CN115015405B - Construction method and application of characteristic spectrum of red bean or pharmaceutical preparation thereof - Google Patents
Construction method and application of characteristic spectrum of red bean or pharmaceutical preparation thereof Download PDFInfo
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- CN115015405B CN115015405B CN202210524043.4A CN202210524043A CN115015405B CN 115015405 B CN115015405 B CN 115015405B CN 202210524043 A CN202210524043 A CN 202210524043A CN 115015405 B CN115015405 B CN 115015405B
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Images
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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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Library & Information Science (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to the technical field of traditional Chinese medicine detection, in particular to a construction method of a characteristic map of red bean or a pharmaceutical preparation thereof and application thereof. The construction method of the characteristic spectrum of the red bean or the pharmaceutical preparation thereof provided by the invention can be used for detecting the red bean (red bean) and the red bean (red bean), and has the advantages of short detection time, comprehensive characteristic peak display and good characteristic peak separation degree.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine detection, in particular to a construction method and application of a characteristic map of red bean or a pharmaceutical preparation thereof.
Background
The semen Phaseoli, chinese medicinal name, is dried mature seed of semen Phaseoli Vigna umbeuata Ohwi et Ohashi or semen Phaseoli Vigna angutaris Ohwi et Ohashi of Leguminosae. The semen Phaseoli (semen Phaseoli) compound granule is prepared by processing dried mature seed of semen Phaseoli Vigna umbeuata Ohwi et Ohashi of Leguminosae and processing according to main quality index of standard decoction. The red bean (red bean) formula granule is prepared by processing dried mature seeds of red bean Vigna angutaris Ohwi et Ohashi of leguminous plant and processing according to the main quality index of standard decoction.
At present, the quality standard of the traditional Chinese medicine formula granule is only the detection of red beans (red beans) or the detection of red beans (red beans), and a method capable of detecting and distinguishing the red beans (red beans) and the red beans (red beans) is not provided, and the detection time is long, the separation degree is poor, and the pharmacodynamic chemical components cannot be displayed more comprehensively.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a construction method of the characteristic map of the red bean or the pharmaceutical preparation thereof, which can detect the red bean (red bean) and the red bean (red bean) or the pharmaceutical preparation thereof, has short detection time and good separation degree, and can display the pharmacodynamic chemical components more comprehensively.
The invention aims to provide a method for distinguishing the red beans with different primordia or the pharmaceutical preparations thereof, which can be used for distinguishing the red beans with different primordia well.
A construction method of a characteristic map of red bean or a pharmaceutical preparation thereof comprises the following steps:
chromatographic conditions and system applicability test, according to high performance liquid chromatography, the chromatographic column uses octadecylsilane chemically bonded silica as filler; methanol is taken as a mobile phase A, 0.07% (v/v) phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the following gradient elution program: 0-8 min, wherein the volume percentage of the mobile phase A is 10%; 8-55 min, wherein the volume percentage of the mobile phase A is 10% -45%; 55-56 min, wherein the volume percentage of the mobile phase A is 45% -10%; 56-60 min, wherein the volume percentage of the mobile phase A is 10%; the column temperature is 18-22 ℃; the flow rate is 0.18-0.22ml per minute; the detection wavelength is 280nm; the theoretical plate number is not less than 5000 calculated according to catechin-7-O-beta-glucopyranoside peak;
preparation of reference solution: a control drug reference solution prepared from red bean control drug; reference solutions of reference substances prepared from catechin-7-O-beta-glucopyranoside, catechin, isoquercitrin, and quercetin-7-O-beta-glucopyranoside, respectively;
preparation of test solution: preparing a sample solution from a sample;
assay: precisely sucking the reference solution and the sample solution, respectively, injecting into a liquid chromatograph, measuring, and recording chromatograms;
the semen Phaseoli comprises semen Phaseoli Vigna umbeuata Ohwi et Ohashi or semen Phaseoli Vigna angutaris Ohwi et Ohashi.
The chromatographic column is selected from Agilent ZORBAX Eclipse Plus C RRHD, waters ACQUITY UPLC BEH C18 or Thermo ScientificTM Hypersil GOLDTM;
alternatively, the chromatographic column is Agilent ZORBAX Eclipse Plus C RRHD, the column length is 150mm, the inner diameter is 2.1mm, and the particle size is 1.8 μm;
optionally, the column temperature is 20 ℃;
optionally, the flow rate is 0.2ml/min.
The construction method of the characteristic spectrum of the red bean or the pharmaceutical preparation thereof comprises the following steps of: taking about 2g of red bean reference medicine, placing the red bean reference medicine into a conical flask with a plug, adding 100ml of water, heating and refluxing for 45 minutes, taking out, cooling, filtering, evaporating filtrate to dryness, adding 15ml of 30% v/v methanol into residues, performing ultrasonic treatment for 30 minutes under the condition of power 300W and frequency 40kHz, taking out, cooling, filtering, and taking out the subsequent filtrate to be used as a reference substance solution of the reference medicine;
optionally, the preparation method of the reference substance solution of the reference substance comprises the following steps: taking a proper amount of catechin-7-O-beta-glucopyranoside, catechin, isoquercitrin and quercetin-7-O-beta-glucoside reference substances, precisely weighing, adding 30-50% v/v methanol to prepare a mixed solution containing 0.15-0.25mg of catechin-7-O-beta-glucopyranoside, 0.045-0.055mg of catechin, 0.045-0.055mg of isoquercitrin and 0.045-0.055mg of quercetin-7-O-beta-glucoside per 1ml of reference substance solution;
optionally, the preparation method of the reference substance solution of the reference substance comprises the following steps: taking a proper amount of catechin-7-O-beta-glucopyranoside, catechin, isoquercitrin and quercetin-7-O-beta-glucoside reference substances, precisely weighing, and adding 30% v/v methanol to prepare a mixed solution containing 0.2mg of catechin-7-O-beta-glucopyranoside, 0.05mg of catechin, 0.05mg of isoquercitrin and 0.05mg of quercetin-7-O-beta-glucoside per 1ml of reference substance solution;
optionally, preparing a test solution: grinding a test sample, taking about 0.5-1.5g, precisely weighing, placing into a conical flask with a plug, precisely adding 25-50ml of 30-50% v/v methanol, weighing, performing ultrasonic treatment for 30-60 minutes under the condition of 200-300W of power and 35-45kHz of frequency, taking out, cooling, weighing again, supplementing the lost weight with 30-50% v/v methanol, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the product;
optionally, preparing a test solution: taking a test sample, grinding, precisely weighing about 1.0g, placing in a conical flask with a plug, precisely adding 25ml of 30% v/v methanol, weighing, performing ultrasonic treatment for 30 minutes under the power of 300W and the frequency of 40kHz, taking out, cooling, weighing again, supplementing the lost weight with 30% v/v methanol, shaking uniformly, filtering, and taking the subsequent filtrate.
