CN114720602B - Method for constructing characteristic spectrum of lithospermum through medicinal material or standard decoction thereof and detection method - Google Patents

Method for constructing characteristic spectrum of lithospermum through medicinal material or standard decoction thereof and detection method Download PDF

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CN114720602B
CN114720602B CN202210383733.2A CN202210383733A CN114720602B CN 114720602 B CN114720602 B CN 114720602B CN 202210383733 A CN202210383733 A CN 202210383733A CN 114720602 B CN114720602 B CN 114720602B
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lithospermum
medicinal material
mobile phase
standard decoction
characteristic
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CN114720602A (en
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梁慧
陶晨璐
范倩
彭致铖
刘艳梅
蔡盛康
吴晓纯
丁青
朱德全
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Guangdong Yifang Pharmaceutical Co Ltd
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    • 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
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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
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract

The invention relates to a characteristic spectrum construction method and a detection method of a medicinal material of lithospermum or a standard decoction thereof. The feature map construction method comprises the following steps: preparing a reference substance solution of a reference medicinal material of the herba Salviae chinensis reference medicinal material; preparing reference solution of caffeic acid reference, chlorogenic acid reference, rosmarinic acid reference, salvianolic acid B reference and sodium salvianic acid A reference; preparing a sample solution from a herba Salviae chinensis or herba Salviae chinensis standard decoction lyophilized powder; and detecting by adopting an ultra-high performance liquid chromatography method, and establishing a characteristic spectrum. The characteristic spectrum constructed by the method calibrates the characteristic peaks of 6 water-soluble components, can rapidly and comprehensively realize quality monitoring of a plurality of characteristic components of the medicinal material of the lithospermum through, provides a more comprehensive, objective and rapid quality evaluation method for the medicinal material of the lithospermum through and the standard decoction of the lithospermum through, and has simple and convenient operation and good repeatability.

Description

Method for constructing characteristic spectrum of lithospermum through medicinal material or standard decoction thereof and detection method
Technical Field
The invention relates to the technical field of traditional Chinese medicines, in particular to a characteristic spectrum construction method and a detection method of a medicinal material of lithospermum through or a standard decoction thereof.
Background
The Chinese pharmacopoeia 1977 edition of Shijingshi Chuan is a Labiatae plant dried aerial parts of Salvia chinensis Benth; cutting in summer and autumn Ji Huaqi, removing impurities, and sun drying; its taste is bitter and pungent and its nature is flat; enter liver, stomach and lung meridians; has effects in clearing away heat and toxic materials, promoting blood circulation, activating qi-flowing, and relieving pain; can be used for treating distending pain in the stomach and hypochondrium, carbuncle, and swelling. The chemical components of the herba Salviae chinensis are mainly phenolic acids, phenolic acid esters and triterpenes, and also comprise acidic polysaccharides, sterols, amino acids, etc. Wherein the phenolic acid and phenolic acid esters have the highest content, such as rosmarinic acid, rosmarinic acid methyl ester, rosmarinic acid ethyl ester, salvianolic acid A, salvianolic acid B, salvianolic acid C methyl ester, salvianic acid A, etc., and most of the substances are water-soluble compounds, have stronger pharmacological activity, and are one of the most main effective components of the lithospermum. In recent years, the Chinese medicinal composition with the Chinese medicinal composition as the anti-tumor medicament has high clinical value.
However, the existing pharmacopoeia standard of the herba Salviae chinensis and the standard of the medicinal materials in each place are relatively simple, and only comprise characters, microscopic and thin-layer identification items, so that the quality of the herba Salviae chinensis cannot be comprehensively evaluated. At present, the literature reports about the penetration of stone are mainly researched on the chemical components and pharmacological actions of the stone, and only a small number of reports about the content measurement of phenolic acid components exist in the aspects of quality standard research. In addition, the herba Salviae chinensis has the characteristics of wide sources and complex components, is mainly clinically used, usually contains various water-soluble components, and is insufficient to fully reflect the inherent quality of herba Salviae chinensis medicinal materials and the clinical effect of herba Salviae chinensis by means of content measurement of simple components or two components, and lacks of specificity, and also cannot reflect the material basic characteristics of the clinical decoction.
Disclosure of Invention
Based on the method, the invention provides a characteristic spectrum construction method and a detection method of a medicinal material of lithospermum or a standard decoction thereof. The method provides a more comprehensive, objective and rapid quality evaluation method for the medicinal materials of the herba Salviae chinensis and the standard decoction thereof.
The specific technical scheme is as follows:
the invention provides a method for constructing a characteristic spectrum of a medicinal material of lithospermum or a standard decoction thereof, which comprises the following steps:
extracting herba Salviae chinensis reference material with organic solvent, filtering, and collecting filtrate to obtain reference solution; dissolving caffeic acid reference substance, chlorogenic acid reference substance, rosmarinic acid reference substance, salvianolic acid B reference substance, and salvianic acid A sodium reference substance in organic solvent to obtain reference substance solution;
extracting herba Salviae chinensis or decoction pieces with organic solvent, filtering, and collecting filtrate to obtain sample solution I; or extracting herba Salviae chinensis lyophilized powder with organic solvent, filtering, and collecting filtrate to obtain sample solution II;
detecting the reference substance solution of the reference medicinal material, the reference substance solution of the reference substance and the solution I of the test sample by adopting an ultra-high performance liquid chromatography, and establishing a characteristic map of the lithospermum through medicinal material; or alternatively, the first and second heat exchangers may be,
Detecting the reference substance solution of the reference medicinal material, the reference substance solution of the reference substance and the solution II of the test substance by adopting an ultra-high performance liquid chromatography, and establishing a characteristic map of the standard decoction of the lithospermum through;
the mobile phase A adopted by the ultra-high performance liquid chromatography is acetonitrile, the mobile phase B is formic acid aqueous solution with the volume fraction of 0.05-0.2%, and the elution mode is gradient elution.
In one embodiment, the gradient elution comprises the following procedure:
0-3 min, wherein the volume percentage of the mobile phase A is kept to be 2%;
3-10 min, wherein the volume percentage of the mobile phase A is increased from 2% to 7%;
10-13 min, wherein the volume percentage of the mobile phase A is increased from 7% to 9%;
13-30 min, wherein the volume percentage of the mobile phase A is increased from 9% to 20%;
30-36 min, wherein the volume percentage of the mobile phase A is increased from 20% to 26%;
36-40 min, wherein the volume percentage of the mobile phase A is increased from 26% to 60%.
In one embodiment, the ultra performance liquid chromatography further comprises the following detection conditions:
chromatographic column: octadecylsilane chemically bonded silica is used as a filler;
flow rate: 0.2-0.6 mL/min;
column temperature: 35-45 ℃;
Detection wavelength: 280 nm-300 nm.
In one embodiment, the organic solvent is 40% -60% methanol aqueous solution by volume fraction.
