CN113514576B - Method for establishing fingerprint of bupleurum medicinal material, extract and single preparation - Google Patents
Method for establishing fingerprint of bupleurum medicinal material, extract and single preparation Download PDFInfo
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- G—PHYSICS
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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
- G01N30/02—Column chromatography
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—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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- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
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Abstract
The invention relates to the technical field of bupleurum medicinal material quality analysis, in particular to a method for establishing fingerprint patterns of bupleurum medicinal materials, extracts and single preparations. The method for establishing the fingerprint of the bupleurum medicinal material, the extract and the single preparation comprises the following steps: detecting the sample solution by adopting an HPLC-CAD method to obtain a fingerprint spectrum containing 22 common characteristic peaks; the detection conditions of the HPLC-CAD method comprise: and (3) carrying out gradient elution by taking formic acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B. The invention establishes the HPLC-CAD fingerprint spectrum measuring method of the bupleurum medicinal material, the extract and the single preparation, realizes the good separation of chromatographic peaks, and 22 common characteristic peaks not only contain the components with ultraviolet absorption, but also contain the components with weak ultraviolet absorption and the components without ultraviolet absorption, thereby being capable of more comprehensively representing the material composition and the relative content of the bupleurum medicinal material, the extract and the single preparation.
Description
Technical Field
The invention relates to the technical field of bupleurum medicinal material quality analysis, in particular to a method for establishing fingerprint patterns of bupleurum medicinal materials, extracts and single preparations.
Background
Radix bupleuri is dry root of Bupleurum chinense of Umbelliferae Bupleurum chinense DC or Bupleurum scorzonerifolium Bupleurum scorzonerifolium willd, and is collected in spring and autumn to remove stem and leaf and silt, and dried. The bupleurum medicinal materials are mainly produced in mountain western, shaanxi, gansu, hebei and other places, and have the effects of dispelling heat, soothing liver, relieving depression, lifting yang qi and the like.
The bupleurum contains mainly saponin, flavone, volatile oil, polysaccharide and other components, and the quality of bupleurum is evaluated with saikosaponin at present, and under the medicine item of bupleurum in Chinese pharmacopoeia, saikosaponin a and saikosaponin d are used as content control indexes and no fingerprint method exists. The traditional Chinese medicine has complex components, the clinical efficacy is the result of the combined action of the chemical components, the chemical components directly influence the quality of the medicinal materials, and the content measurement of one component and two components hardly truly reflects the quality of the traditional Chinese medicine, so that a fingerprint spectrum method is required to be established for analyzing the whole chemical components of the traditional Chinese medicine, the quality of the medicinal materials is scientifically and comprehensively evaluated, and a scientific basis is provided for the clinical application of the traditional Chinese medicine.
At present, the researches on the fingerprints of bupleurum medicinal materials in literature mostly adopt Ultraviolet (UV) or Evaporative Light Scattering (ELSD) detectors. The UV detector can only detect the ultraviolet absorbing component, and the chromophore-free substance cannot be detected without a response; although ELSDs are general-purpose detectors, due to the disadvantages of low detection sensitivity, poor reproducibility, etc., it is difficult to achieve good detection results for some components with low content in traditional Chinese medicine. Therefore, a fingerprint detection method of a universal detector with high sensitivity and good reproducibility needs to be established.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a method for establishing fingerprint of bupleurum medicinal materials, extracts and single preparations, which solves the technical problem that the quality of bupleurum medicinal materials cannot be comprehensively represented in the prior art.
In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:
the method for establishing the fingerprint of the bupleurum medicinal material, the extract and the single preparation comprises the following steps:
detecting the sample solution by adopting an HPLC-CAD method to obtain a fingerprint spectrum containing 22 common characteristic peaks;
the detection conditions of the HPLC-CAD method comprise:
carrying out gradient elution by taking formic acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B; the gradient elution includes: 0-20 min, the volume fraction of the mobile phase A is changed from 88-92% to 68-72%; 20-35 min, the volume fraction of the mobile phase A is changed from 68-72% to 63-67%; the volume fraction of the mobile phase A is changed from 63 to 67 percent to 58 to 62 percent within 35 to 60 minutes; 60-65 min, the volume fraction of the mobile phase A is changed from 58-62% to 53-57%; 65-70 min, the volume fraction of the mobile phase A is 53-57%; 70-85 min, and changing the volume fraction of the mobile phase A from 53-57% to 33-37%; 85-90 min, the volume fraction of the mobile phase A is changed from 33-37% to 13-17%; 90-100 min, the volume fraction of the mobile phase A is changed from 13-17% to 8-12%; 100-105 min, the volume fraction of the mobile phase A is changed from 8-12% to 3-7%.