In the method, 1-3 mu l of reference substance solution and 3-5 mu l of sample solution of a reference substance are respectively sucked precisely, and injected into a liquid chromatograph;
optionally, in the assay, 2 μl of each of the reference solution and the solution of the test sample is precisely sucked, and 4 μl of the reference solution of the reference material is injected into the liquid chromatograph.
The construction method of the characteristic spectrum of the red bean or the pharmaceutical preparation thereof comprises the use of the characteristic spectrum of the red bean medicinal material, the red bean decoction pieces, the red bean decoction piece standard decoction or the red bean prescription granule;
the semen Phaseoli comprises semen Phaseoli Vigna umbeuata Ohwi et Ohashi or semen Phaseoli Vigna angutaris Ohwi et Ohashi;
the red bean pharmaceutical preparation comprises a pharmaceutical preparation prepared from red bean Vigna umbeuata Ohwi et Ohashi or red bean Vigna angutaris Ohwi et Ohashi.
According to the method for quality detection of the construction method of the characteristic spectrum of the red bean or the pharmaceutical preparation thereof, 10 characteristic peaks are shown in the characteristic spectrum of a test sample, the retention time of the 10 characteristic peaks in a reference chromatogram of a reference substance of a reference medicine is corresponding to the retention time of the 10 characteristic peaks of the reference substance of the reference medicine, wherein the 1, 3, 8 and 9 peaks are respectively corresponding to the retention time of the corresponding reference substance peaks, the 1 peak is catechin-7-O-beta-glucopyranoside, the 3 peak is catechin, the 4 peak is epicatechin-5-O-beta-glucopyranoside, the 8 peak is quercetin-7-O-beta-glucoside, the 9 peak is isoquercitrin, the corresponding peak of the reference substance peak of the catechin-7-O-beta-glucopyranoside is S peak, and the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within +/-10% of a specified value, and the specified value is: peak 2 was 1.26, peak 4 was 1.85, peak 5 was 2.72, peak 6 was 2.84, peak 7 was 2.90, and peak 10 was 3.60;
the test sample comprises semen Phaseoli or its pharmaceutical preparation; the semen Phaseoli or its pharmaceutical preparation comprises semen Phaseoli medicinal material, semen Phaseoli decoction pieces, semen Phaseoli decoction piece standard decoction or semen Phaseoli granule;
the semen Phaseoli comprises semen Phaseoli Vigna umbeuata Ohwi et Ohashi or semen Phaseoli Vigna angutaris Ohwi et Ohashi;
the red bean pharmaceutical preparation comprises a pharmaceutical preparation prepared from red bean Vigna umbeuata Ohwi et Ohashi or red bean Vigna angutaris Ohwi et Ohashi;
when the test sample is red bean Vigna angutaris Ohwi et Ohashi or a pharmaceutical preparation thereof, the characteristic spectrum of the test sample does not contain peak 6.
A method for distinguishing red beans with different primordia or pharmaceutical preparations thereof, which comprises a construction method utilizing characteristic patterns of the red beans or the pharmaceutical preparations thereof;
when the peak 6 is deleted in the obtained characteristic map and the relative peak area of the peak 7 and the S peak is smaller than 0.14, the basic source of the sample is red bean Vigna angularis Ohwi et Ohashi;
when the obtained characteristic spectrum does not lack peak 6, and the relative peak area of peak 7 and S peak is more than or equal to 0.14, the basic source of the sample is red bean Vigna umbellata Ohwi et Ohashi.
In the invention, for better distinguishing the red bean Vigna umbellata Ohwi et Ohashi and the red bean Vigna angularis Ohwi et Ohashi-based red bean or the medicinal preparation thereof, the medicinal materials, decoction pieces, decoction piece standard decoction or formula particles of the red bean Vigna umbellata Ohwi et Ohashi are abbreviated as corresponding medicinal materials, decoction pieces, decoction piece standard decoction or formula particles of the red bean (red bean); the medicinal materials, decoction pieces, decoction piece standard decoction or formula particles of the red beans Vigna angularis Ohwi et Ohashi are abbreviated as corresponding medicinal materials, decoction pieces, decoction piece standard decoction or formula particles of the red beans (red beans).
The technical scheme of the invention has the following advantages:
1. the construction method of the characteristic spectrum of the red bean or the pharmaceutical preparation thereof provided by the invention can be used for detecting the red bean or the pharmaceutical preparation thereof, and also can be used for detecting the red bean or the pharmaceutical preparation thereof, and has the advantages of shorter detection time, more comprehensive characteristic peak display and better characteristic peak separation degree.
