CN111189952A - Method for measuring special content of verbena and controlling quality of verbena - Google Patents
Method for measuring special content of verbena and controlling quality of verbena Download PDFInfo
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- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
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Abstract
The invention discloses a method for measuring the special content of verbena and controlling the quality of the verbena, wherein the content measuring and controlling method adopts a high performance liquid chromatography to detect the content of the special components of the verbena, and the conditions of the high performance liquid chromatography are as follows: a chromatographic column: orca C18Chromatography column, 250mm × 4.6mm, 5 μm; mobile phase: the mobile phase A is acetonitrile, the mobile phase B is 0.05 percent phosphoric acid water solution, and gradient elution is carried out, wherein the flow rate is as follows: 1.0 mL/min; column temperature: 25-35 ℃; ultraviolet detection wavelength: 200 nm and 600nm, the invention establishes a quality control method of verbena HPLC fingerprint, establishes a verbena comparison fingerprint common mode through a system clustering analysis and similarity evaluation system, and can classify the verbena through the clustering analysis and the similarity analysis, thereby controlling the quality of the verbena.
Description
Technical Field
The invention relates to the technical field of drug analysis and detection, in particular to a method for determining the special content of verbena and controlling the quality of the verbena.
Background
The herba Verbenae is dry aerial part of herba Verbenae of Verbenaceae, is wild resource, and is mainly produced in Shandong, Jiangsu, Guangxi, Guizhou, Hubei, Henan, Sichuan and other places. Has effects of promoting blood circulation, removing blood stasis, removing toxic substance, promoting diuresis, eliminating jaundice, and preventing malaria. Can be used for treating abdominal mass, dysmenorrhea, amenorrhea, sore throat, carbuncle, edema, jaundice, and malaria. Modern pharmacological research shows that the water extract, the alcohol extract, the volatile oil and the like of the verbena medicinal material have the effects of resisting tumors, inflammation, pain, bacteria, protecting nerves, regulating the immunocompetence, resisting oxidation, resisting early pregnancy and the like, and the main components of the verbena medicinal material comprise volatile oil, triterpenes, iridoid glycosides and flavonoids, and in addition, a small amount of phenethyl alcohol glycosides are contained. Wherein the iridoid glycoside is a characteristic component of verbena and is also one of the effective components of verbena.
The verbena is an adjuvant drug in the wind-dispelling and toxin-removing capsule formula, is one of important medicinal materials, and is used for controlling the quality of finished products in order to ensure the quality, safety and effectiveness of the wind-dispelling and toxin-removing capsule, the quality of raw medicinal materials is controlled firstly. The Chinese pharmacopoeia (2010 edition) controls the content of oleanolic acid and ursolic acid in verbena medicinal materials, and the method takes ELSD as a detector, so that the sample preparation is complicated, and the specificity is poor, and therefore, a method for measuring the specific content and controlling the quality of the verbena is needed.
Disclosure of Invention
The invention aims to provide a method for determining the special content of verbena and controlling the quality of the verbena, which can solve the following problems:
1. measuring contents of three special components of verbascoside, verbascoside and verbascoside in the verbena medicinal material by an HPLC method, and controlling by a gradient mobile phase to clearly separate chromatographic peaks of the components;
2. the quality of the verbena can be simply and effectively evaluated by establishing a quality control method of the verbena HPLC fingerprint spectrum, so that the quality of the verbena medicinal material is controlled.
The purpose of the invention can be realized by the following technical scheme:
a method for measuring the special content of verbena comprises the following steps of adopting a high performance liquid chromatography to detect the content of special components of the verbena, wherein the conditions of the high performance liquid chromatography are as follows:
a chromatographic column: orca C18Chromatography column, 250mm × 4.6mm, 5 μm;
mobile phase: the mobile phase A is acetonitrile, the mobile phase B is 0.05 percent phosphoric acid water solution, gradient elution is carried out, the proportions of the mobile phases are as follows, wherein the proportions of the mobile phases are volume percent:
0-30min, 5-25% of mobile phase A and 95-75% of mobile phase B;
for 40-65min, the mobile phase A is 25% -35%, and the mobile phase B is 75% -65%;
65-70min, 35-80% of mobile phase A and 65-20% of mobile phase B;
flow rate: 1.0 mL/min;
column temperature: 30-35 ℃;
ultraviolet detection wavelength: 200-600 nm.
