CN117007700A - Construction method and application of ginseng leaf characteristic spectrum - Google Patents
Construction method and application of ginseng leaf characteristic spectrum Download PDFInfo
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- CN117007700A CN117007700A CN202310668215.XA CN202310668215A CN117007700A CN 117007700 A CN117007700 A CN 117007700A CN 202310668215 A CN202310668215 A CN 202310668215A CN 117007700 A CN117007700 A CN 117007700A
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- SPFXZQZPHXUJSR-UHFFFAOYSA-N ginsenoside-Rc Natural products CC(=CCCC(C)(OC1OC(CO)C(O)C(O)C1OC2OC(CO)C(O)C2O)C3CCC4(C)C3C(O)CC5C6(C)CCC(OC7OC(CO)C(O)C(O)C7OC8OC(CO)C(O)C(O)C8O)C(C)(C)C6CCC45C)C SPFXZQZPHXUJSR-UHFFFAOYSA-N 0.000 description 1
- ZTQSADJAYQOCDD-UHFFFAOYSA-N ginsenoside-Rd2 Natural products C1CC(C2(CCC3C(C)(C)C(OC4C(C(O)C(O)C(CO)O4)O)CCC3(C)C2CC2O)C)(C)C2C1C(C)(CCC=C(C)C)OC(C(C(O)C1O)O)OC1COC1OCC(O)C(O)C1O ZTQSADJAYQOCDD-UHFFFAOYSA-N 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 229940035034 maltodextrin Drugs 0.000 description 1
- 238000003808 methanol extraction Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- IFGCUJZIWBUILZ-UHFFFAOYSA-N sodium 2-[[2-[[hydroxy-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyphosphoryl]amino]-4-methylpentanoyl]amino]-3-(1H-indol-3-yl)propanoic acid Chemical compound [Na+].C=1NC2=CC=CC=C2C=1CC(C(O)=O)NC(=O)C(CC(C)C)NP(O)(=O)OC1OC(C)C(O)C(O)C1O IFGCUJZIWBUILZ-UHFFFAOYSA-N 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
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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
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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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
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
- G01N30/8634—Peak quality criteria
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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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention belongs to the technical field of medicine analysis, and particularly relates to a construction method and application of a ginseng leaf characteristic spectrum. The construction method comprises the following steps: preparation of the sample solution, preparation of the reference solution, and determination according to high performance liquid chromatography, and defining a mobile phase A of acetonitrile and a mobile phase B of phosphoric acid aqueous solution and a specific gradient elution procedure. The separation degree of each characteristic peak in the characteristic spectrum obtained by the construction method is good, the spectrum specificity is strong, the implementation is easy, and a basis is provided for effectively controlling and scientifically evaluating the quality of ginseng leaf medicinal materials and preparations thereof. The method provided by the invention has the advantages of simple operation, accurate result and good result reproducibility.
Description
Technical Field
The invention belongs to the technical field of medicine analysis, and particularly relates to a construction method and application of a ginseng leaf characteristic spectrum.
Background
The ginseng leaf is a dried leaf of Panax ginseng C.A. Mey. The ginseng has a plurality of medicinal parts, including ginseng, ginseng leaves, ginseng flowers and the like, the chemical components of different parts of ginseng are similar, mainly ginsenoside components are used as main components, and the chemical components of American ginseng leaves of the same genus are also used as main components, and the ginseng leaves and other ginseng plants are difficult to distinguish by pharmacopoeia standards due to the similarity of the chemical components of the ginseng, the traditional Chinese medicine formula particles are prepared by adding water into traditional Chinese medicine decoction pieces, extracting, drying, granulating and other processes, the traditional Chinese medicine formula particles lose the characteristic identification characteristics of the raw medicinal materials, and cannot be checked and identified from the aspects of shape, size, texture and the like of the medicinal materials. Therefore, a method for identifying ginseng leaves is required to be established, and a basis is provided for effectively controlling and scientifically evaluating the quality of ginseng leaves. The characteristic spectrum of the ginseng leaf obtained by the prior art has the problems of poor separation degree between characteristic peaks, insufficient spectrum specificity and the like.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to overcome the defects of poor separation degree, weak specificity and the like of characteristic peaks in the constructed atlas when evaluating the quality of ginseng leaves in the prior art, thereby providing a construction method and application of the ginseng leaf characteristic atlas.
For this purpose, the invention provides the following technical scheme.
The invention provides a construction method of a ginseng leaf characteristic map, which comprises the following steps:
preparation of test solution: preparing a sample solution from a sample;
preparation of reference solution: preparing reference solution from reference materials;
and (3) measuring: according to high performance liquid chromatography;
wherein the chromatographic conditions of the high performance liquid chromatography include: acetonitrile is taken as a mobile phase A, phosphoric acid aqueous solution is taken as a mobile phase B, and gradient elution is carried out;
the gradient elution procedure included: 0-10min, 5-20% mobile phase A, 95-80% mobile phase B;10-18min, 20-22% mobile phase A, 80-78% mobile phase B;18-20min, 22-31% mobile phase A, 78-69% mobile phase B;20-30min, 31-33% mobile phase A, 69-67% mobile phase B;
the gradient elution procedure further comprises: 30-40min, 33-60% mobile phase A, 67-40% mobile phase B;40-45min, 60-90% mobile phase A, 40-10% mobile phase B; or 30-50min, 33-80% mobile phase A, 67-20% mobile phase B.
The chromatographic conditions of the high performance liquid chromatography further include: octadecylsilane chemically bonded silica gel column is used as filler, and the specification of the chromatographic column is as follows: the inner diameter is 4.6mm, the column length is 150mm, and the grain diameter is 2.7 mu m; and/or the number of the groups of groups,
the column temperature is 23-27 ℃; and/or the number of the groups of groups,
the wavelength is 201-205nm; and/or the number of the groups of groups,
the sample injection amount is 5-15 mu L; and/or the number of the groups of groups,
the flow rate is 0.9-1.1ml/min; and/or the number of the groups of groups,
taking 0.08-0.12% phosphoric acid aqueous solution as mobile phase B.
The sample is at least one of folium Ginseng decoction pieces, folium Ginseng granule, folium Ginseng standard decoction lyophilized powder, folium Ginseng extract powder and folium Ginseng medicinal material.
