redissolving the residue with water, passing through SP825 chromatographic column, eluting with water and methanol respectively, collecting methanol elution part, removing solvent to obtain solid, and dissolving the solid with methanol water solution to obtain solution;
preferably, the method further comprises the following steps: filtering the obtained solution by a microporous filter membrane, and taking a subsequent filtrate as the test solution;
directly obtaining corresponding liquid as a test solution for a liquid formulation of the astragalus extract and a liquid formulation of the single astragalus preparation;
preferably, the single astragalus preparation comprises any one of an astragalus injection, an astragalus essence oral liquid, an astragalus granule, an astragalus tablet, an astragalus essence granule and an astragalus tablet.
7. The method according to claim 6, wherein the time of ultrasonic extraction is 45-120 min; the power of the ultrasonic wave is 400-500W, and the frequency of the ultrasonic wave is 38-40 kHz;
preferably, the ultrasonic extraction time is 45-60 min.
8. The method according to claim 6, wherein the extraction solvent is 70-90% methanol aqueous solution by volume fraction;
preferably, the ratio of the astragalus mongholicus to the extraction solvent is 1 g/20-30 mL.
9. The method according to any one of claims 1 to 8, wherein the relative retention time and the relative peak area of the 31 common characteristic peaks are as follows, respectively, using peak number 21 as a reference peak:
peak No. 1: the relative retention time is 0.17-0.19, and the relative peak area is 0.21-0.94;
peak No. 2: the relative retention time is 0.28-0.30, and the relative peak area is 0.01-0.19;
peak No. 3: the relative retention time is 0.33-0.35, and the relative peak area is 0.14-1.86;
peak No. 4: the relative retention time is 0.40-0.42, and the relative peak area is 0.07-0.47;
peak No. 5: the relative retention time is 0.44-0.46, and the relative peak area is 0.01-0.13;
peak No. 6: the relative retention time is 0.45-0.47, and the relative peak area is 0.01-0.09;
peak No. 7: the relative retention time is 0.47-0.49, and the relative peak area is 0.12-0.86;
peak No. 8: the relative retention time is 0.50-0.52, and the relative peak area is 0.11-1.17;
peak No. 9: the relative retention time is 0.52-0.54, and the relative peak area is 0.02-0.17;
peak No. 10: the relative retention time is 0.52-0.54, and the relative peak area is 0.05-0.82;
peak No. 11: the relative retention time is 0.54-0.56, and the relative peak area is 0.01-0.12;
peak No. 12: the relative retention time is 0.56-0.58, and the relative peak area is 0.01-1.97;
peak No. 13: the relative retention time is 0.58-0.60, and the relative peak area is 0.01-0.79;
peak No. 14: the relative retention time is 0.70-0.72, and the relative peak area is 0.04-0.30;
peak No. 15: the relative retention time is 0.74-0.76, and the relative peak area is 0.01-0.25;
peak 16: the relative retention time is 0.75-0.77, and the relative peak area is 0.06-0.19;
peak No. 17: the relative retention time is 0.80-0.82, and the relative peak area is 0.13-0.63;
peak No. 18: the relative retention time is 0.83-0.85, and the relative peak area is 0.26-0.50;
peak No. 19: the relative retention time is 0.85-0.87, and the relative peak area is 0.09-0.40;
peak No. 20: the relative retention time is 0.88-0.90, and the relative peak area is 0.01-0.12;
peak No. 21: the relative retention time is 1.00, and the relative peak area is 1.00;
peak No. 22: the relative retention time is 1.04-1.06, and the relative peak area is 0.06-0.18;
peak No. 23: the relative retention time is 1.17-1.19, and the relative peak area is 0.01-0.20;
peak No. 24: the relative retention time is 1.19-1.21, and the relative peak area is 0.01-0.90;
peak No. 25: the relative retention time is 1.30-1.32, and the relative peak area is 0.16-0.85;
peak number 26: the relative retention time is 1.45-1.47, and the relative peak area is 0.01-0.10;
peak No. 27: the relative retention time is 1.52-1.55, and the relative peak area is 0.03-0.20;
peak number 28: the relative retention time is 1.53-1.56, and the relative peak area is 0.10-1.47;
peak No. 29: the relative retention time is 1.59-1.61, and the relative peak area is 0.22-2.73;
peak No. 30: the relative retention time is 1.64-1.66, and the relative peak area is 0.08-0.57;
peak No. 31: the relative retention time is 1.65-1.67, and the relative peak area is 0.04-0.56.
10. The method as claimed in claim 9, wherein in the fingerprint, peak 1 is calycosin glucoside, peak 4 is formononetin, peak 7 is pterocarpan glycoside, peak 8 is astragaloside IV, peak 15 is astragaloside IV, peak 16 is formononetin, peak 17 is 7,2' -dihydroxy-3 ',4' -dimethoxy isoflavan, peak 18 is astragaloside II, peak 20 is isoastragaloside II, peak 21 is astragaloside I, and peak 22 is isoastragaloside I.
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