In one embodiment, the method of extraction is ultrasonic extraction, including the following conditions: the time is 20-40 min, the power is 240-260W, and the frequency is 30-50 kHz.
In one embodiment, the characteristic pattern includes 6 characteristic peaks.
In one embodiment, the method for constructing the characteristic spectrum of the lithospermum through medicinal material or the standard decoction thereof further comprises the following steps:
and identifying the characteristic peaks by adopting mass spectrometry.
In one embodiment, the mass spectrometry comprises the following detection conditions:
the ionization mode is heating electrospray ionization; the flow rate of sheath gas is 30 arb-40 arb; the flow rate of the auxiliary gas is 5 arb-15 arb; the spraying voltage is 3.5 kV-4 kV; the S-lens voltage is 40V-60V; the heating temperature is 300-400 ℃; the temperature of the capillary tube is 300-400 ℃; the scanning mode is an anion mode; the scanning range is 100-1500; the normalized collision energy is 35-45.
In a second aspect of the present invention, a method for detecting a medicinal material of herba Salviae chinensis or a standard decoction thereof is provided, comprising the steps of:
Extracting herba Salviae chinensis or decoction pieces with organic solvent, filtering, and collecting filtrate to obtain sample solution I; or extracting the freeze-dried powder of the standard decoction of the herba Salviae chinensis to be detected with an organic solvent, filtering, and collecting the filtrate to prepare a sample solution II to be detected;
detecting the sample solution I or the sample solution II to be detected by adopting an ultra-high performance liquid chromatography;
the mobile phase A adopted by the ultra-high performance liquid chromatography is acetonitrile, the mobile phase B is formic acid aqueous solution with the volume fraction of 0.05-0.2%, and the elution mode is gradient elution.
In one embodiment, the gradient elution comprises the following procedure:
0-3 min, wherein the volume percentage of the mobile phase A is kept to be 2%;
3-10 min, wherein the volume percentage of the mobile phase A is increased from 2% to 7%;
10-13 min, wherein the volume percentage of the mobile phase A is increased from 7% to 9%;
13-30 min, wherein the volume percentage of the mobile phase A is increased from 9% to 20%;
30-36 min, wherein the volume percentage of the mobile phase A is increased from 20% to 26%;
36-40 min, wherein the volume percentage of the mobile phase A is increased from 26% to 60%.
In one embodiment, the ultra performance liquid chromatography further comprises the following detection conditions:
Chromatographic column: octadecylsilane chemically bonded silica is used as a filler;
flow rate: 0.2-0.6 mL/min;
column temperature: 35-45 ℃;
detection wavelength: 280 nm-300 nm.
In one embodiment, the organic solvent is 40-60% methanol aqueous solution by volume fraction.
In one embodiment, the method of extraction is ultrasonic extraction, including the following conditions: the time is 20-40 min, the power is 240-260W, and the frequency is 30-50 kHz.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, the proper reference object is selected, and the characteristic spectrum of the herba Salviae chinensis or the standard decoction thereof is constructed by adopting certain ultra-high performance liquid chromatography (UPLC) conditions, so that the constructed characteristic spectrum is calibrated with the characteristic peaks of 6 water-soluble components, the quality monitoring of a plurality of characteristic components of the herba Salviae chinensis can be rapidly and comprehensively realized, and the quality of the standard decoction prepared from the herba Salviae chinensis can be accurately reflected, so that a relatively comprehensive, objective and rapid quality evaluation method is provided for the herba Salviae chinensis and the standard decoction thereof, and the method is simple and convenient to operate and good in repeatability.
Drawings
FIG. 1 is a full-wavelength scan of the medicinal material of the herba Salviae chinensis in example 1;
FIG. 2 is a chromatogram of the herba Salviae chinensis at different detection wavelengths in example 1 (S1: 325nm; S2:290nm; S3:254 nm);
FIG. 3 is a chromatogram of the use of different mobile phases B in example 1;
FIG. 4 is a chromatogram of the use of different chromatographic columns in example 1;
FIG. 5 is a characteristic spectrum of 15 batches of radix Salviae Miltiorrhizae in example 1;
FIG. 6 is a characteristic spectrum of the herba Salviae chinensis control material in example 1;
FIG. 7 is a characteristic spectrum of a standard decoction corresponding to 15 batches of herba Salviae chinensis in example 1;
FIG. 8 is a UV and total ion flow chromatogram of the medicinal material (S1) of the herba Salviae chinensis in example 1;
FIG. 9 is a characteristic peak confirmation chart of the characteristic pattern of the stone-like puncture in example 1;
FIG. 10 is a chromatogram for specificity investigation of a characteristic spectrum of a medicinal material of herba Salviae chinensis in example 1;
FIG. 11 is a graph of the characteristics of the herba Salviae chinensis herb (Peak 1: salvianic acid A; peak 3: caffeic acid; peak 4: chlorogenic acid; peak 5 (S): rosmarinic acid; peak 6: salvianolic acid B) of example 1;
FIG. 12 is a graph of the features of the standard decoction for use in example 1 (Peak 1: salvianic acid A; peak 3: caffeic acid; peak 4: chlorogenic acid; peak 5 (S): rosmarinic acid; peak 6: salvianolic acid B);
FIG. 13 is a graph showing the comparison of the characteristic pattern of the herba Salviae chinensis and the characteristic pattern of the herba Salviae chinensis standard decoction in example 1;
Fig. 14 is a UPLC chromatogram of the medicinal material of the lithospermum to be tested in example 2.
Detailed Description
In order that the invention may be understood more fully, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended claims. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other.
In the present invention, "first aspect", "second aspect", etc. are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor as implying an importance or quantity of the indicated technical features. Moreover, "first," "second," etc. are for non-exhaustive list description purposes only, and it should be understood that no closed limitation on the number is made.
In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.
In the present invention, the numerical ranges are referred to as continuous, and include the minimum and maximum values of the ranges, and each value between the minimum and maximum values, unless otherwise specified. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range description features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to include any and all subranges subsumed therein.
The temperature parameter in the present invention is not particularly limited, and may be a constant temperature treatment or a treatment within a predetermined temperature range. The constant temperature process allows the temperature to fluctuate within the accuracy of the instrument control.