The preparation method of the sample solution comprises the following steps: extracting the sample with solvent, and filtering;
preferably, in preparing the sample solution, the extraction solvent is at least one of water, methanol and ethanol;
preferably, the extraction solvent is methanol;
preferably, in preparing the sample solution, the extraction method is reflux extraction or ultrasonic extraction.
When the sample is at least one of ginseng leaf prescription granule, ginseng leaf standard decoction freeze-dried powder and ginseng leaf extract powder, the ratio of the mass of the sample to the volume of the extraction solvent is 1g (45-55 ml) when the sample solution is prepared.
The preparation method of the reference substance solution comprises the following steps: extracting reference materials with water, filtering, and evaporating to dryness to obtain water extract; adding extraction solvent into the water extract, extracting, and filtering;
preferably, in preparing the reference solution, the extraction solvent is at least one of water, methanol and ethanol;
preferably, the extraction solvent is methanol;
preferably, the ratio of the quality of the reference medicinal material to the volume of the extraction solvent is 1g (15-19 ml);
preferably, in preparing the reference solution, the extraction method is reflux extraction or ultrasonic extraction.
The construction method also comprises the preparation of a reference substance solution of the reference substance;
the reference substance is at least one of kaempferol-3-O-sophoroside reference substance, ginsenoside Rg1 reference substance, ginsenoside Re reference substance and ginsenoside Rd reference substance.
The characteristic map obtained by the construction method comprises 14 characteristic peaks;
the relative retention time of peak No. 1, with peak No. 2 as reference peak No. 1, was defined as: 0.58;
the relative retention time values of the reference peaks No. 2, no. 5, no. 6, no. 7, no. 8, no. 9, no. 10, no. 12, no. 13 and No. 14 were respectively: 1.11, 1.25, 1.29, 1.35, 1.43, 1.48, 1.72, 1.76, and 1.79;
wherein the relative retention time of the characteristic peak is within + -10% of the prescribed value.
The invention also provides a method for identifying the ginseng leaves, which adopts the construction method to obtain the characteristic spectrum of the to-be-detected sample, and utilizes the areas of the No. 3 peak and the No. 11 peak to identify whether the to-be-detected sample is the ginseng leaves.
In the identification method, when a is 0.43-1.5, the sample to be detected is ginseng leaf;
wherein,
S peak No. 11 Peak area of No. 11 peak, S Peak 3 Peak area of peak No. 3.
The technical scheme of the invention has the following advantages:
1. the construction method of the ginseng leaf characteristic map provided by the invention comprises the following steps: preparation of the sample solution, preparation of the reference solution, and determination according to high performance liquid chromatography, and defining a mobile phase A of acetonitrile and a mobile phase B of phosphoric acid aqueous solution and a specific gradient elution procedure. The separation degree of each characteristic peak in the characteristic spectrum obtained by the construction method is good, the spectrum specificity is strong, the implementation is easy, and a basis is provided for effectively controlling and scientifically evaluating the quality of ginseng leaf medicinal materials and preparations thereof. The method provided by the invention has the advantages of simple operation, accurate result and good result reproducibility.
2. The identification method of ginseng leaves provided by the invention can be used for distinguishing ginseng leaves from other medicinal parts of ginseng by utilizing the characteristic spectrum obtained by the construction method provided by the invention, and distinguishing ginseng leaves from plants of the same genus.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a characteristic map of 18 batches of ginseng leaf standard decoction lyophilized powder and 3 batches of ginseng leaf formula granules in example 1 of the present invention;
FIG. 2 is a control map of ginseng leaf according to example 1 of the present invention;
FIG. 3 is a characteristic spectrum of ginseng leaf formula particles obtained by different gradient elution procedures in experimental example 1 of the present invention;
FIG. 4 is a characteristic spectrum of ginseng leaf formula particles obtained at different flow rates in experimental example 1 of the present invention;
FIG. 5 is a characteristic spectrum of ginseng leaf formula particles obtained at different column temperatures in experimental example 1 of the present invention;
FIG. 6 is a characteristic spectrum of ginseng leaf formula particles obtained from different mobile phases B in experimental example 1 of the present invention;
FIG. 7 is a characteristic spectrum of ginseng leaf formula particles obtained at different mobile phase B concentrations in experimental example 1 of the present invention;
FIG. 8 is a characteristic spectrum of ginseng leaf formula particles obtained by different chromatographic columns in experimental example 1 of the present invention;
FIG. 9 is a characteristic spectrum of ginseng leaf formula granules obtained by different batch chromatographic columns in experimental example 1 of the present invention;
FIG. 10 is a characteristic spectrum of a sample obtained by different extraction methods in Experimental example 2 of the present invention;
FIG. 11 is a characteristic spectrum of a sample obtained at different extraction times in Experimental example 2 of the present invention;
FIG. 12 is a characteristic spectrum of a sample obtained by different extraction solvents in Experimental example 2 of the present invention;
FIG. 13 is a characteristic spectrum of a sample obtained by extracting solvents at different concentrations in Experimental example 2 of the present invention;
FIG. 14 is an adaptive signature of the chromatographic condition system of Experimental example 3 of the present invention;
FIG. 15 is a graph showing a negative sample of Experimental example 3 of the present invention;
FIG. 16 is a characteristic spectrum of a different sample to be tested according to example 2 of the present invention.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.
The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
The apparatus and reagents used in the examples below:
instrument: chromatograph 1: the Waters e2695 chromatographic system comprises a quaternary gradient infusion pump (Alliance 2695), a 120-bit high-performance automatic sampler, an original inlet chromatographic column incubator, a Waters 2998 diode array ultraviolet detector and an Empower chromatographic management system; chromatograph 2: the Shimadzu chromatographic system comprises an LC-20AT type quaternary pump, an SIL-20AC type autosampler, an SPD-M20A type PDA diode array detector, a CTO-20AC type column incubator, a CBM-20A system controller and an Empower chromatographic management system; chromatograph 3: the Daian UltiMate 3000 chromatographic system comprises an LPG-3400A quaternary pump, a WPS-3000TSL autosampler, a PDA diode array detector and a chromatographic workstation.