Preferably, the method for detecting the special content comprises the following specific steps:
a1, respectively placing 2.00mg of each of verbascoside, verbascoside and verbascoside reference substances into 10mL measuring bottles, and adding 50% methanol to a constant volume to obtain reference substance stock solution with a mass concentration of 200 μ g/mL;
a2, pulverizing herba Verbenae, sieving with 40 mesh sieve, collecting powder 0.5g, adding 50% methanol 20mL, placing in a round bottom flask, weighing, reflux extracting for 45min, cooling, weighing, supplementing with 50% methanol, shaking, filtering with 0.45 μm microporous membrane, and collecting filtrate to obtain sample solution;
a3, diluting the reference stock solution obtained in A1 by 10 times with 50% methanol, sucking 5 μ L of each stock solution, injecting into a high performance liquid chromatograph, and detecting to obtain liquid chromatogram of verbascoside, pennyoside and verbascoside;
a4, sucking 10 mu L of sample solution obtained from A2, injecting the sample solution into a high performance liquid chromatograph for detection, comparing the obtained liquid chromatogram with the liquid chromatogram obtained from A3 to obtain the contents of verbena major components, namely verbascoside, verbascoside and verbascoside, and measuring by the high performance liquid chromatography according to the following experimental conditions:
a chromatographic column: diamonsil II C18Chromatography column, 250mm × 4.6mm, 5 μm;
mobile phase: the mobile phase A is acetonitrile, the mobile phase B is 0.05 percent phosphoric acid water solution, gradient elution is carried out, the proportions of the mobile phases are as follows, wherein the proportions of the mobile phases are volume percent:
0-30min, 5-25% of mobile phase A and 95-75% of mobile phase B;
for 40-65min, the mobile phase A is 25% -35%, and the mobile phase B is 75% -65%;
65-70min, 35-80% of mobile phase A and 65-20% of mobile phase B;
flow rate: 1.0 mL/min;
column temperature: 30 ℃;
ultraviolet detection wavelength: 230 nm.
A verbena quality control method adopts high performance liquid chromatography to detect the content of the main components of the verbena, wherein the conditions of the high performance liquid chromatography comprise:
a chromatographic column: orca C18Chromatography column, 250mm × 4.6mm, 5 μm;
mobile phase: the mobile phase A is acetonitrile, the mobile phase B is 0.05 percent phosphoric acid water solution, gradient elution is carried out, the proportions of the mobile phases are as follows, wherein the proportions of the mobile phases are volume percent:
0-30min, 5-25% of mobile phase A and 95-75% of mobile phase B;
for 40-65min, the mobile phase A is 25% -35%, and the mobile phase B is 75% -65%;
65-70min, 35-80% of mobile phase A and 65-20% of mobile phase B;
flow rate: 1.0 mL/min;
column temperature: 25-35 ℃;
ultraviolet detection wavelength: 200-600 nm.
Preferably, the verbena quality control method specifically comprises the following steps:
b1, pulverizing the medicinal material of the verbena, sieving with a 40-mesh sieve, taking 1g of powder, adding 20mL of 50% methanol, placing in a round-bottom flask, weighing, extracting under reflux for 45min, cooling, weighing, supplementing with 50% methanol, shaking up, filtering with a 0.45-micrometer microporous membrane, and taking the subsequent filtrate to obtain a sample solution;
b2, precisely absorbing 10 mu L of test solution, injecting the test solution into a high performance liquid chromatograph, and measuring according to the following chromatographic conditions to obtain a fingerprint spectrum:
a chromatographic column: orca C18Chromatography column, 250mm × 4.6mm, 5 μm;
mobile phase: the mobile phase A is acetonitrile, the mobile phase B is 0.05 percent of phosphoric acid aqueous solution, and the proportion of the mobile phase is subjected to gradient elution, wherein the mobile phase proportion is as follows, and the mobile phase proportion is volume percentage:
0-30min, 5-25% of mobile phase A and 95-75% of mobile phase B;
for 40-65min, the mobile phase A is 25% -35%, and the mobile phase B is 75% -65%;
flow rate: 1 mL/min;
column temperature: 25 ℃;
ultraviolet detection wavelength: 230 nm;
b3, taking 14 batches of verbena medicinal herbs, establishing a fingerprint by a method B1 and B2, introducing fingerprint data into similarity software of a 2004A edition of traditional Chinese medicine chromatogram fingerprint similarity evaluation system for analysis, determining 5 main chromatogram characteristic peaks, selecting a No. 4 peak verbascoside peak as a reference peak, wherein the relative peak area of each peak relative to the No. 4 peak is as follows:
peak 1: 0.06-0.75; peak 2: 0.03-0.23; peak 3: 0.44-1.49; peak 4: 1.00; peak 5: 0.50-1.36;
b4, performing cluster analysis on the quantized peak areas by using SPSS software, generating a comparison fingerprint, comparing the sample with the comparison fingerprint by using calculation software 2004B of 'traditional Chinese medicine chromatogram fingerprint similarity evaluation system' to obtain a similarity evaluation result, screening verbena medicinal materials according to the similarity evaluation result and the cluster analysis result, and screening out medicinal materials in batches with lower similarity, thereby finishing quality control.