The invention provides a method for constructing a characteristic spectrum of a medicinal material of lithospermum or a standard decoction thereof, which comprises the following steps:
extracting herba Salviae chinensis reference material with organic solvent, filtering, and collecting filtrate to obtain reference solution; dissolving caffeic acid reference substance, chlorogenic acid reference substance, rosmarinic acid reference substance, salvianolic acid B reference substance, and salvianic acid A sodium reference substance in organic solvent to obtain reference substance solution;
extracting herba Salviae chinensis or decoction pieces with organic solvent, filtering, and collecting filtrate to obtain sample solution I; or extracting herba Salviae chinensis lyophilized powder with organic solvent, filtering, and collecting filtrate to obtain sample solution II;
detecting the reference substance solution of the reference medicinal material, the reference substance solution of the reference substance and the solution I of the test sample by adopting an ultra-high performance liquid chromatography method, and establishing a characteristic map of the lithospermum through medicinal material; or alternatively, the first and second heat exchangers may be,
detecting the reference substance solution of the reference medicinal material, the reference substance solution of the reference substance and the solution II of the test sample by adopting an ultra-high performance liquid chromatography, and establishing a characteristic map of the standard decoction of the lithospermum through;
The mobile phase A adopted by the ultra-high performance liquid chromatography is acetonitrile, the mobile phase B is formic acid aqueous solution with the volume fraction of 0.05-0.2%, and the elution mode is gradient elution.
It can be appreciated that the characteristic spectrum is a comprehensive and quantifiable modern mass analysis method, and can realize comprehensive evaluation of the integrity of the Chinese medicine. In the present stage, aiming at the diversity and complexity of the effective components of the traditional Chinese medicine, the establishment of the characteristic spectrum of the traditional Chinese medicine can greatly improve the technical level and the technological content of the quality control of the traditional Chinese medicine. The traditional characteristic spectrum is completed through HPLC, the time consumption is long, and the UPLC is adopted to construct the characteristic spectrum, so that the method has the advantages of high analysis speed and high sensitivity compared with HPLC.
In one example, the volume fraction of the aqueous formic acid solution includes, but is not limited to: 0.05%, 0.07%, 0.09%, 0.1%, 0.11%, 0.12%, 0.14%, 0.16%, 0.18%, 0.2%.
In one example, gradient elution includes the following procedure:
0-3 min, wherein the volume percentage of the mobile phase A is kept to be 2%;
3-10 min, wherein the volume percentage of the mobile phase A is increased from 2% to 7%;
the volume percentage of the mobile phase A is increased from 7% to 9% after 10 min-13 min;
13-30 min, wherein the volume percentage of the mobile phase A is increased from 9% to 20%;
30-36 min, wherein the volume percentage of the mobile phase A is increased from 20% to 26%;
36-40 min, the volume percentage of the mobile phase A is increased from 26% to 60%.
In one example, the ultra performance liquid chromatography further includes the following detection conditions: the chromatographic column uses octadecylsilane chemically bonded silica gel as filler. Further, the column was a Waters BEH C18 (100 mm. Times.2.1 mm,1.7 μm) column.
In one example, the ultra performance liquid chromatography further includes the following detection conditions: the flow rate is 0.2 mL/min-0.6 mL/min. Further, flow rates include, but are not limited to: 0.2mL/min, 0.25mL/min, 0.3mL/min, 0.35mL/min, 0.4mL/min, 0.45mL/min, 0.5mL/min, 0.55mL/min, 0.6mL/min.
In one example, the ultra performance liquid chromatography further includes the following detection conditions: the column temperature is 35-45 ℃. Further, column temperatures include, but are not limited to: 35 ℃, 37 ℃, 39 ℃, 40 ℃, 41 ℃, 43 ℃, 45 ℃.
In one example, the ultra performance liquid chromatography further includes the following detection conditions: the ultraviolet detection wavelength is 280 nm-300 nm. Further, ultraviolet detection wavelengths include, but are not limited to: 280nm, 285nm, 290nm, 295nm, 300nm.
Specifically, in one example, the ultra performance liquid chromatography further includes the following detection conditions:
chromatographic column: octadecylsilane chemically bonded silica is used as a filler;
flow rate: 0.2-0.6 mL/min;
column temperature: 35-45 ℃;
detection wavelength: 280 nm-300 nm.
In one example, the organic solvent is 40% -60% aqueous methanol by volume. Further, the volume fraction of the aqueous methanol solution includes, but is not limited to: 40%, 45%, 47%, 50%, 53%, 55%, 60%.
In one example, the method of extraction is ultrasonic extraction, including the following conditions: the time is 20-40 min, the power is 240-260W, and the frequency is 30-50 kHz. Further, the time of ultrasonic extraction includes, but is not limited to: 20min, 25min, 29min, 30min, 31min, 35min, and 40min; the power of ultrasound extraction includes, but is not limited to: 240W, 245W, 247W, 250W, 253W, 255W, 260W; the frequency of ultrasonic extraction includes, but is not limited to: 30kHz, 35kHz, 40kHz, 45kHz, 50kHz.
In one example, the method for preparing the reference substance solution comprises the following steps:
taking a caffeic acid reference substance, a chlorogenic acid reference substance, a rosmarinic acid reference substance and a salvianolic acid B reference substance, precisely weighing, adding 40-60% methanol water solution in volume fraction to prepare reference substance solutions containing 50 mug of caffeic acid, 50 mug of chlorogenic acid, 100 mug of rosmarinic acid and 50 mug of salvianolic acid B in each 1 mL;
Taking sodium salvianic acid A reference substance, precisely weighing, and adding 40% -60% methanol water solution to prepare reference substance solution containing 50 μg (equivalent to 45 μg of salvianic acid A in each 1 mL) per 1 mL.
In one example, the feature map includes 6 feature peaks.
According to the invention, by selecting a proper reference object and adopting a certain ultra-high performance liquid chromatography (UPLC) condition, the characteristic spectrum of the herba Salviae chinensis and the standard decoction thereof can be constructed, the characteristic spectrum of the herba Salviae chinensis reference medicinal material, the characteristic spectrum of the herba Salviae chinensis medicinal material and the characteristic spectrum of the herba Salviae chinensis standard decoction are compared, 6 common peaks are determined, and all 6 common peaks can be stably transmitted from the herba Salviae chinensis medicinal material to the herba Salviae chinensis standard decoction, so that the 6 common peaks are set as the characteristic peaks of the herba Salviae chinensis medicinal material or the standard decoction thereof. Further, the present invention combines mass spectrometry to identify and confirm characteristic peaks, and clarifies chemical components thereof.
In one example, the method for constructing the characteristic spectrum of the lithospermum through medicinal material or the standard decoction thereof further comprises the following steps:
and identifying characteristic peaks by adopting a mass spectrometry method.
In one example, mass spectrometry includes the following detection conditions:
The ionization mode is heating electrospray ionization; the flow rate of sheath gas is 30 arb-40 arb; the flow rate of the auxiliary gas is 5 arb-15 arb; the spraying voltage is 3.5 kV-4 kV; the S-lens voltage is 40V-60V; the heating temperature is 300-400 ℃; the temperature of the capillary tube is 300-400 ℃; the scanning mode is an anion mode; the scanning range is 100-1500; the Normalized Collision Energy (NCE) is 35 to 45.
In one example, the sheath gas flow rate of mass spectrometry includes, but is not limited to: 30arb, 31arb, 32arb, 33arb, 34arb, 35arb, 36arb, 37arb, 38arb, 39arb, 40arb.