Chromatographic column: (1) Agilent ZORBAX SB-Aq, 4.6mm.times.150mm, 3.5 μm; (2) CAPCELL CORE C18, 4.6mm.times.150mm, 2.7 μm; (3) CORTECS T3,4.6 mm. Times.150 mm,2.7 μm.
Reagent: acetonitrile is chromatographic pure, and water is ultrapure water; the other reagents were all analytically pure.
Reagent: kaempferol-3-O-sophoroside (lot number: 19895-95-5 for content determination, calculated as 90.8%) was purchased from Shanghai Hongyang Biotechnology Co., ltd; ginsenoside Rg1 (batch No. 110703-201933, for content determination, calculated as 93.4%) was purchased from China food and drug inspection institute; ginsenoside Re (batch No. 110754-202028, calculated as 93.9%) is purchased from Chinese food and drug assay institute; ginsenoside Rd (lot number: 111818-201603 for content measurement, calculated as 92.1%) was purchased from China food and drug inspection institute; ginseng leaf control (lot number: 37006-202101; purchased from Shanghai HongYongsheng biotechnology Co., ltd.).
Ginseng leaf formula granules (batch numbers: 2110001Y, 2110002Y, 2110003Y).
Ginseng leaf decoction pieces (lot numbers: 2010001Y, 2010002Y, 2010003Y, 2010004Y, 2010005Y, 2010006Y, 2010008Y, 2010009Y, 2010010Y, 2010012Y, 2010013Y, 2010017Y, 2010018Y, 2010019Y, 2010020Y, 210801Y, 210802Y, 210803Y).
Ginseng leaf standard decoction freeze-dried powder: the 18 batches of ginseng leaf decoction pieces are used as raw materials, and the method comprises the following steps: soaking appropriate amount of folium Ginseng decoction pieces for 30min, decocting for 2 times, decocting with strong fire (500W) for 12 times, decocting with slow fire (300W) for 30min, decocting with strong fire (500W) for 10 times, decocting with strong fire (300W) for 25min, mixing filtrates, concentrating at 65deg.C to a feed liquid ratio of about 1:1 (relative density of 1.06-1.12; deg.C), and lyophilizing to obtain 18 batches of folium Ginseng standard decoction lyophilized powder with batch numbers corresponding to 2010001D, 2010002D, 2010003D, 2010004D, 2010005D, 2010006D, 2010008D, 2010009D, 2010010D, 2010012D, 2010013D, 2010017D, 2010018D, 2010019D, 2010020D, 210801D, 210802D, 210803D respectively.
Example 1
The embodiment provides a construction method of a ginseng leaf characteristic map, which comprises the following steps:
preparation of test solution: extracting the sample with extraction solvent, and filtering to obtain sample solution. Specifically, when the test sample is decoction pieces or medicinal materials, the preparation of the test sample solution: about 1.5g of the sample is taken and placed in a conical flask with a plug, 50ml of water is added, heating reflux is carried out for 1h, filtering is carried out, filtrate is evaporated to dryness, 25ml of 80% methanol is added into residues, sealing is carried out, ultrasonic treatment is carried out for 30min, the power is 250W, the frequency is 40kHz, cooling is carried out, shaking is carried out, filtering is carried out, and subsequent filtrate is taken, thus obtaining the sample solution. When the test sample is prescription granule, standard decoction lyophilized powder and extract powder, preparing a test sample solution: taking a proper amount of a test sample, grinding, taking about 0.5g, placing into a conical flask with a plug, adding 25ml of 80% methanol, sealing, performing ultrasonic treatment for 30min, performing ultrasonic power of 250W and frequency of 40kHz, cooling, filtering, and taking a subsequent filtrate to obtain a test sample solution.
Preparation of reference solution: taking about 1.5g of ginseng leaf reference medicine, placing the ginseng leaf reference medicine into a conical flask with a plug, adding 5ml of water, heating and refluxing for 1h, filtering, evaporating filtrate to dryness, adding 25ml of 80% methanol into residues, sealing, performing ultrasonic treatment for 30min, performing ultrasonic power of 250W, and obtaining a reference substance solution by cooling, shaking uniformly, filtering, and taking the subsequent filtrate.
Preparation of a control solution: taking a proper amount of kaempferol-3-O-sophoroside reference substance, ginsenoside Rg1 reference substance, ginsenoside Re reference substance and ginsenoside Rd reference substance, precisely weighing, adding methanol to prepare a mixed solution containing 0.1mg of kaempferol-3-O-sophoroside, 10.25mg of ginsenoside Rg, 0.5mg of ginsenoside Re and 0.2mg of ginsenoside Rd in each 1ml of the mixed solution respectively, and obtaining reference substance solution of the reference substance.
And (3) measuring: the measurement is carried out by high performance liquid chromatography (the rule 0512 of the edition of Chinese pharmacopoeia 2020). Chromatographic conditions: octadecylsilane chemically bonded silica gel column is used as filler, and the specification of the chromatographic column is as follows: the inner diameter is 4.6mm, the column length is 150mm, and the grain diameter is 2.7 mu m; the flow rate is 1.0ml/min; column temperature 25 ℃; the detection wavelength is 203nm; acetonitrile as mobile phase a,0.1% phosphoric acid aqueous solution as mobile phase B, gradient elution procedure: 0-10min, 5-20% mobile phase A, 95-80% mobile phase B;10-18min, 20-22% mobile phase A, 80-78% mobile phase B;18-20min, 22-31% mobile phase A, 78-69% mobile phase B;20-30min, 31-33% mobile phase A, 69-67% mobile phase B;30-40min, 33-60% mobile phase A, 67-40% mobile phase B;40-45min, 60-90% mobile phase A, 40-10% mobile phase B; the theoretical plate number is not lower than 1500 calculated according to ginsenoside Re.
Precisely sucking 10 μl of each of the sample solution, the reference solution and the reference solution, and injecting into a liquid chromatograph for measurement.
Identification of characteristic peaks
Taking 18 batches of ginseng leaf decoction piece standard decoction freeze-dried powder and 3 batches of ginseng leaf formula granules, respectively preparing a ginseng leaf decoction piece test sample solution and a ginseng leaf formula granule test sample solution according to the steps, and measuring according to the chromatographic conditions to obtain characteristic patterns of each test sample solution, wherein the result is shown in figure 1. In fig. 1, S1-S18 correspond to 18 batches of ginseng leaf standard decoction freeze-dried powder characteristic patterns, and S19-S21 correspond to 3 batches of ginseng leaf formula granule characteristic patterns.