2. The method for distinguishing the red bean formula particles with different base sources can strictly distinguish the red beans (red beans) with different base sources or the pharmaceutical preparations thereof from the red beans (red beans) or the pharmaceutical preparations thereof.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a comparative characteristic map of red bean (red bean) in example 1 of the present invention;
FIG. 2 is a characteristic map of the red bean (red bean) control drug in example 1 of the present invention;
FIG. 3 is a characteristic map of 15 batches of red bean (red bean) decoction pieces standard decoction (lyophilized powder) in example 1 of the present invention;
FIG. 4 is a characteristic spectrum of 3 lot of red bean (red bean) formula particles in example 1 of the present invention;
FIG. 5 is a chromatogram showing the location of catechin-7-O-beta-glucopyranoside, catechin, epicatechin-5-O-beta-glucopyranoside, as a control of the red bean (red bean) pellet formulation in example 1 of the present invention;
FIG. 6 is a localization chromatogram of quercetin-7-O-beta-glucoside as a control of the red bean (red bean) formulation granule in example 1 of the present invention;
FIG. 7 is a localization chromatogram of isoquercitrin as a control of the Red Bean (Red Bean) formulation in example 1 of the present invention;
FIG. 8 is a characteristic spectrum of a comparison of the standard decoction of semen Phaseoli (semen Phaseoli) and semen Phaseoli (semen Phaseoli) decoction pieces in example 2 of the present invention; wherein, S1 (7): the standard decoction control map of semen Phaseoli decoction pieces; s2 (7): the standard decoction of semen Phaseoli (semen Phaseoli) decoction pieces is compared with the map;
FIG. 9 is a chromatogram of comparative example 1 of the present invention;
FIG. 10 is a chromatogram of comparative example 2 of the present invention;
FIG. 11 is a chromatogram of comparative example 3 of the present invention;
FIG. 12 is a chromatogram of comparative example 4 of the present invention;
FIG. 13 is a chromatogram of comparative example 5 of the present invention;
FIG. 14 is a chromatogram of comparative example 6 of the present invention;
FIG. 15 is a chromatogram of comparative example 7 of the present invention;
FIG. 16 is a chromatogram of comparative example 8 of the present invention;
FIG. 17 is a chromatogram of comparative example 9 of the present invention;
FIG. 18 is a chromatogram of pediatric theanine-7-O-beta-glucopyranoside in Experimental example 1 of the present invention, verification of 1, chromatography conditions and system applicability;
FIG. 19 is a graph showing the "1", verification of chromatographic conditions and system suitability "of the pediatric tea extract of Experimental example 1 of the invention;
FIG. 20 is a chromatogram of epicatechin-5-O-beta-glucopyranoside in Experimental example 1 of the present invention, "1, verification of chromatographic conditions and System applicability";
FIG. 21 is a chromatogram of the formulation of red bean (red bean) in "1, verification of chromatographic conditions and system applicability" in Experimental example 1 of the present invention;
FIG. 22 is a chromatogram of the precision of Experimental example 2 of the present invention;
FIG. 23 is a repetitive chromatogram of Experimental example 2 of the present invention;
FIG. 24 is an intermediate precision (personnel) chromatogram in Experimental example 2 of the present invention;
FIG. 25 is a chromatogram of a negative blank sample in the specificity of Experimental example 2 of the present invention;
FIG. 26 is a chromatogram in the stability study in Experimental example 2 of the present invention;
FIG. 27 is a chromatogram of the durability of the present invention in terms of flow rate of 0.18 ml/min;
FIG. 28 is a chromatogram of the durability of the present invention in terms of flow rate of 0.22 ml/min;
FIG. 29 is a chromatogram of the durability center column temperature of 18℃in Experimental example 2 of the present invention;
FIG. 30 is a chromatogram of the present invention in Experimental example 2 at a column temperature of 22 ℃;
FIG. 31 is a chromatogram of a high performance liquid chromatography apparatus Waters H-CLASS UPLC for durability in Experimental example 2 of the present invention;
FIG. 32 is a chromatogram of a Thermo UPLC as a high performance liquid phase instrument of durability in Experimental example 2 of the present invention;
FIG. 33 is a chromatogram of Waters ACQUITY UPLC BEH C18 in durability in Experimental example 2 of the present invention;
FIG. 34 is a chromatogram of Thermo ScientificTM Hypersil GOLDTM in durability in Experimental example 2 of the present invention.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are intended to limit the scope of the invention, any and all products that come within the meaning of the invention or that combine the features of the invention with other prior art are within the scope of the invention.
The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
The following examples and experimental examples relate to the apparatus and reagents
Instrument apparatus:
chromatograph 2: thermo Vanquish Flex UHPLC chromatography system comprising quaternary solvent manager (Vanquish Quaternary P. Mu. Mp F VF-P20-A), autosampler (Vanquish Split Sampler FT VF-A10-A-02), as-is inlet chromatography column incubator (Vanquish Col. Mu. Mn Compartment H VH-C10-A-02), DAD detector (Vanquish VF-D40-A);
an electronic balance: METTER TOLEDO (Metrehler Switzerland) ME36S, XS, XS205, XSE205 (one ten thousandth)
Ultrasonic instrument: SK5200H Shanghai Kogyo ultrasonic instruments Co.
Chromatographic column:
(1)Agilent ZORBAX Eclipse Plus C18 RRHD 2.1×150mm,1.8μm
(2)Waters ACQUITY UPLC BEH C18 2.1x150mm 1.7μm
(3)Thermo ScientificTM Hypersil GOLDTM 2.1x150mm 1.9μm
reagent:
methanol is chromatographic pure, and water is ultrapure water; the other reagents were all analytically pure.
Reagent:
catechin-7-O-beta-glucopyranoside (lot number 22022102, for content determination, calculated as 99.1%) was purchased from Shanghai Shidand standard technical service Co.
epicatechin-5-O-beta-glucopyranoside (lot 22022104, for content determination, calculated as 99.1%) is purchased from Shanghai shidand standard technical services limited.
Catechin (lot 110877-202005, calculated as 95.1%) was purchased from the national food and drug verification institute for content determination.
The medicinal materials or decoction pieces of semen Phaseoli (semen Phaseoli Vigna umbeuata Ohwi et Ohashi or semen Phaseoli Vigna angutaris Ohwi et Ohashi) are all commercially available products.