The invention has the beneficial effects that:
1. the invention establishes a method for measuring the contents of three special components of verbascoside, verbascoside and verbascoside in the verbena medicinal material by adopting an HPLC method, has the advantages of simple and accurate method and good repeatability, achieves clear separation of chromatographic peaks of all components by controlling a gradient mobile phase, lays a foundation for the comprehensive quality control of the verbena medicinal material and the research of the compatibility rule of the verbena medicinal material in a compound, and provides reference for the component analysis and identification of other compound preparations;
2. the invention establishes a quality control method of verbena HPLC fingerprint, establishes a verbena comparison fingerprint common mode through a system clustering analysis and similarity evaluation system, can judge the quality of each batch of medicinal materials through a similarity evaluation result, classifies the verbena with larger similarity difference, can control the quality of the verbena, is stable, reliable and good in reproducibility, provides a basis for the quality evaluation of the verbena medicinal materials, and ensures the quality of the wind-dispelling and detoxifying capsules taking the verbena medicinal materials as main components.
Drawings
In order to facilitate understanding for those skilled in the art, the present invention will be further described with reference to the accompanying drawings.
FIG. 1 is a superimposed HPLC chromatogram for precision measurement;
FIG. 2 is a diagram showing an overlay of HPLC chromatograms for stability tests;
FIG. 3 is a diagram showing a chromatogram overlay of an HPLC chromatogram for a reproducibility test;
FIG. 4 is an HPLC fingerprint of M1 to M15;
FIG. 5 is a graph of cluster analysis of M1 through M15;
FIG. 6 is a control fingerprint of M1 to M15.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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 Verbena drug tested used in the following examples was provided by Anhui Ji pharmaceutical industries, Inc., and was identified as Verbena officinalis L by Zhang Jun investigator, the specific lot numbers are given in the following Table:
other instruments and reagents:
agilent 1100-high performance liquid chromatograph, AB204-N electronic balance (METTLER TOLEDO), Autoscience AS3120 ultrasonic instrument, electric heating thermostat water bath (Jiangsu province medical instrument factory), METTLER TOLEDOPB303-N electronic balance (METTLER TOLEDO, Switzerland).
Methanol, acetonitrile (chromatographically pure) Tianjin Kancoded science and technology, Inc.; ethanol, phosphoric acid (analytical grade), Tianjin chemical reagent factory; water, purified water; verbascoside (J140314001), verbascoside (J140314001) and verbascoside (JL130312002) have purity of more than 98%, and are available from Shanghai future industries, Ltd.