In one example, the auxiliary gas flow rate of mass spectrometry includes, but is not limited to: 5arb, 6arb, 7arb, 8arb, 9arb, 10arb, 11arb, 12arb, 13arb, 14arb, 15arb.
In one example, the spray voltage of mass spectrometry includes, but is not limited to: 3.5kV, 3.6kV, 3.7kV, 3.8kV, 3.9kV and 4kV.
In one example, the S-lens voltages of mass spectrometry include, but are not limited to: 40V, 42V, 44V, 46V, 48V, 50V, 52V, 54V, 56V, 58V, 60V.
In one example, the heating temperature of mass spectrometry includes, but is not limited to: 300 ℃, 320 ℃, 340 ℃, 360 ℃, 380 ℃, 400 ℃.
In one example, the capillary temperature of mass spectrometry includes, but is not limited to: 300 ℃, 320 ℃, 340 ℃, 360 ℃, 380 ℃, 400 ℃.
In one example, the Normalized Collision Energy (NCE) of mass spectrometry includes, but is not limited to: 35. 36, 37, 38, 39, 40, 41, 42, 43, 44, 45.
The invention also provides a detection method of the lithospermum through medicinal material or the standard decoction thereof, which comprises the following steps:
extracting herba Salviae chinensis or decoction pieces with organic solvent, filtering, and collecting filtrate to obtain sample solution I; or extracting the freeze-dried powder of the standard decoction of the herba Salviae chinensis to be detected with an organic solvent, filtering, and collecting the filtrate to prepare a sample solution II to be detected;
detecting the sample solution I to be detected or the sample solution II to be detected by adopting an ultra-high performance liquid chromatography;
the mobile phase A adopted by the ultra-high performance liquid chromatography is acetonitrile, the mobile phase B is formic acid aqueous solution with the volume fraction of 0.05-0.2%, and the elution mode is gradient elution.
It can be understood that the detection of the sample solution to be detected by using the ultra-high performance liquid chromatography method further comprises comparing the detection result with the feature map constructed by the feature map construction method.
Further, the detection of a single sample to be detected can be performed, and the single sample to be detected can be compared with the characteristic spectrum established by the characteristic spectrum establishment method so as to obtain the quality of the single sample to be detected; or detecting a plurality of batches of samples to be detected, and comparing the patterns of the batches of samples to be detected to obtain the similarity among the batches of samples to be detected.
It can be understood that the technical scheme of the detection method is similar to the characteristic spectrum construction method of the lithospermum or the standard decoction thereof, wherein the preparation of the sample solution to be detected is the same as the preparation of the sample solution, and the description is omitted here.
The present invention will be described in further detail with reference to specific examples. The raw materials, reagents and the like used in the following examples are commercially available products unless otherwise specified.
Example 1
The embodiment provides a feature map construction method of a herba Salviae chinensis and a standard decoction thereof, which comprises the following steps:
1. instrument, reagent and reagent
(1) Instrument for measuring and controlling the intensity of light
Waters high performance liquid chromatograph (Waters H-Class, waters corporation), thermo high performance liquid chromatograph (Thermo Vanquish, siemens flying corporation), agilent high performance liquid chromatograph (Agilent 1290, agilent corporation); thermo Scientific TM Q Exactive TM Focus combined quadrupole Orbitrap mass spectrometer (Siemens, USA); mzVault Mass Spectrometry database (Sieimer, USA); waters BEH C18 column (100 mm. Times.2.1 mm,1.7 μm), YMC Triart C18 column (100 mm. Times.2.1 mm,1.9 μm), agilent SB C18 column (100 mm. Times.2.1 mm,1.8 μm); one parts per million balance (ME 204E, mertler-tolidol corporation), one parts per million flat (XP 26, mertler-tolidol corporation); digital control ultrasonic cleaner (KQ 500DE, kunshan ultrasonic instruments Co., ltd.), thermostatic water bath (HWS 28, shanghai-He technology Co., ltd.) ultrapure water system (Milli-Q Direct, merck Co., ltd.).
(2) Reagent(s)
Methanol (analytically pure, the company of the sciences, the company of the ridge);
acetonitrile for liquid phase (chromatographic grade, merck, inc.);
formic acid (HPLC chromatographic grade, mitsunobu chemical company, division of the paris);
the water was ultrapure water (laboratory homemade).
(3) Reagent
The herba Salviae chinensis contrast medicinal material (batch number: 121579-201802, china food and drug inspection institute); sodium salvianic acid A control (batch number: 110855-201915, content: 97.8%, national food and drug verification institute); caffeic acid reference substance (batch number: 110885-201703, content: 99.7%, national food and drug verification institute); chlorogenic acid reference (batch No. 110753-202018, content 96.1%, national food and drug institute); rosmarinic acid reference (lot number: 111871-202007, content: 98.1%, national food and drug verification institute); salvianolic acid B reference substance (lot number: 111562-201917, content: 96.6%, national food and drug verification institute);
15 batches of herba Salviae chinensis were identified as dry aerial parts of Salvia chinensis Benth of Labiatae by mass center of Guangdong party pharmaceutical Co., ltd, and the production place and number information are shown in Table 1.
Table 1 sample information table
Figure BDA0003589938260000091
2. Preparation of reference and test solutions
2.1 preparation of reference solution of reference medicinal material
Taking 1.0g of a reference medicinal material of the lithospermum through, precisely weighing, placing into a conical flask with a plug, precisely adding 25mL of 50% methanol by volume fraction, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 30min, shaking uniformly, filtering, and taking the subsequent filtrate as a reference solution of the reference medicinal material.
2.2 preparation of reference solutions for controls
Taking a proper amount of caffeic acid reference substance, chlorogenic acid reference substance, rosmarinic acid reference substance and salvianolic acid B reference substance, precisely weighing, adding 50% methanol to prepare a solution containing 50 mug of caffeic acid, 50 mug of chlorogenic acid, 100 mug of rosmarinic acid and 50 mug of salvianolic acid B in each 1mL, and obtaining a corresponding reference substance solution;
taking a proper amount of sodium salvianic acid A reference substance, precisely weighing, adding 50% methanol to prepare a solution containing 50 mug per 1mL (equivalent to 45 mug per 1 mL) of salvianic acid A, and obtaining the reference substance solution of salvianic acid A.