The following examples or experimental examples refer to the following raw materials:
the preparation method of the red bean decoction piece standard decoction comprises the following steps: soaking semen Phaseoli decoction pieces 100g in 800ml water for 30min, boiling with strong fire, decocting with slow fire for 30min, filtering, collecting filtrate, adding 600ml water into the residue, boiling with strong fire, decocting with slow fire for 30min, filtering, mixing filtrates, concentrating under reduced pressure, and lyophilizing. ( Standard decoction lot of semen Phaseoli (semen Phaseoli) decoction pieces: 200901Y … … 2009015Y )
The preparation method of the red bean formula granule comprises the following steps: reflux extracting semen Phaseoli decoction pieces with heat for at least 1 time, adding at least 2 times of water for at least 30min, filtering, mixing filtrates, concentrating filtrate to relative density of 1.05-1.10 at 60deg.C, adding conventional adjuvants, and making into clinically acceptable granule by conventional process. ( Lot of red bean (red bean) formula particles: 2005001Y, 2005002Y, 2005003Y )
Example 1 construction method of characteristic Spectrum of Red Bean or its pharmaceutical preparation
The embodiment provides a construction method of a characteristic spectrum of red bean or a pharmaceutical preparation thereof, which comprises the following steps:
chromatographic conditions and system applicability test, according to high performance liquid chromatography (China Pharmacopeia 2020 edition rule 0512), the chromatographic column uses octadecylsilane chemically bonded silica gel as filler, the chromatographic column is Agilent ZORBAX Eclipse Plus C RRHD, the column length is 150mm, the inner diameter is 2.1mm, and the particle diameter is 1.8 μm; gradient elution was performed using methanol as mobile phase A and 0.07% (v/v) phosphoric acid solution as mobile phase B according to the gradient elution procedure shown in Table 1 below; the column temperature is 20 ℃; the flow rate is 0.2ml per minute; the detection wavelength is 280nm; the theoretical plate number is not less than 5000 according to catechin-7-O-beta-glucopyranoside peak;
TABLE 1
Preparation of reference solution: about 2g of red bean reference medicine is taken, placed in a conical flask with a plug, 100ml of water is added, heating reflux is carried out for 45 minutes, the mixture is taken out, cooled, filtered, the filtrate is evaporated to dryness, 30% methanol 15ml is added to residues, ultrasonic treatment (power 300W, frequency 40 kHz) is carried out for 30 minutes, the mixture is taken out, cooled, filtered, and the subsequent filtrate is taken as reference medicine solution. Taking a proper amount of catechin-7-O-beta-glucopyranoside, catechin, isoquercitrin and quercetin-7-O-beta-glucopyranoside reference substances, precisely weighing, adding 30% methanol to prepare a mixed solution containing 0.2mg of catechin-7-O-beta-glucopyranoside, 0.05mg of catechin, 0.05mg of isoquercitrin and 0.05mg of quercetin-7-O-beta-glucopyranoside per 1ml, and taking the mixed solution as a reference substance solution;
preparation of test solution: grinding a test sample, taking about 1.0g, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 30% methanol, weighing, performing ultrasonic treatment (power 300W, frequency 40 kHz) for 30 minutes, taking out, cooling, weighing again, supplementing the lost weight with 30% methanol, shaking uniformly, filtering, and collecting the subsequent filtrate;
assay: precisely sucking 2 μl of reference substance solution and 4 μl of reference substance solution, respectively, injecting into liquid chromatograph, measuring, and recording chromatogram;
when the sample is semen Phaseoli Vigna umbeuata Ohwi et Ohashi or its pharmaceutical preparation, taking semen Phaseoli Vigna umbeuata Ohwi et Ohashi as reference material to prepare reference material solution;
when the sample is semen Phaseoli Vigna angutaris Ohwi et Ohashi or its pharmaceutical preparation, semen Phaseoli Vigna angutaris Ohwi et Ohashi is used as control material to prepare reference solution.
In the embodiment, 18 batches of red bean (red bean) medicinal preparations are selected as the samples to be tested to generate characteristic patterns, the generated characteristic patterns are analyzed, fingerprint similarity evaluation software '2012 edition of traditional Chinese medicine chromatographic fingerprint similarity evaluation system' compiled by pharmacopoeia committee is adopted, and a Mark peak fitting mode is adopted to generate a comparison characteristic pattern; after the chromatographic matching, 10 common characteristic peaks with proper response values (peak areas), good separation degree and high purity are obtained, and the chromatographic peak numbers of the chromatographic peaks are rearranged according to the sequence of the chromatographic peaks and are from peak 1 to peak 10, as shown in figure 1. Wherein the relative retention times and relative peak areas of the control profile are shown in tables 2 and 3 below.
Table 2, relative retention time of the characteristic pattern of Red Bean (Red Bean)
Table 2 comparative characteristic pattern relative retention time of continuous and small red beans (red beans)
TABLE 3 comparison of characteristic patterns against peak areas of Red Bean (Red Bean)
TABLE 3 comparative peak areas of continuous and small red beans (red beans) control feature patterns
The characteristic patterns of the red bean (red bean) control medicinal materials are shown in fig. 2, and the relative retention time and the relative peak area are shown in tables 4 and 5 below. The characteristic patterns of the 18 lot of red bean (red bean) pharmaceutical preparations are shown in fig. 3 and 4, the relative retention times and the relative peak areas are shown in tables 6 and 7 below, and the similarity results are shown in table 8.