The fingerprint similarity evaluation software compiled by the pharmacopoeia committee is adopted to perform fitting by adopting a mode of 'multi-point correction and MARK peak matching' to generate a comparison characteristic spectrum, and 14 characteristic chromatographic peaks are displayed in total in the spectrum as shown in figure 2 and table 1. The relative retention time of the characteristic spectrum is determined according to the research result: the characteristic patterns of the ginseng leaf comprise 14 characteristic peaks which are respectively 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14, wherein the 2, 3,4 and 11 peaks respectively correspond to the retention time of reference substances of kaempferol-3-O-sophoroside, ginsenoside Rg1, ginsenoside Re and ginsenoside Rd, and the components of the 2, 3,4 and 11 peaks are sequentially kaempferol-3-O-sophoroside, ginsenoside Rg1, ginsenoside Re and ginsenoside Rd; calculating the relative retention time of each characteristic peak by sections by adopting double reference peaks, taking the characteristic peak of kaempferol-3-O-sophoroside as a reference object peak 1 (S1), and defining the relative retention time of a No. 1 peak to be 0.58; the relative retention time of the reference peak 2 (S2) was defined as the characteristic peak of ginsenoside Re, and the specific values of the relative retention time of the peak No. 5, the peak No. 6, the peak No. 7, the peak No. 8, the peak No. 9, the peak No. 10, the peak No. 12, the peak No. 13 and the peak No. 14 were: 1.11, 1.25, 1.29, 1.35, 1.43, 1.48, 1.72, 1.76 and 1.79.
TABLE 1 retention time of each characteristic peak of ginseng leaf standard decoction lyophilized powder and formulated granule
Note that: t/min is the retention time and t/ts is the relative retention time.
Identification of characteristic peaks
The ginseng leaves have 14 characteristic peaks, and identification and assignment of the characteristic peaks are adopted by HPLC and LC/MS/MS. The results are shown in the table below, wherein the peak 1 is L-tryptophan, the peak 2 is kaempferol-3-O-sophoroside, the peak 3 is ginsenoside Rg1, the peak 4 is ginsenoside Re, the peak 5 is Vietnam ginsenoside R4, the peak 6 is ginsenoside F5, the peak 7 is ginsenoside Rb1, the peak 8 is ginsenoside Rc, the peak 9 is ginsenoside Rb2, the peak 10 is ginsenoside F1, the peak 11 is ginsenoside Rd, the peak 12 is ginsenoside F4, the peak 13 is 20 (S) -ginsenoside F2 and the peak 14 is ginsenoside Rh 4.
TABLE 2 Components corresponding to characteristic peaks
Experimental example 1 determination of chromatographic conditions
1.1 gradient elution procedure
The same batch of ginseng leaf formula particles was taken, 4 parts of test solution was prepared according to the "preparation of test solution" item in example 1, the gradient elution procedure was the same as example 1 except that the gradient elution procedure was different, and the results are shown in the following table of fig. 3:
TABLE 3 gradient elution procedure 1
| Time (min) | Flow rate (ml/min) | Mobile phase a (%) | Mobile phase B (%) |
| 0～10 | 1 | 5→20 | 95→80 |
| 10～20 | 1 | 20→25 | 80→75 |
| 20～35 | 1 | 25→35 | 75→65 |
| 35～45 | 1 | 35→80 | 65→20 |
| 45～50 | 1 | 80→90 | 20→10 |
| 40～45 | 1 | 90 | 10 |
TABLE 4 gradient elution procedure 2
| Time (min) | Flow rate (ml/min) | Mobile phase a (%) | Mobile phase B (%) |
| 0～10 | 1.0 | 5→20 | 95→80 |
| 10～18 | 1.0 | 20→22 | 80→78 |
| 18～20 | 1.0 | 22→31 | 78→69 |
| 20～30 | 1.0 | 31→33 | 69→67 |
| 30～50 | 1.0 | 33→80 | 67→20 |
TABLE 5 gradient elution procedure 3
| Time (min) | Flow rate (ml/min) | Mobile phase a (%) | Mobile phase B (%) |
| 0～10 | 1.0 | 5→20 | 95→80 |
| 10～18 | 1.0 | 20→22 | 80→78 |
| 18～20 | 1.0 | 22→31 | 78→69 |
| 20～30 | 1.0 | 31→33 | 69→67 |
| 30～40 | 1.0 | 33→60 | 67→40 |
| 40～45 | 1.0 | 60→90 | 40→10 |
From the above results, it can be seen that the gradient elution procedure 1 was unable to effectively separate the characteristic peaks due to the difference in the degree of separation between the characteristic peaks between 20 and 30 minutes. The gradient elution procedures 2 and 3 have good separation degree between characteristic peaks of the obtained atlas, and can be used for constructing ginseng leaf characteristic atlas, but compared with the gradient elution procedures 2 and 3, the gradient elution procedure 3 has short time, early peak-out time and better gradient elution procedure 3.
1.2 flow Rate
Taking the same batch of ginseng leaf formula particles, preparing 3 parts of test solution according to the preparation of the test solution in the embodiment 1, and obtaining characteristic maps of different test solutions according to the measurement item in the embodiment 1 by taking the flow rate as a variable; the flow rates were 0.8ml/min, 1.0ml/min and 1.2ml/min, respectively, and the separation of chromatographic peaks and the system adaptability parameters were detected as evaluation indexes, and the results are shown in the following table and FIG. 4.
TABLE 6 sample patterns obtained at various flow rates
From the above results, it can be seen that the retention time and peak area of each characteristic peak are affected by different flow rates, and when the flow rate is 1.0ml/min, the separation effect of the No. 7 peak, the No. 11 peak and the surrounding small peaks is better, and the flow rate is 1.0ml/min, which is the optimal scheme of the flow rate.