Example 1
The method for detecting the special content comprises the following specific steps:
a1, respectively placing 2.00mg of each of verbascoside, verbascoside and verbascoside reference substances into 10mL measuring bottles, and adding 50% methanol to a constant volume to obtain reference substance stock solution with a mass concentration of 200 μ g/mL;
a2, pulverizing herba Verbenae, sieving with 40 mesh sieve, collecting powder 0.5g, adding 50% methanol 20mL, placing in a round bottom flask, weighing, reflux extracting for 45min, cooling, weighing, supplementing with 50% methanol, shaking, filtering with 0.45 μm microporous membrane, and collecting filtrate to obtain sample solution;
a3, diluting the reference stock solution obtained in A1 by 10 times with 50% methanol, sucking 5 μ L of each stock solution, injecting into a high performance liquid chromatograph, and detecting to obtain liquid chromatogram of verbascoside, pennyoside and verbascoside;
a4, sucking 10 mu L of sample solution obtained from A2, injecting the sample solution into a high performance liquid chromatograph for detection, comparing the obtained liquid chromatogram with the liquid chromatogram obtained from A3 to obtain the contents of verbena major components, namely verbascoside, verbascoside and verbascoside, and measuring by the high performance liquid chromatography according to the following experimental conditions:
a chromatographic column: diamonsil II C18Chromatography column, 250mm × 4.6mm, 5 μm;
mobile phase: the mobile phase A is acetonitrile, the mobile phase B is 0.05 percent phosphoric acid water solution, gradient elution is carried out, the proportions of the mobile phases are as follows, wherein the proportions of the mobile phases are volume percent:
0-30min, 5-25% of mobile phase A and 95-75% of mobile phase B;
for 40-65min, the mobile phase A is 25% -35%, and the mobile phase B is 75% -65%;
65-70min, 35-80% of mobile phase A and 65-20% of mobile phase B;
flow rate: 1.0 mL/min;
column temperature: 30 ℃;
ultraviolet detection wavelength: 230 nm.
The results of measuring the content of the specific components in 14 batches of the verbena medicinal materials by the above method are shown in the following table:
example 2
Establishing a fingerprint of verbena by adopting an HPLC method:
b1, pulverizing the medicinal material of the verbena, sieving with a 40-mesh sieve, taking 1g of powder, adding 20mL of 50% methanol, placing in a round-bottom flask, weighing, extracting under reflux for 45min, cooling, weighing, supplementing with 50% methanol, shaking up, filtering with a 0.45-micrometer microporous membrane, and taking the subsequent filtrate to obtain a sample solution;
b2, precisely absorbing 10 mu L of test solution, injecting the test solution into a high performance liquid chromatograph, and measuring according to the following chromatographic conditions to obtain a fingerprint spectrum:
a chromatographic column: orca C18Chromatography column, 250mm × 4.6mm, 5 μm;
mobile phase: the mobile phase A is acetonitrile, the mobile phase B is 0.05 percent of phosphoric acid aqueous solution, and the proportion of the mobile phase is subjected to gradient elution, wherein the mobile phase proportion is as follows, and the mobile phase proportion is volume percentage:
0-30min, 5-25% of mobile phase A and 95-75% of mobile phase B;
for 40-65min, the mobile phase A is 25% -35%, and the mobile phase B is 75% -65%;
flow rate: 1 mL/min;
column temperature: 25 ℃;
ultraviolet detection wavelength: 230 nm;
example 3
Methodology investigation of fingerprint establishment method:
(1) precision test
Precisely absorbing the same test solution by adopting the operation and conditions the same as those in the example 2, continuously feeding for 6 times, determining an HPLC chromatogram, inspecting the consistency of the relative retention time and the relative peak area of a chromatographic peak, taking the No. 4 peak verbascoside peak as a reference peak, calculating the relative retention time and the relative peak area of each chromatographic peak, and obtaining an HPLC chromatogram overlay chart of a precision test as shown in the figure 1;
the result shows that the relative retention time of each spectrum peak and the RSD value of the relative peak area are both less than 5 percent, and the requirements of the fingerprint spectrum are met.
(2) Stability test
Preparing a test solution by adopting the same operation and conditions as the example 2, sealing and placing at room temperature, detecting a fingerprint spectrum at time intervals of 0, 3, 6, 9, 12 and 24 hours, respectively, calculating the relative retention time and the relative peak area of each chromatographic peak by taking the No. 4 peak verbascoside as a reference peak, and showing an HPLC chromatogram superposition chart of a stability test as shown in FIG. 3;
the result shows that the relative retention time of each chromatographic peak and the RSD value of the relative peak area are both less than 5 percent, the requirement of the fingerprint is met, and the test solution is stable within 24 hours.
(3) Repeatability test
The same batch of samples are taken by adopting the same operation and conditions as the example 2 to prepare 6 parts of test solution, the No. 4 peak verbascoside peak is taken as a reference peak, the relative retention time and the relative peak area of each chromatographic peak are calculated, and the HPLC chromatogram map overlay of the repeatability test is shown in figure 4;
the result shows that the relative retention time of each spectrum peak and the RSD value of the relative peak area are both less than 5 percent, and the requirements of the fingerprint spectrum are met.