2.3 preparation of sample solutions
(1) The preparation of the freeze-dried powder of the standard decoction of the herba Salviae chinensis is carried out: taking herba Salviae chinensis, removing impurities, spraying clear water, slightly moistening, cutting, and drying to obtain herba Salviae chinensis decoction pieces. Taking 100g of lithospermum decoction pieces, placing the lithospermum decoction pieces in an electroceramic pot, and adding water for decoction twice: soaking in 12 times of water (by mass ratio) for 30min, boiling with strong fire (500W), keeping micro-boiling with slow fire (200W) for 30min, filtering the decoction with 350 mesh sieve, and cooling the filtrate with cold water; adding 10 times of water (by mass ratio) for the second time, boiling with strong fire, keeping micro-boiling with slow fire for 25min, filtering the decoction with 350 mesh sieve, and cooling the filtrate with cold water. Mixing the two decoctions, concentrating to 90-110 mL of extract, subpackaging into 6-14 mL containers, wherein the subpackaging volume of each bottle is 2-3 mL, freeze-drying after subpackaging, taking out, and rolling an aluminum cover to obtain the herba Salviae chinensis standard decoction freeze-dried powder.
(2) Taking 1.0g of herba Salviae chinensis powder (sieving with a third sieve), precisely weighing, placing into a conical flask with a stopper, precisely adding 25mL of 50% methanol, weighing, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 30min, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking, filtering, and collecting the filtrate to obtain the sample solution of herba Salviae chinensis.
(3) And (3) taking 0.2g of the freeze-dried powder of the herba Salviae chinensis standard decoction prepared in the step (1), precisely weighing, placing in a conical bottle with a plug, precisely adding 25mL of 50% methanol, weighing, performing ultrasonic treatment (with power of 250W and frequency of 40 kHz) for 30min, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the sample solution of the herba Salviae chinensis standard decoction.
Determination of 3-chromatography conditions
(1) Determination of detection wavelength
A Waters BEH C18 (100 mm. Times.2.1 mm,1.7 μm) column was used; acetonitrile as mobile phase a and 0.1% formic acid aqueous solution as mobile phase B, gradient elution was performed as specified in table 2 below; the flow rate is 0.4mL/min; column temperature is 40 ℃; the sample loading was 1. Mu.L. Taking the sample solution of the lithospermum through medicine (S1), carrying out full-wavelength scanning, examining the conditions of different chromatographic wavelengths, and selecting the optimal detection wavelength, wherein the result is shown in figures 1 and 2.
TABLE 2 gradient elution table
Figure BDA0003589938260000101
The result shows that when 290nm is selected as the detection wavelength, each characteristic chromatographic peak has better response and complete peak information, so 290nm is selected as the detection wavelength.
(2) Determination of mobile phase
Based on the gradient conditions in Table 2, the detection wavelength was selected to be 290nm, the chromatographic column was Waters BEH C18 (100 mm. Times.2.1 mm,1.7 μm), acetonitrile was used as mobile phase A, and an aqueous solution of phosphoric acid with a volume fraction of 0.1%, an aqueous solution of acetic acid with a volume fraction of 0.1% and an aqueous solution of formic acid with a volume fraction of 0.1% were used as mobile phase B, and the effect of three different mobile phases B on the chromatographic peak of the lithospermum was examined, and the results are shown in FIG. 3.
The results showed that the aqueous formic acid solution with a volume fraction of 0.1% was used as mobile phase B, and each of the chromatographic peaks and separations was preferred, so that the aqueous formic acid solution with a volume fraction of 0.1% was selected as mobile phase B and acetonitrile as mobile phase a.
(3) Determination of chromatographic column
Based on the gradient conditions of Table 2, the effect of the Waters BEH C18 column (100 mm. Times.2.1 mm,1.7 μm), YMC Triart C18 column (100 mm. Times.2.1 mm,1.9 μm) and Agilent SB C18 column (100 mm. Times.2.1 mm,1.8 μm) on the chromatographic peak of the medicinal material was examined with acetonitrile as mobile phase A and 0.1% formic acid aqueous solution as mobile phase B, respectively, and the results are shown in FIG. 4.
The results show that when the Waters BEH C18 chromatographic column is adopted, the peak type and the separation degree of each chromatographic peak are optimal, so the Waters BEH C18 chromatographic column is selected as the characteristic spectrum chromatographic column of the medicinal herb.
(4) Determination of chromatographic conditions
From the foregoing examination, the chromatographic conditions can be determined as:
chromatographic column: waters BEH C18 (100 mm. Times.2.1 mm,1.7 μm) column with octadecylsilane chemically bonded silica as filler;
mobile phase: acetonitrile as mobile phase a and 0.1% formic acid aqueous solution as mobile phase B, and gradient elution was performed as specified in table 2;
flow rate: 0.4mL/min; column temperature: 40 ℃; ultraviolet detection wavelength: 290nm.
4. Determination of characteristic peaks
Taking 15 batches of the herba lycopodii medicinal materials in table 1, preparing a sample solution I of the herba lycopodii medicinal materials according to the method under the item of 2.3, taking the sample solution I and a reference medicinal material solution, and measuring according to the chromatographic conditions determined under the item of 3, so as to obtain a characteristic spectrum of 15 batches of the herba lycopodii medicinal materials (figure 5) and a characteristic spectrum of the herba lycopodii medicinal materials (figure 6).
Taking 15 batches of herba Salviae chinensis medicinal materials in table 1, preparing a herba Salviae chinensis standard decoction and a corresponding sample solution II according to the method under item "2.3", and measuring the sample solution II according to the chromatographic conditions determined under item "3" to obtain a characteristic spectrum of the corresponding standard decoction of 15 batches of herba Salviae chinensis medicinal materials (figure 7).
By comparing fig. 5 to 7, 6 common peaks can be determined, and each of the 6 common peaks can be stably transmitted from the medicinal material of the lithospermum through to the medicinal material of the lithospermum through standard decoction, so the 6 common peaks are set as characteristic peaks of the medicinal material of the lithospermum through, and further the characteristic peaks are identified and confirmed to determine the chemical components of the medicinal material of the lithospermum through standard decoction.
5. Identification and confirmation of characteristic peaks
5.1 identification of characteristic peaks
And (3) performing chemical component identification on characteristic peaks in the characteristic spectrum of the lithospermum through UPLC-QE-Orbitrap-MS.
(1) UPLC chromatographic conditions: the chromatographic conditions were as defined under item "3".
(2) Mass spectrometry conditions: specific parameters are shown in table 3 below.
Table 3 mass spectral parameters
Figure BDA0003589938260000121
(3) Precisely sucking 1 μl of the sample solution of herba Salviae chinensis (S1), and injecting into the LC-MS system to obtain ultraviolet and total ion flow chromatograms (figure 8). The acquired data are analyzed by adopting an mzVault mass spectrum database, the accurate mass number is obtained from a high resolution system, the element composition analysis is carried out on the data, the chemical formula of the compound is obtained, and according to the secondary mass spectrum information of the compound, the composition information of 6 characteristic peaks in the stone penetration is estimated by combining the present geologic spectrum database and related literature analysis, as shown in the following table 4.