1.3 column temperature
Taking the same batch of ginseng leaf formula particles, preparing 3 parts of test solution according to the preparation of the test solution in the embodiment 1, and obtaining characteristic patterns of different test solutions according to the measurement item in the embodiment 1 by taking column temperature as a variable; the column temperatures were 20 ℃,25 ℃ and 30 ℃ respectively, and the separation conditions of chromatographic peaks and the system adaptability parameters were detected as evaluation indexes, and the results are shown in the following table and fig. 5.
TABLE 7 spectra of test samples obtained at various column temperatures
From the above results, it can be seen that peak No. 7 is greatly disturbed when the column temperature is 30 ℃; when the column temperature is 25 ℃, the separation effect of the No. 7 peak and the No. 12 peak is better, and the separation condition of the characteristic peak and the system adaptability parameter are better. Thus, 25 ℃ is the optimal column temperature.
1.4 mobile phase B
Taking the same batch of ginseng leaf formula particles, preparing 3 parts of test solution according to the preparation of the test solution in the embodiment 1, and obtaining characteristic patterns of different test solutions according to the measurement item in the embodiment 1 by taking a mobile phase B as a variable; the mobile phases were acetonitrile-0.1% phosphoric acid aqueous solution, acetonitrile-0.1% formic acid aqueous solution, acetonitrile-0.1% acetic acid aqueous solution, respectively, and the separation of chromatographic peaks and the system adaptability parameters were detected as evaluation indexes, and the results are shown in the following table and fig. 6. As a result, it can be seen that phosphoric acid was used as the mobile phase B, the obtained spectrum had a relatively smooth base line, and the separation degree between characteristic peaks of the No. 5 peak, the No. 11 peak and the No. 13 peak was excellent. Thus, acetonitrile was used as mobile phase a and aqueous phosphoric acid as mobile phase B.
TABLE 8 spectra of test samples obtained from different mobile phases B
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1.5 Mobile phase B concentration
Taking the same batch of ginseng leaf formula particles, preparing 3 parts of test solution according to the preparation of the test solution in the embodiment 1, and obtaining different test solution characteristic maps according to the measurement item in the embodiment 1 by taking the concentration of the mobile phase B as a variable; mobile phase B concentrations were 0.05%, 0.1% and 0.2%, respectively, to detect separation of chromatographic peaks and system adaptation parameters as evaluation indicators, the results are shown in the following table and fig. 7. As a result, it was found that the symmetry of the obtained spectrum was excellent with 0.1% phosphoric acid aqueous solution as mobile phase B, and 0.1% phosphoric acid aqueous solution was preferable as mobile phase B.
TABLE 9 spectra of samples obtained from mobile phases B at different concentrations
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1.6 chromatography column
Taking the same batch of ginseng leaf formula particles, preparing 3 parts of test solution according to the preparation of the test solution in the embodiment 1, and obtaining characteristic patterns of different test solutions according to the measurement item in the embodiment 1 by taking a chromatographic column as a variable; the columns were (1)Agilent ZORBAX SB-Aq, 4.6mm.times.150mm, 3.5 μm; (2) CAPCELL CORE C18,4.6 mm. Times.150 mm,2.7 μm; (3) CORTECS T3,4.6 mm. Times.150 mm,2.7 μm. The results are shown in the following table and FIG. 8.
TABLE 10 retention time of the spectra of the test samples obtained on different chromatographic columns
TABLE 11 retention time versus peak area of the spectra of the test samples obtained with different chromatographic columns
From the above results, it can be seen that the separation effect of the spectrum obtained by CORTECS T3,4.6 mm. Times.150 mm,2.7 μm as a column is optimal, and therefore, the column is preferable in the present invention.
The effect of different batch chromatographic columns (CORTECS T3,4.6 mm. Times.150 mm,2.7 μm) on the spectra was further examined. The same batch of ginseng leaf formula particles is taken, 3 parts of test sample solutions are prepared according to the test sample solution preparation in the embodiment 1, three chromatographic columns with different batch numbers (marked as a column 1, a column 2 and a column 3) are adopted, and characteristic patterns of different test sample solutions are obtained according to the measurement item in the embodiment 1, and the results are shown in the following table and fig. 9.
TABLE 12 comparison of the results of relative retention times for different lots of columns
TABLE 13 comparison of the results of the relative peak areas for different lots of columns
As can be seen from the above results, the deviation of the relative retention time of each characteristic peak is less than 2%, the characteristic spectrum result does not change with the batch number of the chromatographic column, and the retention time of each characteristic chromatographic peak does not change much, so that the method is recommended to be preferably used as a chromatographic column for subsequent research and investigation, namely, a chromatographic column CORTECS T3,4.6mm multiplied by 150mm and 2.7 mu m.
In summary, the optimal chromatographic conditions are determined: octadecylsilane chemically bonded silica is used as filler (column length is 150mm, inner diameter is 4.6mm, and particle diameter is 2.7 μm); acetonitrile as mobile phase a,0.1% phosphoric acid as mobile phase B, gradient elution procedure: 0-10min, 5-20% mobile phase A, 95-80% mobile phase B;10-18min, 20-22% mobile phase A, 80-78% mobile phase B;18-20min, 22-31% mobile phase A, 78-69% mobile phase B;20-30min, 31-33% mobile phase A, 69-67% mobile phase B;30-40min, 33-60% mobile phase A, 67-40% mobile phase B;40-45min, 60-90% mobile phase A, 40-10% mobile phase B; the detection wavelength is 203nm; the theoretical plate number is not lower than 1500 calculated according to ginsenoside Re.
Experimental example 2 determination of preparation method of sample solution
2.1 extraction method of sample solution
Taking the same batch of ginseng leaf formula particles, extracting test samples by adopting different methods, and obtaining characteristic patterns of different test sample solutions according to the measurement item in the embodiment 1; the first method is the same as in example 1, "preparation of test sample solution"; the second method adopts reflux extraction, and specifically comprises the following steps: taking a proper amount of the product, grinding, taking about 0.5g, placing into a conical flask with a plug, adding 25ml of 80% methanol, heating and refluxing for 30min, cooling, filtering, and taking the subsequent filtrate to obtain a sample solution. The results are shown in the following table and fig. 10.
TABLE 14 spectra obtained by different sample solution extraction modes
From the above results, ultrasonic extraction and reflux extraction have little influence on the feature map, and the ultrasonic extraction method is preferable in view of the simplicity of the experimental method.