Table 4, relative retention time of the characteristic spectrum of the red bean (red bean) against the medicinal material
Table 4 comparative retention time of characteristic patterns of continuous and small red beans (red beans)
Table 5, comparative peak area of characteristic patterns of Red Bean (Red Bean) as reference
Table 5 comparative peak area of characteristic spectrum of continuous and small red bean (small red bean)
Table 6, 18 lot of red bean (red bean) pharmaceutical preparation characteristic spectrum determination relative retention time results
Table 6 results of determination of relative retention time for continuous, 18 lot of pharmaceutical formulations of red bean (red bean)
Table 7, 18 lot of red bean (red bean) pharmaceutical preparation characteristic spectrum determination relative peak area results
TABLE 7 characterization of continuous 18 batches of red bean (red bean) pharmaceutical formulations relative peak area results
Table 8, 18 lot of red bean (red bean) pharmaceutical preparation characteristic map measurement similarity results
Identification and assignment of characteristic peaks by HPLC and LC/MS/MS confirmed that peak 1 was catechin-7-O-beta-glucopyranoside, peak 3 was catechin, peak 4 was epicatechin-5-O-beta-glucopyranoside, peak 8 was quercetin-7-O-beta-glucoside, and peak 9 was isoquercitrin. The positioning chromatograms of the control were performed by HPLC and are shown in figures 5-7.
From the above results, the characteristic spectrum of the red bean (red bean) formula granule should show 10 characteristic peaks, which should correspond to 10 characteristic peaks in the characteristic spectrum of the control medicinal material. Wherein, the retention time of the peaks 1, 3, 8 and 9 respectively correspond to the retention time of the corresponding reference peak of the reference substance; and calculating the relative retention time of each characteristic peak and the S peak by taking the peak corresponding to the reference peak of the catechin-7-O-beta-glucopyranoside reference substance as the S peak, wherein the relative retention time is within +/-10% of a specified value. The specified value is: 1.26 (Peak 2), 1.85 (Peak 4), 2.72 (Peak 5), 2.84 (Peak 6), 2.90 (Peak 7), 3.60 (Peak 10).
According to the peak identification and peak selection results of characteristic peaks in the semen Phaseoli (semen Phaseoli) formula granule and decoction piece standard decoction, the chromatographic peak of the reference substance is catechin-7-O-beta-glucopyranoside, catechin and isoquercitrin. In the characteristic spectrum of the red bean (red bean) decoction piece standard decoction, catechin-7-O-beta-glucopyranoside has higher response and is the main chemical component of the red bean (red bean) decoction piece standard decoction, and in summary, catechin-7-O-beta-glucopyranoside is taken as the S peak of the characteristic spectrum. The relative retention time of each characteristic peak was calculated.
Example 2A method of differentiating Red beans of different base sources
This example provides a method of differentiating red beans of different base sources, tested according to the method of example 1;
when the obtained characteristic spectrum lacks peak 6 and the relative peak area of peak 7 and S peak is lower than (<) 0.14, the basic source of the sample is red bean Vigna angularis Ohwi et Ohashi;
when the obtained characteristic spectrum does not lack peak 6, and the relative peak area of peak 7 and S peak is not less than (more than or equal to) 0.14, the basic source of the sample is semen Phaseoli Vigna umbellata Ohwi et Ohashi.
In this example, the test sample was selected from 15 batches of red bean (red bean) decoction pieces standard decoction (lyophilized powder) and 15 batches of red bean (red bean) decoction pieces standard decoction (lyophilized powder). The relative peak areas of the characteristic patterns of the 15 batches of red bean (red bean) decoction pieces standard decoction (freeze-dried powder) are shown in the following table 9. The relative peak areas of the characteristic patterns of 15 batches of red bean (red bean) decoction pieces standard decoction (freeze-dried powder) are shown in table 7 in example 1.
Table 9, 15 batches of red bean decoction pieces (lyophilized powder) relative peak area determination results
TABLE 9 measurement of relative Peak area of decoction pieces of 15 batches of red beans (red beans)
As is clear from the above results and fig. 8, the characteristic patterns of the red bean (red bean) and the red bean (red bean) decoction pieces were different, and the peak area differences were shown in table 10 below with respect to the peak 6 of the red bean (red bean) decoction piece standard decoction (lyophilized powder) and the peak 2 and peak 7 relative peak areas of the red bean (red bean) decoction pieces standard decoction (lyophilized powder). In summary, the peak with the larger difference in relative peak area was peak 7. Thus, the red beans (red beans) and the different primordia of red beans can be distinguished by:
when the obtained characteristic spectrum lacks a peak 6 and the relative peak area of the peak 7 and the S peak is lower than 0.14, the basic source of the sample is red bean Vigna angularis Ohwi et Ohashi;
when the obtained characteristic spectrum does not lack peak 6 and the relative peak area of peak 7 and S peak is not less than 0.14, the basic source of the sample is semen Phaseoli Vigna umbellata Ohwi et Ohash.
Table 10
Comparative example 1
The comparative example differs from example 1 in that octadecylsilane chemically bonded silica was used as a filler (Agilent Eclipse Plus RRHD C, column length: 100mm, inner diameter: 2.1mm, particle diameter: 1.8 μm) in chromatographic conditions and system suitability test; using methanol as mobile phase A and 0.1% (v/v) phosphoric acid solution as mobile phase B, performing gradient elution according to the UPLC method elution program in Table 11; the flow rate is 0.3ml per minute; column temperature 20 ℃; the detection wavelength was 280nm. The sample is semen Phaseoli decoction piece standard decoction (lyophilized powder).
TABLE 11 UPLC method elution procedure
As shown in FIG. 9, the characteristic spectrum effect of the standard decoction (lyophilized powder) of semen Phaseoli (semen Phaseoli) decoction pieces is not ideal.
Comparative example 2
The difference between this comparative example and comparative example 1 is that the flow rate was 0.3ml/min in the chromatographic conditions and system suitability test, the column temperature was 25℃and the elution procedure was as shown in Table 12 below. The liquid chromatograph adopts an ultra-high performance liquid chromatograph.
TABLE 12 UPLC method elution procedure (flow rate 0.3ml/min, column temperature 25 ℃ C.)