In a specific embodiment of the present invention, the gradient elution comprises: 0-20 min, wherein the volume fraction of the mobile phase A is changed from 90% to 70%; 20-35 min, the volume fraction of the mobile phase A is changed from 70% to 65%; 35-60 min, the volume fraction of the mobile phase A is changed from 65% to 60%; 60-65 min, the volume fraction of the mobile phase A is changed from 60% to 55%; 65-70 min, wherein the volume fraction of the mobile phase A is 55%; 70-85 min, wherein the volume fraction of the mobile phase A is changed from 55% to 35%; 85-90 min, the volume fraction of the mobile phase A is changed from 35% to 15%; 90-100 min, the volume fraction of the mobile phase A is changed from 15% to 10%; 100-105 min, the volume fraction of the mobile phase A is changed from 10% to 5%.
According to the invention, 22 common characteristic peaks are provided in the fingerprint of the bupleurum medicinal material, wherein the common characteristic peaks not only contain ultraviolet absorption components (such as flavonoids and the like) but also include weak ultraviolet absorption components (such as saponins and the like) and partial non-ultraviolet absorption components, so that the quality of the bupleurum medicinal material can be comprehensively represented and evaluated through multiple types of components, a scientific basis is provided for full utilization of bupleurum resources, and the problems of single quality evaluation index and the like of the bupleurum medicinal material are solved. The method also has the advantages of high sensitivity, good reproducibility and the like.
In a specific embodiment of the present invention, further comprising: detecting the reference substance solution by adopting the HPLC-CAD method; the reference substance comprises any one or more of saikosaponin a, saikosaponin c, saikosaponin d, saikosaponin e and saikosaponin f. Preferably, the reference substances comprise saikosaponin a, saikosaponin c, saikosaponin d, saikosaponin e and saikosaponin f.
In a specific embodiment of the present invention, the volume fraction of formic acid in the formic acid aqueous solution is 0.05% to 0.15%. Further, the mobile phase A is a formic acid aqueous solution with the volume fraction of 0.1%.
In a specific embodiment of the present invention, the detection flow rate is 1.0 to 1.5mL/min, preferably 1.4mL/min.
As in the various embodiments, the detection flow rate may be 1.0mL/min, 1.1mL/min, 1.2mL/min, 1.3mL/min, 1.4mL/min, 1.5mL/min, and so forth.
By adopting the detection flow velocity, the chromatographic peak separation degree and the detection efficiency can be further improved.
In a specific embodiment of the present invention, the column temperature of the chromatographic column is 30 to 40 ℃, preferably 40 ℃.
As in various embodiments, the column temperature of the chromatographic column may be 30 ℃, 32 ℃, 34 ℃, 35 ℃, 36 ℃, 38 ℃,40 ℃, etc.
In a specific embodiment of the invention, the HPLC-CAD method employs a C18 column, preferably a Agilent ZORBAX SB-C18 column (150X 4.6mm,3.5 μm).
In a specific embodiment of the present invention, the detection conditions of the HPLC-CAD method include:
the mobile phase A is formic acid aqueous solution with the volume fraction of 0.1%, and the mobile phase B is acetonitrile;
the chromatographic column was Agilent ZORBAX SB-C18 (150X 4.6mm,3.5 μm);
column temperature is 40 ℃;
the flow rate is 1.4mL/min;
the temperature of the CAD drift tube is 35 ℃ or 50 ℃, and the gas flow rate is 2.2-2.6L/min.
As in the various embodiments, the gas flow rate may be 2.2L/min, 2.3L/min, 2.4L/min, 2.5L/min, 2.6L/min, etc., such as 2.4L/min, among other CAD parameters.
In a specific embodiment of the present invention, the amount of sample introduced is 10 to 20. Mu.L, preferably 20. Mu.L.
In a specific embodiment of the present invention, the method for preparing a sample solution includes:
when the substance to be detected is bupleurum medicinal material, the bupleurum medicinal material is subjected to ultrasonic extraction or reflux extraction by taking methanol aqueous solution as an extraction solvent, the extraction solvent is used for supplementing the weight of the bupleurum medicinal material after loss, and the bupleurum medicinal material is filtered, and the subsequent filtrate is taken as a solution of the sample.
For the bupleurum extract in solid or semisolid dosage form, the preparation method of the sample solution of the fingerprint spectrum of the bupleurum single preparation is the same as that of the bupleurum medicinal material. The method specifically comprises the following steps: and (3) performing ultrasonic extraction or reflux extraction on the bupleurum extract or the bupleurum single preparation by taking a methanol aqueous solution as an extraction solvent, supplementing the reduced weight by the extraction solvent, and filtering to obtain a subsequent filtrate as a sample solution.