2.2 extraction time
Taking the same batch of ginseng leaf formula particles, preparing 4 parts of test solution according to the preparation of the test solution in the embodiment 1 by taking ultrasonic treatment time as a variable, and obtaining characteristic patterns of different test solutions according to the measurement item in the embodiment 1; the ultrasonic time was 15min, 30min, 45min and 60min, respectively. The results are shown in FIG. 11. As can be seen from the experimental results, the total peak area of the characteristic peaks is not greatly different with the extension of the extraction time, and each characteristic component is completely extracted within this range, and it is preferable that 30 minutes is taken as the extraction time in consideration of the extraction efficiency.
2.3 ultrasonic Power
Taking the same batch of ginseng leaf formula particles, preparing 3 parts of test solution according to the preparation of the test solution in the embodiment 1, and obtaining characteristic maps of different test solutions according to the measurement item in the embodiment 1 by taking ultrasonic power as a variable; the ultrasonic power is 200W, 250W and 300W respectively. During the experiment, it was found that the ultrasonic power had little effect on the experimental results.
2.4 extraction solvent
Taking the same batch of ginseng leaf formula particles, preparing 3 parts of test solution according to the preparation of the test solution in the embodiment 1 by taking an extraction solvent as a variable, and obtaining characteristic patterns of different test solutions according to the measurement item in the embodiment 1; in preparing the test sample solutions, the extraction solvents were water, methanol and ethanol, respectively, and the results are shown in the following table and fig. 12. As can be seen from fig. 12, methanol is used as the extraction solvent, the obtained characteristic spectrum has a relatively stable base line, less interference of impurity peaks, and methanol is preferred as the extraction solvent.
TABLE 15 spectra of samples obtained from different extraction solvents
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2.5 concentration of extraction solvent
Taking the same batch of ginseng leaf formula particles, preparing 4 parts of test solution according to the preparation of the test solution in the embodiment 1 by taking the concentration of an extraction solvent as a variable, and obtaining characteristic maps of different test solutions according to the measurement item in the embodiment 1; in preparing the test sample solutions, the extraction solvent concentrations were 40% aqueous methanol solution, 60% aqueous methanol solution, 80% aqueous methanol solution and methanol, respectively, and the results are shown in the following table and fig. 13.
TABLE 16 retention time and peak area of sample solution patterns obtained at different extraction solvent concentrations
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From the above results and fig. 13, it can be seen that there is no significant difference in peak area of characteristic peaks in characteristic patterns obtained from aqueous methanol solutions of different concentrations, and that 80% methanol is preferable as an extraction solvent for the sample solution in combination with the dissolution effect of ginseng leaf formula particles.
2.6 extraction solvent volume
Taking the same batch of ginseng leaf formula particles, preparing 3 parts of test solution according to the preparation of the test solution in the embodiment 1 by taking the volume of an extraction solvent as a variable, and obtaining characteristic patterns of different test solutions according to the measurement item in the embodiment 1; when preparing the test solution, the volumes of the extraction solvents are respectively 10ml, 25ml and 50ml, and the result shows that the response value of each chromatographic peak exhibited by about 0.5g of test formulation particles corresponding to 25ml of 80% methanol extraction is relatively good, so that the test selects the volume of the extraction solvent to be 25ml for subsequent investigation.
2.7 sample amount of sample
Taking the same batch of ginseng leaf formula particles, taking the sampling amount of a sample to be tested as a variable, preparing 5 parts of sample solution according to the preparation of the sample solution in the embodiment 1, and obtaining characteristic maps of different sample solutions according to the measurement item in the embodiment 1; in the preparation of the sample solution, the sample amounts were 0.20g, 0.40g, 0.50g, 0.60g and 0.80g, respectively. As a result, as the sample amount of the sample increases, the cover level of each characteristic peak exhibits a corresponding multiple increase, indicating that each characteristic peak is completely extracted. When the sampling amount is 0.5g, the peak height and peak width of the characteristic peak are relatively moderate, and 0.5g is preferable as the sampling amount of the sample.
In summary, the optimal preparation method of the sample solution is determined, specifically, when the sample is a formulation granule or a standard decoction lyophilized powder, the preparation method comprises: grinding the above materials, collecting about 0.5g, placing into conical flask with plug, adding 80% methanol 25ml, sealing, ultrasonic treating (power 250W, frequency 40 kHz) for 30min, cooling, filtering, and collecting filtrate. When the test sample is a medicinal material or decoction piece, the preparation method comprises the following steps: about 1.5g of the sample is taken and put into a conical flask with a plug, 50ml of water is added, heating reflux is carried out for 1h, filtering is carried out, filtrate is evaporated to dryness, 25ml of 80wt% methanol is added into residues, sealing is carried out, ultrasonic treatment is carried out for 30min, the power is 250W, the frequency is 40kHz, cooling is carried out, shaking is carried out, filtering is carried out, and subsequent filtrate is taken, thus obtaining the product.
Experimental example 3 chromatographic conditions and System applicability
(1) HPLC analysis was performed according to the test solution preparation method determined in Experimental example 2 and the chromatographic conditions determined in Experimental example 1, and the chromatographic conditions and system suitability of the ginseng leaf formula particles were verified, and chromatograms were recorded as shown in the following Table and FIG. 14.