The results are shown in fig. 10, and characteristic peaks of the characteristic map spectrum of the red bean (red bean) decoction piece standard decoction (freeze-dried powder) mainly appear in the first 25 minutes, and no characteristic peaks exist after 25 minutes.
Comparative example 3
The difference between this comparative example and comparative example 2 is the elution procedure in the chromatographic conditions and system suitability test as shown in Table 13 below.
TABLE 13 UPLC method elution procedure (flow rate 0.3ml/min, column temperature 25 ℃ C.)
The results are shown in fig. 11, and characteristic peaks of the standard decoction pieces (lyophilized powder) of red bean (red bean) at 14 minutes and 30 minutes are not completely separated.
Comparative example 4
The difference between this comparative example and comparative example 3 is that the chromatographic column was Waters ACQUITY BEH C, 2.1X105 mm,1.7 μm in chromatographic conditions and system suitability test.
As shown in FIG. 12, the characteristic spectrum of the standard decoction (lyophilized powder) of semen Phaseoli (semen Phaseoli) has good peak separation effect, but the characteristic peak separation degree is poor in 8-16 min and 34 min.
Comparative example 5
The difference between this comparative example and comparative example 4 is that the flow rate was 0.25ml/min and the column temperature was 20℃in the chromatographic conditions and system suitability test.
The results are shown in fig. 13, and the characteristic spectrum of the red bean (red bean) decoction piece standard decoction (freeze-dried powder) is found in the later investigation process, the column pressure is higher under the condition, and the instrument system is easy to be overloaded.
Comparative example 6
The difference between this comparative example and comparative example 4 is that the flow rate was 0.20ml/min and the column temperature was 25℃in the chromatographic conditions and system suitability test.
The results are shown in fig. 14, and the characteristic spectrum effect of the red bean (red bean) decoction piece standard decoction (freeze-dried powder) is not ideal.
Comparative example 7
The difference between this comparative example and comparative example 5 is that the chromatographic column Agilent Eclipse Plus RRHD C (2.1X105 mm,1.8 μm) was used in the chromatographic conditions and system suitability test.
As shown in fig. 15, the characteristic spectrum of the standard decoction pieces of red bean (red bean) was better separated as a whole, but the two characteristic peaks at 10 minutes were worse.
Comparative example 8
The difference between this comparative example and comparative example 7 is that the flow rate was 0.20ml/min in the chromatographic conditions and system suitability test.
As shown in FIG. 16, the characteristic spectrum of the standard decoction (lyophilized powder) of the semen Phaseoli decoction pieces has good overall separation, but the separation degree of two characteristic peaks at 12-14 minutes still does not meet the requirement.
Comparative example 9
The difference between this comparative example and comparative example 8 is the elution procedure in the chromatographic conditions and system suitability test as shown in Table 14 below.
TABLE 14 UPLC method elution procedure
As shown in FIG. 17, the characteristic spectrum of the standard decoction (lyophilized powder) of semen Phaseoli (semen Phaseoli) decoction pieces shows good overall separation of the chromatographic peaks, but the separation effect of part of characteristic peaks and some small impurity peaks is poor.
Experimental example 1
1. Verification of chromatographic conditions and System applicability
Test and control solutions were prepared as in example 1, and HPLC analysis was performed under the chromatographic conditions described in example 1, with the test being red bean (red bean) formulation granules (lot 2005001Y). The chromatogram is recorded.
Parameters of catechin-7-O-beta-glucopyranoside chromatogram are shown in Table 15 below and in FIG. 18.
TABLE 15
Parameters of catechin chromatograms are shown in Table 16 below and FIG. 19.
Table 16
Parameters of epicatechin-5-O-beta-glucopyranoside chromatogram are shown in Table 17 below and in FIG. 20.
TABLE 17
Parameters of the red bean (red bean) formula particle chromatogram are shown in table 18 below, and the chromatogram is shown in fig. 21.
TABLE 18
From the above, the chromatographic method in the embodiment 1 of the invention has good system adaptability and specificity, and can be used as a detection method for the characteristic spectrum of the red bean (red bean) formula particle.
Experimental example 2 methodological verification
1. Precision test
The same sample solution (2005001Y, prepared according to example 1) was taken and the sample was repeatedly introduced 6 times under the chromatographic conditions of example 1 to determine the relative retention time and relative peak area of 9 common peaks, and the RSD of the relative retention time of each characteristic peak and the reference S peak (No. 1 peak) was less than 2%. The results are shown in tables 19-20 below, and FIG. 22, which shows that the instrument precision is good.
TABLE 19 results of Instrument precision versus retention time test
Table 19 results of continuous and instrumental precision versus retention time test
Table 20, results of Instrument precision similarity test
2. Method repeatability test
6 parts of the same sample (2005001Y, prepared in accordance with example 1) were taken, and the relative retention time and the relative peak area of 10 common peaks were measured under the chromatographic conditions of example 1, respectively, and the relative retention time of each characteristic peak and the reference S peak (No. 1 peak) was less than 2%. The results are shown in tables 21-22 below, and FIG. 23, which demonstrate that the process is reproducible.
TABLE 21 method repeatability vs. retention time test results
TABLE 21 results of relative retention time test for procedure repeatability
Table 22 results of the repeatability similarity test
3. Intermediate precision (different operators)
The same sample solution (lot: 2005001Y, prepared as in example 1) of red bean (red bean) was taken at different times by three inspectors, and the relative retention time and relative peak area of each common peak were measured according to the chromatographic conditions of example 1 and using the same equipment. The relative average deviation of the relative retention time of each characteristic peak from the reference S peak (peak No. 1) was less than 2%. The results are shown in tables 23-24 and FIG. 24, which demonstrate that the intermediate precision of the present method is good.