Example 4
The establishment of the quality control method comprises the following steps:
analyzing and measuring M1-M14 verbena medicinal materials according to the method in the embodiment 2 to obtain fingerprints, importing AIA data files of the fingerprints of 14 batches of medicinal materials into similarity software 2004A edition' evaluation system for traditional Chinese medicine chromatogram fingerprint similarity, and analyzing and determining 5 main chromatogram characteristic peaks as common peaks. Selecting a No. 4 peak with large peak area and moderate and stable peak-off time as a reference peak, calculating the peak area ratio of each spectrum peak relative to the peak area ratio, wherein HPLC fingerprint spectrums of 14 batches of medicinal materials are shown in figure 4, and the test data are shown in the following table:
using 'SPSS software' to perform cluster analysis on quantized peak areas, wherein a cluster analysis chart is shown in fig. 5, the cluster analysis divides 14 batches of verbena officinalis samples into two categories, samples with batch numbers 1, 2, 3, 4, 7, 8, 9, 11, 13 and 14 belong to class I, samples with batch numbers 5, 6, 10 and 12 belong to class II, a comparison fingerprint is generated from chromatograms of 10 batches of medicinal materials of the class I samples, the comparison fingerprint is shown in fig. 6, using calculation software 2004 version B of the traditional Chinese medicine chromatogram fingerprint similarity evaluation system to compare the 14 batches of samples with the comparison fingerprint, and similarity evaluation is performed, wherein the similarity evaluation results are shown in the following table:
the similarity evaluation result in the research shows that the HPLC chromatograms of the medicinal material verbena are very similar, but the clustering analysis can see that the verbena in different batches have certain difference in the content of the main components, and the verbena can be divided into two categories according to the difference, so that the quality of the verbena is controlled.
The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise forms disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.
Claims (6)
1. A method for measuring the special content of verbena is characterized in that the special content detection method adopts a high performance liquid chromatography to detect the content of special components of the verbena, and the conditions of the high performance liquid chromatography are as follows:
a chromatographic column: orca C18Chromatography column, 250mm × 4.6mm, 5 μm;
mobile phase: the mobile phase A is acetonitrile, the mobile phase B is 0.05 percent phosphoric acid water solution, gradient elution is carried out, the proportions of the mobile phases are as follows, wherein the proportions of the mobile phases are volume percent:
0-30min, 5-25% of mobile phase A and 95-75% of mobile phase B;
for 40-65min, the mobile phase A is 25% -35%, and the mobile phase B is 75% -65%;
65-70min, 35-80% of mobile phase A and 65-20% of mobile phase B;
flow rate: 1.0 mL/min;
column temperature: 30-35 ℃;
ultraviolet detection wavelength: 200-600 nm.
2. The method for determining the special content of verbena officinalis according to claim 1, wherein the method for detecting the special content comprises the following steps:
a1, respectively placing 2.00mg of each of verbascoside, verbascoside and verbascoside reference substances into 10mL measuring bottles, and adding 50% methanol to a constant volume to obtain reference substance stock solution with a mass concentration of 200 μ g/mL;
a2, pulverizing herba Verbenae, sieving with 40 mesh sieve, collecting powder 0.5g, adding 50% methanol 20mL, placing in a round bottom flask, weighing, reflux extracting for 45min, cooling, weighing, supplementing with 50% methanol, shaking, filtering with 0.45 μm microporous membrane, and collecting filtrate to obtain sample solution;
a3, diluting the reference stock solution obtained in A1 by 10 times with 50% methanol, sucking 5 μ L of each stock solution, injecting into a high performance liquid chromatograph, and detecting to obtain liquid chromatogram of verbascoside, pennyoside and verbascoside;
a4, sucking 10 mu L of sample solution obtained from A2, injecting the sample solution into a high performance liquid chromatograph for detection, and comparing the obtained liquid chromatogram with the liquid chromatogram obtained from A3 to obtain the contents of verbena major components, namely verbascoside, verbascoside and verbascoside.