Table 17 system adaptation profile
| Sequence number | Retention time | Area of | % area | Height | Width of (L) | Degree of separation | s/n | Symmetry factor | Number of theoretical plates of USP |
| 1 | 6.819 | 644723 | 5.49 | 135937 | 14 | 31.42 | 1.6 | 49552 | |
| 2 | 11.701 | 3638637 | 30.97 | 1063977 | 9.8 | 46.89 | 245.96 | 1.3 | 274628 |
| 3 | 20.546 | 1061897 | 9.04 | 166729 | 29 | 71.38 | 38.54 | 1.1 | 256212 |
| 4 | 20.914 | 2221159 | 18.9 | 494197 | 17.9 | 2.72 | 114.24 | 1.15 | 541605 |
| 5 | 23.187 | 150318 | 1.28 | 38339 | 14.1 | 22.15 | 8.86 | 1.55 | 899827 |
| 6 | 26.196 | 250188 | 2.13 | 41948 | 28 | 25.22 | 9.7 | 1.39 | 515684 |
| 7 | 26.926 | 646493 | 5.5 | 95148 | 15 | 4.38 | 22 | 0.98 | 334842 |
| 8 | 28.303 | 338909 | 2.88 | 44535 | 29.4 | 7.19 | 10.3 | 1 | 316606 |
| 9 | 30.03 | 424758 | 3.61 | 47736 | 29.9 | 8.15 | 11.03 | 0.91 | 246521 |
| 10 | 30.912 | 377049 | 3.21 | 44063 | 28.1 | 3.91 | 10.19 | 1.18 | 307452 |
| 11 | 33.474 | 1228445 | 10.45 | 171306 | 31.5 | 12.79 | 39.6 | 0.85 | 453028 |
| 12 | 35.924 | 86882 | 0.74 | 25193 | 13.8 | 18.62 | 5.82 | 1.37 | 2807559 |
| 13 | 36.867 | 520585 | 4.43 | 132926 | 20 | 10.49 | 30.73 | 1.26 | 2271068 |
| 14 | 37.289 | 160746 | 1.37 | 44392 | 16.2 | 4.6 | 10.26 | 1.91 | 2834581 |
From the results, the separation degree of each characteristic peak is larger than 1.5, and the number of theoretical plates of the characteristic peak is larger than 1500, which indicates that the method has good system adaptability and can be used as a detection method of the characteristic spectrum of the ginseng leaf formula particle.
(2) Negative samples were prepared according to the test sample solution preparation method determined in experimental example 2, and HPLC analysis was performed according to the chromatographic conditions determined in experimental example 1, and the results are shown in FIG. 15. The result shows that the negative sample has no interference to the spectrum, and the method has good system adaptability and specificity, and can be used as a detection method of the characteristic spectrum of the ginseng leaf formula particle. Wherein the preparation method of the negative sample comprises the following steps: the auxiliary material maltodextrin dry preparation is used for obtaining a negative sample, grinding the negative sample, taking about 0.5g, placing the negative sample into a conical bottle with a plug, adding 25ml of 80% methanol, sealing, performing ultrasonic treatment for 30min at the power of 250W and the frequency of 40kHz, cooling, filtering, and taking a subsequent filtrate to obtain the negative sample.
Experimental example 4 analytical method verification
4.1 precision of instrument
Preparation of sample solution according to "preparation of sample solution" in example 1, sample injection was repeated 6 times according to "measurement" item in example 1, and the relative retention time and relative peak area of each characteristic peak were measured, which revealed that the relative retention time RSD of each characteristic peak was less than 2%, and the relative peak area RSD of each characteristic peak was less than 2% except for peak 14. Indicating that the instrument has good precision.
4.2 repeatability experiments
Taking the same batch of ginseng leaf formula particles, preparing 6 parts of test solution according to the preparation of the test solution in the embodiment 1, and obtaining characteristic patterns of different test solutions according to the measurement item in the embodiment 1; the relative retention time and relative peak area of each characteristic peak were measured, and the result showed that the relative retention time RSD of each characteristic peak was less than 2%, and the relative peak area RSD of each characteristic peak was less than 2% except for peak 14. The results indicate that the reproducibility of the process is good.
4.3 different operator precision
Taking the same batch of ginseng leaf formula particles at different times by three different inspectors respectively, preparing test solution according to the test solution preparation in the embodiment 1, and obtaining characteristic patterns of different test solutions by adopting the same equipment according to the test item in the embodiment 1; the relative retention time and relative peak area of each characteristic peak were measured, and the results showed that the relative retention time and relative peak area of each characteristic peak were less than 2%, except for peak 14. Indicating that the intermediate precision of the method is good.
4.4 different intermediate precision of high Performance liquid chromatography
Taking the same batch of ginseng leaf formula particles at different times by the same inspector, preparing 3 parts of test solution according to the preparation of the test solution in the embodiment 1, and obtaining characteristic patterns of different test solutions according to the measurement item in the embodiment 1 by taking a high performance liquid chromatograph as a variable; the relative retention time and the relative peak area of each characteristic peak are measured, wherein three high performance liquid chromatographs are respectively: waters e2695, shimadzu, dyan UltiMate 3000. From the results, it can be seen that the relative retention time of each characteristic peak has a relative deviation of less than 3%, but the relative peak area of the characteristic peak is greatly affected by the instrument. Indicating that the relative retention times of the characteristic peaks are less affected by the different instruments.
4.5 stability investigation
Taking the same batch of ginseng leaf formula particles, preparing 6 parts of test solution according to the preparation of the test solution in the embodiment 1, respectively placing for 0h, 4h, 8h, 12h, 18h and 24h at room temperature, obtaining a characteristic map of the test solution according to the measurement item in the embodiment 1, calculating the relative peak area and the relative retention time, calculating RSD, and indicating that the relative retention time RSD of each characteristic peak is less than 2 percent, and the relative peak area RSD of each characteristic peak is less than 2 percent except for the peak 14, so that the test solution is stable within 24h and meets the measurement requirement.
Example 2
The embodiment provides a method for identifying ginseng leaves, which comprises the following steps:
preparing a test sample solution from a test sample according to the method provided in example 1;
the test solution is detected according to the 'determination' item of the embodiment 1 to obtain a characteristic map of the test solution, firstly observing whether a No. 3 peak and a No. 14 peak appear, if so, adopting the following formula to identify, and if a is 0.43-1.5, the test solution is ginseng leaves, and if not, indicating that the test solution is not ginseng leaves.
S Peak No. 11 Peak area of No. 11 peak, S Peak 3 Peak area of peak No. 3.
Verification
The ginseng, ginseng flower, ginseng stem and American ginseng leaf are prepared into test solution according to example 1, and then detected according to the "measurement" item of example 1, and the characteristic patterns of the ginseng, ginseng flower, ginseng stem and American ginseng leaf are obtained, see fig. 16.
As can be seen from fig. 16, there are no peaks No. 12 and no peak No. 14 in the ginseng flower characteristic map, and the response values of peaks No. 1, no. 2 and No. 3 are lower than those of ginseng leaves; the peak area ratio of peak 11 to peak 3 is 1.54-7.61.