Table 23, results of intermediate precision relative retention time test (different operators)
Table 23 results of the continuous, intermediate precision versus retention time test (different operators)
TABLE 24 intermediate precision similarity test results
4. Specialization of
Preparation of negative blank samples: taking about 1g of blank auxiliary material maltodextrin, precisely weighing, placing into a cone-shaped bottle with a plug, precisely adding 25ml of 30% methanol, sealing, performing ultrasonic treatment (power is 250W and frequency is 40 kHz) for 30 minutes, taking out, cooling, shaking uniformly, filtering, and taking out subsequent filtrate.
2 μl of each of the sample solution to be tested (2005001Y) and the negative blank sample solution was precisely sucked, and the samples were respectively injected into a high performance liquid chromatograph, and tested under the chromatographic conditions of example 1, the result of the negative blank sample being shown in FIG. 25, and the chromatogram of the sample solution to be tested (2005001Y) being shown in FIG. 21, indicating that the negative sample solution was not disturbed.
5. Stability investigation
Sample solutions (2005001Y, prepared according to example 1) of the same batch were taken and were sampled at 0, 4, 8, 12, 18 and 24 hours according to the chromatographic conditions of example 1, and the relative retention time and the relative peak area of 10 common peaks were measured, wherein the relative retention time of each characteristic peak and the reference S peak was less than 2%. The results are shown in tables 25-26 below and FIG. 26, which demonstrate that the test solutions are stable over 24 hours and meet the assay requirements.
TABLE 25 stability versus retention time test results
Table 25 results of continuous, stability versus retention time test
Table 26 stability similarity test results
6. Durability of
6.1 investigation of different flow Rate
Sample solutions (2005001Y, prepared according to example 1) of the same lot were taken and subjected to the chromatographic conditions of example 1 at flow rates of 0.18ml/min, 0.2ml/min, 0.22ml/min, respectively
TABLE 27 relative retention time results for different flow rates
TABLE 27 continuous, different flow rate versus retention time results
6.2 investigation of different column temperatures
The same batch of sample solution (2005001Y, prepared according to example 1) was taken, the chromatographic conditions of example 1 were followed, the column temperatures were set at 18℃and 20℃and 22℃respectively, and the separation effect of each characteristic peak and the relative peak area at the time of the change in the column temperature were examined, and the results were shown in tables 29 to 30 below, and FIGS. 29, 21 and 30, showing that the separation of the different columns Wen Shifeng, 9 and 10 was affected, but within an acceptable range, the effect of the column temperature of 20℃was optimal. Therefore, the fixed column temperature of the present invention was measured at 20 ℃.
Table 28 comparison of results of measurements of different column temperatures versus retention time
Table 28 comparison of results of measurements of relative retention times for different column temperatures
6.3 investigation of different instruments
The same batch of sample solution (2005001Y, prepared according to example 1) was taken, the chromatographic conditions of example 1 were followed, and different types of high performance liquid chromatography apparatuses were set, namely Waters H-CLASS UPLC and Thermo UPLC chromatography, respectively, to examine the separation effect of each characteristic peak when the high performance liquid chromatography apparatus was changed. The results are shown in Table 29, and in FIGS. 31 and 32, which show that the separation of characteristic peaks of the red bean (red bean) formulation particles is good between different instruments, and the difference between different instruments should be considered when specifying the relative retention time.
TABLE 29 determination of the relative retention time of characteristic spectra of different HPLC instruments
Table 29 results of measuring the relative retention time of characteristic maps of different high performance liquid instruments
6.4 investigation of different chromatographic columns
The same batch of sample solution (2005001Y, prepared according to example 1) was taken, chromatographic conditions of example 1 were operated, chromatographic columns of different types were set as Agilent ZORBAX Eclipse Plus C RRHD, waters ACQUITY UPLC BEH C, thermo ScientificTM Hypersil GOLDTM respectively, and the separation effect of each characteristic peak was examined when the chromatographic column was changed. The results are shown in Table 30 below and in FIGS. 21, 33 and 34, which show that the characteristic chromatographic peak retention times change when the chromatographic column changes, indicating that the characteristic chromatographic peak retention times are affected by the change in the chromatographic column type, especially the chromatographic peak type on the chromatographic column 3 (Waters ACQUITY UPLC CSHTM C) is poor, and therefore the method fixes the chromatographic column Agilent ZORBAX Eclipse Plus C RRHD as a chromatographic column for subsequent study.
TABLE 30 comparison of the results of the relative retention time measurements for different chromatographic columns
TABLE 30 comparison of results of relative retention time determinations for different chromatographic columns
It should be apparent that the above embodiments are merely examples for clarity of illustration and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary or exhaustive of all embodiments. While obvious variations or modifications of the embodiments herein are within the scope of the present invention.