Table 4 identification information of characteristic peaks of medicinal material of lithospermum
Figure BDA0003589938260000122
Figure BDA0003589938260000131
5.2 confirmation of characteristic peaks
Preparing reference substance solutions of caffeic acid, chlorogenic acid, rosmarinic acid, salvianolic acid B and salvianic acid according to the identification information in Table 4 under the method of item "2.2", taking the reference substance solutions of the reference substance, the herba Salviae chinensis medicinal material and the sample solution of the standard decoction thereof, and detecting according to the chromatographic conditions determined under the method of item "3", wherein the result is shown in figure 9.
The chemical components of 5 characteristic peaks in the lithospermum through comparison with the retention time of a reference substance and an ultraviolet-visible 3D spectrum can be primarily confirmed, wherein: characteristic peak 1 is salvianic acid, characteristic peak 3 is caffeic acid, characteristic peak 4 is chlorogenic acid, characteristic peak 5 is rosmarinic acid, characteristic peak 6 is salvianolic acid B, and characteristic peak 2 is C according to mass spectrum information 7 H 5 O 3 The compound is a compound of a parent nucleus, and the compound has a plurality of types and cannot be confirmed temporarily.
By combining the identification information of Table 4 with characteristic patterns (fig. 5 and 7) of 15 batches of radix et rhizoma Rhei-see-through medicinal materials and standard decoction thereof, it can be found that the peak areas of characteristic peak 5 in the radix et rhizoma Rhei-see-through medicinal materials and standard decoction are stable, and the reference substance is easily obtained, so that characteristic peak 5, namely rosmarinic acid chromatographic peak, is selected as reference peak.
6. Examination of methodology
6.1 investigation of precision
Taking a medicinal material (S1) of herba Salviae chinensis, preparing a sample solution according to the method under the item "2.3", repeatedly sampling and measuring for 6 times according to the chromatographic conditions determined under the item "3", taking rosmarinic acid peak as reference peak S, and calculating the relative retention time of 6 characteristic peaks and S peak and RSD of the relative peak area. The results show that the relative retention time and relative peak area RSD of the 6 characteristic peaks are less than 3% (the results are shown in table 5 and table 6), which indicates that the instrument precision is good.
TABLE 5 precision investigation experiment results (relative retention time)
Figure BDA0003589938260000141
Table 6 precision investigation experiment results (relative peak area)
Figure BDA0003589938260000142
6.2 repeatability investigation
Taking the same batch of the lithospermum samples (S1), preparing 6 parts of sample solutions in parallel according to the method under the item of 2.3, determining chromatographic conditions under the item of 3, carrying out sample injection measurement, taking rosmarinic acid peak as a reference peak S, and calculating the relative retention time of 6 characteristic peaks and S peak and RSD of the relative peak area. The results showed that the relative retention time and relative peak area RSD of the 6 characteristic peaks were less than 3% (the results are shown in tables 7 and 8), indicating that the reproducibility of the method was good.
TABLE 7 repeatability test results (relative retention time)
Figure BDA0003589938260000143
Figure BDA0003589938260000151
Table 8 results of the repeatability test (relative peak areas)
Figure BDA0003589938260000152
6.3 stability investigation
Taking the same sample solution of the medicinal material (S1) of the herba Salviae chinensis, determining chromatographic conditions under the item of 3, respectively sampling at 0h, 2h, 4h, 6h, 8h, 12h and 24h, taking rosmarinic acid peak as reference peak S, and calculating relative retention time of 6 characteristic peaks and S peak and RSD of relative peak area. The results showed that the relative retention time and relative peak area RSD for the 6 characteristic peaks were less than 3% (results shown in table 10, 9) indicating that the test sample solution was stable over 24 h.
TABLE 9 stability investigation experiment results (relative retention time)
Figure BDA0003589938260000153
Table 10 stability investigation experiment results (relative peak area)
Figure BDA0003589938260000154
Figure BDA0003589938260000161
6.4 investigation of specificity
Taking herba Salviae chinensis (S15), preparing a test solution according to the method under item "2.3", precisely sucking 1 μl of the test solution, the reference solution for reference under item "2.2" and the blank solvent, and performing sample injection analysis according to the chromatographic conditions determined under item "3" (the result is shown in FIG. 10). The results show that the sample and the reference substance have the same chromatographic peak at the corresponding retention time, and the blank solvent has no interference, thus indicating that the established method has good specificity.
6.5 investigation of durability
(1) Investigation of different flow rates
Taking a sample solution of the medicinal material (S1) to be processed, comparing chromatograms at the flow rates of 0.38mL/min, 0.40mL/min and 0.42mL/min on the basis of the chromatographic condition of 3 items, and calculating the relative retention time between the 6 characteristic peaks and the S peak and the RSD of the relative peak area by taking the rosmarinic acid peak as a reference peak S. The results show that the relative retention time and relative peak area RSD of the 6 characteristic peaks are less than 5% (the results are shown in table 11 and table 12), and the established method has good flow velocity durability, and the influence of the smaller flow velocity variation on the map is smaller.
TABLE 11 flow rate investigation experiment results (relative retention time)
Figure BDA0003589938260000162
Table 12 flow Rate investigation experiment results (relative peak area)
Figure BDA0003589938260000163
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Figure BDA0003589938260000171
(2) Investigation of different column temperatures
Taking a sample solution of a medicinal material (S1) to be tested, comparing chromatograms of the medicinal material at 38 ℃ and 40 ℃ and 42 ℃ on the basis of chromatographic conditions of 3 items, and calculating relative retention time of 6 characteristic peaks and S peaks and RSD of relative peak areas by taking rosmarinic acid peaks as reference peaks S. The results show that the relative retention time and the relative peak area RSD of the 6 characteristic peaks are less than 5 percent (the results are shown in tables 13 and 14), and the method has good column temperature durability and less influence on the map caused by column temperature fluctuation at +/-2 ℃.
TABLE 13 column temperature investigation experimental results (relative retention time)
Figure BDA0003589938260000172
Table 14 column temperature investigation experiment results (relative peak area)
Figure BDA0003589938260000173
(3) Inspection of different brands of chromatographs
Taking a sample solution of a medicinal material (S1) to be tested, comparing chromatograms collected by three different brands of instruments, namely Waters H-Class, thermo Vanquish and Agilent 1290, based on the chromatographic condition of 3 items, and calculating the relative retention time of 6 characteristic peaks and S peaks and RSD of the relative peak areas by taking rosmarinic acid peaks as reference peaks S. The results show that the characteristic spectrum of the medicinal material of the lithospermum through can be well reproduced on three different brands of instruments, the relative retention time of 6 characteristic peaks and the relative peak area RSD are less than 5 percent (the results are shown in tables 15 and 16), and the method has good durability on different brands of chromatographs.
Table 15 chromatograph examines the experimental results (relative retention time)
Figure BDA0003589938260000174
Figure BDA0003589938260000181
Table 16 chromatograph examines the experimental results (relative peak area)
Figure BDA0003589938260000182
7. Establishment of characteristic spectrum of lithospermum through medicinal material
Analyzing the characteristic spectrum of 15 batches of herba Salviae chinensis, taking rosmarinic acid peak as reference peak S, calculating the relative retention time and relative peak area of each characteristic peak and S peak, and calculating RSD value, and experimental results are shown in tables 17 and 18.