The characteristic patterns of the ginseng stems have no peaks 6, 8, 9, 10 and 12, and the response values of the peaks 1, 2, 3,4, 5, 7, 11, 13 and 14 are lower than those of the ginseng leaves, which indicates that the content of the components corresponding to the characteristic peaks in the ginseng stems is lower. The peak area ratio of peak 11 to peak 3 is 0.12-0.40.
The ginseng characteristic spectrum has no No. 2 peak, no. 13 peak and No. 14 peak, and the response value of each characteristic peak is lower than that of the ginseng leaf sample. The peak area ratio of peak 11 to peak 3 is 0.15-0.20.
No. 5 peak, no. 6 peak and No. 13 peak are absent in the characteristic spectrum of American ginseng leaf. The peak area ratio of peak 11 to peak 3 is 3.36-6.67.
The peak area ratio of No. 11 peak to No. 3 peak in the characteristic spectrum of ginseng leaf is 0.57-1.16, and there is no intersection with the range of the relative peak area ratio of ginseng flower, ginseng stem, ginseng and American ginseng leaf, and obvious difference exists.
Multiplying the upper limit of the ginseng leaf range of 0.57-1.16 by a coefficient of 1.3, namely 1.16x1.3, wherein the upper limit of the specified range is 1.5; the ginseng leaf is discriminated from other parts, american ginseng leaf, etc. by multiplying the lower limit of the ginseng leaf range of 0.57-1.16 by a coefficient of 0.75, i.e., 0.57×0.75, thus defining the peak area ratio of the No. 11 peak to the No. 3 peak in the ginseng leaf characteristic map of 0.43-1.5.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (10)
1. The construction method of the ginseng leaf characteristic map is characterized by comprising the following steps of:
preparation of test solution: preparing a sample solution from a sample;
preparation of reference solution: preparing reference solution from reference materials;
and (3) measuring: according to high performance liquid chromatography;
wherein the chromatographic conditions of the high performance liquid chromatography include: acetonitrile is taken as a mobile phase A, phosphoric acid aqueous solution is taken as a mobile phase B, and gradient elution is carried out;
the gradient elution procedure included: 0-10min, 5-20% mobile phase A, 95-80% mobile phase B;10-18min, 20-22% mobile phase A, 80-78% mobile phase B;18-20min, 22-31% mobile phase A, 78-69% mobile phase B;20-30min, 31-33% mobile phase A, 69-67% mobile phase B;
the gradient elution procedure further comprises: 30-40min, 33-60% mobile phase A, 67-40% mobile phase B;40-45min, 60-90% mobile phase A, 40-10% mobile phase B; or 30-50min, 33-80% mobile phase A, 67-20% mobile phase B.
2. The method of claim 1, wherein the chromatographic conditions of the high performance liquid chromatography further comprise: octadecylsilane chemically bonded silica gel column is used as filler, and the specification of the chromatographic column is as follows: the inner diameter is 4.6mm, the column length is 150mm, and the grain diameter is 2.7 mu m; and/or the number of the groups of groups,
the column temperature is 23-27 ℃; and/or the number of the groups of groups,
the wavelength is 201-205nm; and/or the number of the groups of groups,
the sample injection amount is 5-15 mu L; and/or the number of the groups of groups,
the flow rate is 0.9-1.1ml/min; and/or the number of the groups of groups,
taking 0.08-0.12% phosphoric acid aqueous solution as mobile phase B.
3. The construction method according to claim 1 or 2, wherein the test substance is at least one of ginseng leaf decoction pieces, ginseng leaf formulation granules, ginseng leaf standard decoction freeze-dried powder, ginseng leaf extract powder and ginseng leaf medicinal materials.
4. A method of constructing as claimed in any one of claims 1 to 3 wherein the method of preparing a test solution comprises: extracting the sample with solvent, and filtering;
preferably, in preparing the sample solution, the extraction solvent is at least one of water, methanol and ethanol;
preferably, the extraction solvent is methanol;
preferably, in preparing the sample solution, the extraction method is reflux extraction or ultrasonic extraction.
5. The method according to claim 4, wherein when the sample is at least one of ginseng leaf formulation particles, ginseng leaf standard decoction freeze-dried powder and ginseng leaf extract powder, the ratio of the mass of the sample to the volume of the extraction solvent is 1g (45-55) ml when the sample solution is prepared.
6. The method of constructing a solution according to any one of claims 1 to 5, wherein the method of preparing the reference solution comprises: extracting reference materials with water, filtering, and evaporating to dryness to obtain water extract; adding extraction solvent into the water extract, extracting, and filtering;
preferably, in preparing the reference solution, the extraction solvent is at least one of water, methanol and ethanol;
preferably, the extraction solvent is methanol;
preferably, the ratio of the quality of the reference medicinal material to the volume of the extraction solvent is 1g (15-19 ml);
preferably, in preparing the reference solution, the extraction method is reflux extraction or ultrasonic extraction.
7. The method of any one of claims 1-6, further comprising preparing a reference solution of a control;
the reference substance is at least one of kaempferol-3-O-sophoroside reference substance, ginsenoside Rg1 reference substance, ginsenoside Re reference substance and ginsenoside Rd reference substance.
8. The method according to any one of claims 1 to 7, wherein the profile obtained by the method comprises 14 characteristic peaks;
the relative retention time of peak No. 1, with peak No. 2 as reference peak No. 1, was defined as: 0.58;
the relative retention time values of the reference peaks No. 2, no. 5, no. 6, no. 7, no. 8, no. 9, no. 10, no. 12, no. 13 and No. 14 were respectively: 1.11, 1.25, 1.29, 1.35, 1.43, 1.48, 1.72, 1.76, and 1.79;
wherein the relative retention time of the characteristic peak is within + -10% of the prescribed value.
9. A method for identifying ginseng leaves, which is characterized in that the construction method of any one of claims 1-8 is adopted to obtain a characteristic spectrum of a to-be-detected sample, and the areas of a No. 3 peak and a No. 11 peak are utilized to identify whether the to-be-detected sample is ginseng leaves.
10. The method of claim 9, wherein when a is 0.43-1.5, the sample to be tested is ginseng leaf;
wherein,
S peak No. 11 Peak area of No. 11 peak, S Peak 3 Peak area of peak No. 3.
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