Claims (7)
1. The construction method of the characteristic map of the red bean or the pharmaceutical preparation thereof is characterized by comprising the following steps:
chromatographic conditions and system applicability test, according to high performance liquid chromatography, the chromatographic column uses octadecylsilane chemically bonded silica as filler; methanol is used as a mobile phase A, a 0.07% v/v phosphoric acid solution is used as a mobile phase B, and gradient elution is carried out according to the following gradient elution program: 0-8 min, wherein the volume percentage of the mobile phase A is 10%; 8-55 min, wherein the volume percentage of the mobile phase A is 10% -45%; 55-56 min, wherein the volume percentage of the mobile phase A is 45% -10%; 56-60 min, wherein the volume percentage of the mobile phase A is 10%; the column temperature is 18-22 ℃; the flow rate is 0.18-0.22ml per minute; the detection wavelength is 280nm; the theoretical plate number is not less than 5000 calculated according to catechin-7-O-beta-glucopyranoside peak; the chromatographic column is Agilent ZORBAX Eclipse Plus C RRHD, the column length is 150mm, the inner diameter is 2.1mm, and the particle size is 1.8 mu m; the column temperature is 20 ℃; the flow rate is 0.2ml/min;
preparation of reference solution: the preparation method of the reference substance solution of the reference medicinal material comprises the following steps: taking 2g of red bean reference medicine, placing into a conical flask with a plug, adding 100ml of water, heating and refluxing for 45 minutes, taking out, cooling, filtering, evaporating filtrate to dryness, adding 15ml of 30% v/v methanol into residues, performing ultrasonic treatment for 30 minutes under the condition of power 300W and frequency 40kHz, taking out, cooling, filtering, and taking out subsequent filtrate to be used as reference medicine solution;
taking a proper amount of catechin-7-O-beta-glucopyranoside, catechin, isoquercitrin and quercetin-7-O-beta-glucopyranoside reference substances, precisely weighing, and adding 30% v/v methanol to prepare a mixed solution containing 0.2mg of catechin-7-O-beta-glucopyranoside, 0.05mg of catechin, 0.05mg of isoquercitrin and 0.05mg of quercetin-7-O-beta-glucopyranoside per 1ml of reference substance solution;
preparation of test solution: grinding a test sample, precisely weighing 0.5-1.5g, placing into a conical flask with a plug, precisely adding 25-50ml of 30%v/v methanol, weighing, performing ultrasonic treatment for 30-60 minutes under the condition of 200-300W of power and 35-45kHz of frequency, taking out, cooling, weighing again, supplementing the lost weight with 30-50%v/v methanol, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the product;
assay: precisely sucking the reference solution and the sample solution, respectively, injecting into a liquid chromatograph, measuring, and recording the chromatograms;
the semen Phaseoli comprises semen PhaseoliVigna umbeuata Ohwi et OhashiOr red beanVigna angutaris Ohwi et Ohashi。
2. The method for constructing a characteristic spectrum of red bean or a pharmaceutical preparation thereof according to claim 1, wherein,
preparation of test solution: grinding a test sample, precisely weighing 1.0g, placing into a conical flask with a plug, precisely adding 25ml of 30% v/v methanol, weighing, performing ultrasonic treatment for 30 minutes under the condition of power 300W and frequency 40kHz, taking out, cooling, weighing again, supplementing the lost weight with 30% v/v methanol, shaking uniformly, filtering, and taking the subsequent filtrate.
3. The method for constructing the characteristic spectrum of the red bean or the pharmaceutical preparation thereof according to any one of claims 1 to 2, wherein in the measurement method, 1 to 3 μl of each of a reference substance solution and a test substance solution is precisely sucked, 3 to 5 μl of each of the reference substance solution and the test substance solution, and the reference substance solution is injected into a liquid chromatograph.
4. The method for constructing a characteristic spectrum of red bean or a pharmaceutical preparation thereof according to claim 3,
in the measurement method, 2 mu l of each of a reference substance solution and a test substance solution of a reference substance are respectively and precisely sucked, 4 mu l of the reference substance solution of the reference substance is injected into a liquid chromatograph.
5. The method for constructing a characteristic spectrum of red bean or a pharmaceutical preparation thereof according to any one of claims 1 to 2, wherein the red bean or the pharmaceutical preparation thereof comprises the use of the characteristic spectrum of red bean medicinal materials, red bean decoction pieces, red bean decoction piece standard decoction or red bean formula granules.
6. The method for quality inspection according to any one of claims 1 to 5, wherein 10 characteristic peaks are to be presented in the characteristic spectrum of the sample, corresponding to the retention time of 10 characteristic peaks in the reference chromatogram of the reference sample, wherein 1, 3, 8 and 9 peaks are to be respectively corresponding to the retention time of the corresponding reference sample peaks, peak 1 is catechin-7-O- β -glucopyranoside, peak 3 is catechin, peak 4 is epicatechin-5-O- β -glucopyranoside, peak 8 is quercetin-7-O- β -glucoside, peak 9 is isoquercitrin, the relative retention time of each characteristic peak and S peak is calculated with the peak corresponding to the reference sample peak of catechin-7-O- β -glucopyranoside within ±10% of the specified value, and the specified value is: peak 2 was 1.26, peak 4 was 1.85, peak 5 was 2.72, peak 6 was 2.84, peak 7 was 2.90, and peak 10 was 3.60;
the test sample comprises semen Phaseoli or its pharmaceutical preparation; the semen Phaseoli or its pharmaceutical preparation comprises semen Phaseoli medicinal material, semen Phaseoli decoction pieces, semen Phaseoli decoction piece standard decoction or semen Phaseoli granule;
when the sample is semen PhaseoliVigna angutaris Ohwi et OhashiOr the characteristic spectrum of the test sample does not contain peak 6 when the test sample is prepared into a pharmaceutical preparation.
7. A method for distinguishing between different primordia of red beans or a pharmaceutical preparation thereof, comprising a method for constructing a characteristic map using the red beans or the pharmaceutical preparation thereof according to any one of claims 1 to 5;
when the obtained characteristic spectrum lacks peak 6 and the relative peak area of peak 7 and S peak is less than 0.14, the basic source of the sample is semen PhaseoliVigna angularis Ohwi et Ohashi；
When the obtained characteristic spectrum does not lack peak 6, and the relative peak area of peak 7 and S peak is more than or equal to 0.14, the basic source of the sample is semen PhaseoliVigna umbellata Ohwi et Ohashi。
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| CN113447595A (en) * | 2021-07-21 | 2021-09-28 | 上海和黄药业有限公司 | Detection method of characteristic spectrum of medicinal preparation and application thereof |
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| CN113447595A (en) * | 2021-07-21 | 2021-09-28 | 上海和黄药业有限公司 | Detection method of characteristic spectrum of medicinal preparation and application thereof |
| CN114047268A (en) * | 2021-11-26 | 2022-02-15 | 华润三九医药股份有限公司 | Fingerprint spectrum of Guangdong earthworm medicine preparation, construction method and content determination method thereof |
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| RP-HPLC测定中药赤小豆中儿茶素-7-O-β-D-吡喃葡萄糖苷的含量;穆合塔尔・卡德尔哈孜 等;中国药学杂志;第46卷(第10期);778-780 * |
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