Table 17 Table 15 characteristics of the group of medicinal materials of the group of stone-like see-through herbs and relative retention time
Figure BDA0003589938260000183
Table 18 15 characteristics of the herba Salviae chinensis and relative peak areas
Figure BDA0003589938260000191
The characteristic patterns of 15 batches of the herba Salviae chinensis medicinal materials and the standard decoction thereof are imported into a traditional Chinese medicine chromatographic characteristic pattern similarity evaluation system to respectively generate contrast characteristic patterns of the herba Salviae chinensis medicinal materials and the herba Salviae chinensis standard decoction (figures 11-13).
The results show that 15 batches of the medicinal materials of the lithospermum have 6 common characteristic peaks, and the 6 characteristic peaks can be stably transmitted from the medicinal materials of the lithospermum into the standard decoction of the lithospermum, namely the characteristic spectrum of the lithospermum corresponds to the 6 characteristic peaks in the characteristic spectrum of the standard decoction. The peak 5 rosmarinic acid peak is taken as a reference peak S, the relative retention time RSD of the other 5 characteristic peaks of 15 batches of the characteristic spectrum of the medicinal herb of the lithospermum is 0.13-1.80%, both are less than 3%, the standard requirement of the characteristic spectrum of the medicinal herb of the lithospermum is met, the relative retention time is stable, and the characteristic peaks can be positioned by using the relative retention time. The relative peak areas RSD of 5 other characteristic peaks except the S peak in the characteristic spectrum of 15 batches of the medicinal herb are 30.43-97.40 percent, the relative peak areas have larger difference, the large difference of the proportion of chemical components represented by each characteristic peak of the lithospermum through medicinal materials in different batches is described, and the difference is possibly related to various factors such as production places, growth years, harvest time and the like, so that the relative peak areas are not suitable to be regulated.
In summary, the standard for determining the characteristic spectrum of the medicinal herb of the lithospermum is as follows: the chromatogram of the sample should show 6 characteristic peaks and correspond to the retention time of 6 characteristic peaks in the characteristic chromatogram of the reference of the herba Salviae chinensis; wherein peak 1, peak 3, peak 4, peak 5, and peak 6 should correspond to the retention time of salvianolic acid B reference peak, respectively; calculating the relative retention time of peak 2 with the peak corresponding to the rosmarinic acid reference peak as S peak, wherein the relative retention time is within + -10% of the specified value, and the specified value is: 0.24 (Peak 2).
Through the research, the invention establishes the characteristic spectrum construction method of the herba Salviae chinensis and the standard decoction thereof, and establishes the characteristic spectrum of the herba Salviae chinensis and the standard decoction thereof. The method researches water-soluble characteristic components shared by the herba Salviae chinensis standard decoction and the medicinal materials, and is used as a basis for determining characteristic peaks, so that the mass transfer from medicinal materials to the decoction can be well represented, the basic characteristics of the herba Salviae chinensis clinical decoction are fully displayed, the method has good precision, reproducibility, stability and specificity through methodology investigation, the constructed characteristic map is rich in information, strong in characteristics and good in reproducibility, the quality monitoring of a plurality of characteristic components of the herba Salviae chinensis medicinal materials can be rapidly and comprehensively realized, raw materials meeting the requirements of the herba Salviae chinensis standard decoction are clinically provided, and a scientific new method is provided for the quality control of the herba Salviae chinensis medicinal materials.
Example 2
The embodiment provides a detection method of a medicinal material of lithospermum through
1. Instrument, reagent and reagent
(1) Instrument for measuring and controlling the intensity of light
Waters high Performance liquid chromatography (Waters H-Class, waters corporation), waters BEH C18 column (100 mm 2.1mm,1.7 μm), one-ten-thousandth balance (ME 204E, metrer-Toli Corp.), one-ten-thousandth level (XP 26, metrer-Toli Corp.); digital control ultrasonic cleaner (KQ 500DE, kunshan ultrasonic instruments Co., ltd.), thermostatic water bath (HWS 28, shanghai-He technology Co., ltd.) ultrapure water system (Milli-Q Direct, merck Co., ltd.).
(2) The reagents were as in example 1.
(3) The reagents were the same as in example 1. Wherein, the herba Salviae chinensis medicinal material numbered as S16 in Table 1 is used as herba Salviae chinensis medicinal material to be detected.
2. Preparation of test solutions
A test solution of the medicinal material of the herba Salviae chinensis (S16) to be tested was prepared as in example 1, item 2.3 (2) ".
3. Chromatographic conditions
Gradient elution was performed as specified in Table 2 using Waters BEH C18 (100 mm. Times.2.1 mm,1.7 μm) as the column, acetonitrile as mobile phase A, and 0.1% aqueous formic acid as mobile phase B; flow rate: 0.4mL/min; column temperature: 40 ℃; ultraviolet detection wavelength: 290nm.
4. Measurement
Taking 1 mu L of the sample solution of the herba Salviae chinensis to be detected (S16), injecting into a liquid chromatograph, and measuring according to the chromatographic conditions under the item 3 to obtain a UPLC chromatogram of the herba Salviae chinensis to be detected, wherein the results are shown in FIG. 14 and Table 19.
TABLE 19 relative retention time of the characteristic spectrum of the herba Salviae chinensis
Figure BDA0003589938260000201
Figure BDA0003589938260000211
Comparing the UPLC chromatogram (figure 14) of the herba Salviae chinensis to be detected with the contrast characteristic chromatogram (figure 11) of the herba Salviae chinensis to be detected: by adopting the method to detect the medicinal material of the herba Salviae chinensis to be detected, 6 characteristic peaks can be detected and correspond to 6 characteristic peaks in the contrast characteristic map. The data result shows (table 19) that the relative retention time of the 6 characteristic peaks of the medicinal herb of the lithospermum to be detected is in the range specified by the standard, which indicates that the quality of the batch of medicinal herb of the lithospermum to be detected is qualified, and the use requirement of the clinical decoction is met.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples merely represent a few embodiments of the present invention, which facilitate a specific and detailed understanding of the technical solutions of the present invention, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. It should be understood that, based on the technical solutions provided by the present invention, those skilled in the art may obtain technical solutions through logical analysis, reasoning or limited experiments, which are all within the scope of protection of the appended claims. The scope of the patent is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted as illustrative of the contents of the claims.