3. The method of claim 2, wherein the conditions of the HPLC method include:
a chromatographic column: orca C18Chromatography column, 250mm × 4.6mm, 5 μm;
mobile phase: the mobile phase A is acetonitrile, the mobile phase B is 0.05 percent phosphoric acid water solution, gradient elution is carried out, the proportions of the mobile phases are as follows, wherein the proportions of the mobile phases are volume percent:
0-30min, 5-25% of mobile phase A and 95-75% of mobile phase B;
for 40-65min, the mobile phase A is 25% -35%, and the mobile phase B is 75% -65%;
65-70min, 35-80% of mobile phase A and 65-20% of mobile phase B;
flow rate: 1.0 mL/min;
column temperature: 30 ℃;
ultraviolet detection wavelength: 230 nm.
4. A verbena quality control method is characterized in that the quality control method adopts high performance liquid chromatography to detect the content of the main components of the verbena, wherein the conditions of the high performance liquid chromatography comprise:
a chromatographic column: orca C18Chromatography column, 250mm × 4.6mm, 5 μm;
mobile phase: the mobile phase A is acetonitrile, the mobile phase B is 0.05 percent phosphoric acid water solution, gradient elution is carried out, the proportions of the mobile phases are as follows, wherein the proportions of the mobile phases are volume percent:
0-30min, 5-25% of mobile phase A and 95-75% of mobile phase B;
for 40-65min, the mobile phase A is 25% -35%, and the mobile phase B is 75% -65%;
65-70min, 35-80% of mobile phase A and 65-20% of mobile phase B;
flow rate: 1.0 mL/min;
column temperature: 25-35 ℃;
ultraviolet detection wavelength: 200-600 nm.
5. The method for determining the special content of verbena officinalis and controlling the quality of the verbena officinalis as claimed in claim 4, wherein the method for controlling the quality of the verbena officinalis comprises the following steps:
b1, pulverizing the medicinal material of the verbena, sieving with a 40-mesh sieve, taking 1g of powder, adding 20mL of 50% methanol, placing in a round-bottom flask, weighing, extracting under reflux for 45min, cooling, weighing, supplementing with 50% methanol, shaking up, filtering with a 0.45-micrometer microporous membrane, and taking the subsequent filtrate to obtain a sample solution;
b2, precisely absorbing 10 mu L of test solution, injecting the test solution into a high performance liquid chromatograph, and measuring to obtain a fingerprint:
b3, taking 14 batches of verbena medicinal herbs, establishing a fingerprint by a method B1 and B2, introducing fingerprint data into similarity software of a 2004A edition of traditional Chinese medicine chromatogram fingerprint similarity evaluation system for analysis, determining 5 main chromatogram characteristic peaks, selecting a No. 4 peak verbascoside peak as a reference peak, wherein the relative peak area of each peak relative to the No. 4 peak is as follows:
peak 1: 0.06-0.75; peak 2: 0.03-0.23; peak 3: 0.44-1.49; peak 4: 1.00; peak 5: 0.50-1.36;
b4, performing cluster analysis on the quantized peak areas by using SPSS software, generating a comparison fingerprint, comparing the sample with the comparison fingerprint by using calculation software 2004B of 'traditional Chinese medicine chromatogram fingerprint similarity evaluation system' to obtain a similarity evaluation result, screening verbena medicinal materials according to the similarity evaluation result and the cluster analysis, and screening out medicinal materials in batches with lower similarity, thereby finishing quality control.
6. The method for determining the content and controlling the quality of verbena officinalis according to claim 5, wherein the HPLC conditions in step B2 are as follows:
a chromatographic column: orca C18Chromatography column, 250mm × 4.6mm, 5 μm;
mobile phase: the mobile phase A is acetonitrile, the mobile phase B is 0.05 percent of phosphoric acid aqueous solution, and the proportion of the mobile phase is subjected to gradient elution, wherein the mobile phase proportion is as follows, and the mobile phase proportion is volume percentage:
0-30min, 5-25% of mobile phase A and 95-75% of mobile phase B;
for 40-65min, the mobile phase A is 25% -35%, and the mobile phase B is 75% -65%;
flow rate: 1 mL/min;
column temperature: 25 ℃;
ultraviolet detection wavelength: 230 nm.
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| CN116359418A (en) * | 2023-02-27 | 2023-06-30 | 广东一方制药有限公司 | Method for constructing characteristic spectrum of verbena water extract or preparation thereof and method for measuring index component content thereof |
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