Claims (13)

1. The method for constructing the characteristic spectrum of the medicinal material of the lithospermum or the standard decoction thereof is characterized by comprising the following steps of:
extracting herba Salviae chinensis reference material with organic solvent, filtering, and collecting filtrate to obtain reference solution; dissolving caffeic acid reference substance, chlorogenic acid reference substance, rosmarinic acid reference substance, salvianolic acid B reference substance, and salvianic acid A sodium reference substance in organic solvent to obtain reference substance solution;
extracting herba Salviae chinensis with organic solvent, filtering, and collecting filtrate to obtain sample solution I; or extracting herba Salviae chinensis lyophilized powder with organic solvent, filtering, and collecting filtrate to obtain sample solution II;
the organic solvent is 40% -60% methanol aqueous solution by volume fraction;
detecting the reference substance solution of the reference medicinal material, the reference substance solution of the reference substance and the solution I of the test sample by adopting an ultra-high performance liquid chromatography, and establishing a characteristic map of the lithospermum through medicinal material; or alternatively, the first and second heat exchangers may be,
detecting the reference substance solution of the reference medicinal material, the reference substance solution of the reference substance and the solution II of the test substance by adopting an ultra-high performance liquid chromatography, and establishing a characteristic map of the standard decoction of the lithospermum through;
The mobile phase A adopted by the ultra-high performance liquid chromatography is acetonitrile, the mobile phase B is formic acid aqueous solution with the volume fraction of 0.05% -0.2%, and the elution mode is gradient elution;
the gradient elution included the following procedure:
0 min-3 min, wherein the volume percentage of the mobile phase A is kept to be 2%;
3-10 min, wherein the volume percentage of the mobile phase A is increased from 2% to 7%;
10-13 min, wherein the volume percentage of the mobile phase A is increased from 7% to 9%;
13-30 min, wherein the volume percentage of the mobile phase A is increased from 9% to 20%;
30-36 min, wherein the volume percentage of the mobile phase A is increased from 20% to 26%;
36-40 min, wherein the volume percentage of the mobile phase A is increased from 26% to 60%;
the chromatographic column adopted by the ultra-high performance liquid chromatography is a Waters BEH C18 chromatographic column.
2. The method for constructing a characteristic spectrum of a medicinal material of lithospermum or a standard decoction thereof according to claim 1, wherein the ultra-high performance liquid chromatography further comprises the following detection conditions:
flow rate: 0.2mL/min to 0.6mL/min;
column temperature: 35-45 ℃;
detection wavelength: 280nm to 300nm.
3. The method for constructing the characteristic spectrum of the medicinal material of the lithospermum or the standard decoction thereof according to claim 2, wherein the detection conditions of the ultra-high performance liquid chromatography are as follows:
Flow rate: 0.4mL/min;
column temperature: 40 ℃;
detection wavelength: 290nm.
4. A method for constructing a characteristic spectrum of a medicinal material of herba Salviae chinensis or a standard decoction thereof according to any one of claims 1 to 3, wherein the chromatographic column has a column length of 100mm, an inner diameter of 2.1mm, and an inner filler particle diameter of 1.7 μm.
5. A method for constructing a characteristic spectrum of a medicinal material of lithospermum or a standard decoction thereof according to any one of claims 1 to 3, wherein the extraction method is ultrasonic extraction, and comprises the following conditions: the time is 20-40 min, the power is 240-260W, and the frequency is 30-50 kHz.
6. A method for constructing a characteristic spectrum of a medicinal material of lithospermum or a standard decoction thereof according to any one of claims 1 to 3, wherein the characteristic spectrum comprises 6 characteristic peaks.
7. The method for constructing a characteristic spectrum of a medicinal material of lithospermum or a standard decoction thereof according to claim 6, further comprising the steps of:
and identifying the characteristic peaks by adopting mass spectrometry.
8. The method for constructing a characteristic spectrum of a medicinal material of lithospermum or a standard decoction thereof according to claim 7, wherein the mass spectrometry comprises the following detection conditions:
the ionization mode is heating electrospray ionization; the flow rate of sheath gas is 30 arb-40 arb; the flow rate of the auxiliary gas is 5 arb-15 arb; the spraying voltage is 3.5 kV-4 kV; s-lens voltage is 40V-60V; the heating temperature is 300-400 ℃; the temperature of the capillary tube is 300-400 ℃; the scanning mode is an anion mode; the scanning range is 100-1500.
9. The method for detecting the medicinal material of the lithospermum or the standard decoction thereof is characterized by comprising the following steps of:
extracting herba Salviae chinensis to be detected with organic solvent, filtering, and collecting filtrate to obtain sample solution I; or extracting the freeze-dried powder of the standard decoction of the herba Salviae chinensis to be detected with an organic solvent, filtering, and collecting the filtrate to prepare a sample solution II to be detected;
the organic solvent is 40% -60% methanol aqueous solution by volume fraction;
detecting the sample solution I or the sample solution II to be detected by adopting an ultra-high performance liquid chromatography;
comparing the detection result with a characteristic spectrum constructed by the characteristic spectrum construction method of the lithospermum through medicinal material or the standard decoction thereof according to any one of claims 1-8;
the mobile phase A adopted by the ultra-high performance liquid chromatography is acetonitrile, the mobile phase B is formic acid aqueous solution with the volume fraction of 0.05% -0.2%, and the elution mode is gradient elution;
the gradient elution included the following procedure:
0 min-3 min, wherein the volume percentage of the mobile phase A is kept to be 2%;
3-10 min, wherein the volume percentage of the mobile phase A is increased from 2% to 7%;
10-13 min, wherein the volume percentage of the mobile phase A is increased from 7% to 9%;
13-30 min, wherein the volume percentage of the mobile phase A is increased from 9% to 20%;
30-36 min, wherein the volume percentage of the mobile phase A is increased from 20% to 26%;
36-40 min, wherein the volume percentage of the mobile phase A is increased from 26% to 60%;
the chromatographic column adopted by the ultra-high performance liquid chromatography is a Waters BEH C18 chromatographic column.
10. The method for detecting a medical herb of lithospermum or a standard decoction thereof according to claim 9, wherein the ultra-high performance liquid chromatography further comprises the following detection conditions:
flow rate: 0.2mL/min to 0.6mL/min;
column temperature: 35-45 ℃;
detection wavelength: 280nm to 300nm.
11. The method for detecting the medicinal herb of lithospermum or the standard decoction thereof according to claim 10, wherein the detection conditions of the ultra-high performance liquid chromatography are as follows:
flow rate: 0.4mL/min;
column temperature: 40 ℃;
detection wavelength: 290nm.
12. The method for detecting a medical herb of lithospermum or a standard decoction thereof according to any one of claims 9 to 11, wherein the chromatographic column has a column length of 100mm, an inner diameter of 2.1mm and an inner filler particle diameter of 1.7 μm.
13. The method for detecting the medicinal material of lithospermum or the standard decoction thereof according to any one of claims 9 to 11, wherein the extraction method is ultrasonic extraction, and comprises the following conditions: the time is 20-40 min, the power is 240-260W, and the frequency is 30-50 kHz.
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