For the bupleurum extract and bupleurum single preparation in liquid dosage form, the liquid extract or the liquid preparation can be directly taken as a sample solution for analysis.
In the specific embodiment of the invention, the bupleurum single preparation comprises any one of bupleurum injection, bupleurum oral liquid, bupleurum dripping pill and bupleurum cough relieving tablet. Wherein, the bupleurum drop pill and bupleurum cough relieving tablet are solid preparations, and the bupleurum injection and bupleurum oral liquid are liquid preparations.
The following uses bupleurum medicinal materials as examples to describe the preparation conditions of the sample solution:
in a specific embodiment of the present invention, the ratio of the bupleurum medicinal material to the extraction solvent is 0.5 g/25 mL-1.5 g/25 mL, preferably (0.8-1.2) g/25 mL, such as 1.0 g/25 mL.
In various embodiments, the ratio of the bupleurum medicinal material to the extraction solvent may be 0.5 g/25 mL, 0.6 g/25 mL, 0.7 g/25 mL, 0.8 g/25 mL, 0.9 g/25 mL, 1.0 g/25 mL, 1.1 g/25 mL, 1.2 g/25 mL, 1.3 g/25 mL, 1.4 g/25 mL, 1.5 g/25 mL, etc.
In a specific embodiment of the invention, the extraction mode is ultrasonic extraction. Further, the power of the ultrasonic wave is 300-500W, and the frequency of the ultrasonic wave is 40kHz.
In a specific embodiment of the invention, the extraction time is 30 to 90min, preferably 30 to 60min, such as 30min.
As in the various embodiments, the extraction time may be 30min, 45min, 60min, 75min, 90min, etc.
In a specific embodiment of the present invention, the volume fraction of methanol in the aqueous methanol solution is 70% to 90%, preferably 80%.
In actual operation, the bupleurum medicinal material in powder form is adopted for extraction. Further, the powdery bupleurum medicinal material is powder which is sieved by a No. three sieve.
In a specific embodiment of the present invention, the preparation method of the reference solution includes: precisely weighing saikosaponin a, saikosaponin c, saikosaponin d, saikosaponin e and saikosaponin f, and dissolving the saikosaponin a, the saikosaponin c, the saikosaponin d, the saikosaponin e and the saikosaponin f in a methanol aqueous solution with the volume fraction of 70-90 percent; in the reference solution, the concentrations of saikosaponin a, saikosaponin c, saikosaponin d, saikosaponin e and saikosaponin f are respectively 0.1mg/mL, 50 mug/mL, 0.15mg/mL, 20 mug/mL and 50 mug/mL.
In a specific embodiment of the invention, 13 # The peak is a reference peak, and the relative retention time and the relative peak area of the 22 common characteristic peaks are respectively as follows:
peak No. 1: the relative retention time is 0.04-0.05, and the relative peak area is 0.01-0.15;
peak No. 2: the relative retention time is 0.21-0.23, and the relative peak area is 0.01-0.26;
peak No. 3: the relative retention time is 0.23-0.24, and the relative peak area is 0.01-0.13;
peak No. 4: the relative retention time is 0.30-0.31, and the relative peak area is 0.04-0.26;
peak No. 5: the relative retention time is 0.49-0.50, and the relative peak area is 0.08-0.53;
peak No. 6: the relative retention time is 0.51-0.52, and the relative peak area is 0.21-0.60;
peak No. 7: the relative retention time is 0.54-0.55, and the relative peak area is 0.18-0.95;
peak No. 8: the relative retention time is 0.68-0.69, and the relative peak area is 0.92-1.51;
peak No. 9: the relative retention time is 0.76-0.77, and the relative peak area is 0.10-0.42;
peak No. 10: the relative retention time is 0.78-0.79, and the relative peak area is 0.13-0.59;
peak No. 11: the relative retention time is 0.86-0.87, and the relative peak area is 0.07-0.23;
peak No. 12: the relative retention time is 0.93-0.94, and the relative peak area is 0.03-0.33;
peak No. 13: the relative retention time was 1.00 and the relative peak area was 1.00;
peak No. 14: the relative retention time is 1.14-1.16, and the relative peak area is 0.22-0.62;
peak No. 15: the relative retention time is 1.17-1.19, and the relative peak area is 0.32-1.18;
peak No. 16: the relative retention time is 1.32-1.34, and the relative peak area is 0.27-1.35;
peak No. 17: the relative retention time is 1.67-1.70, and the relative peak area is 0.02-0.16;
peak No. 18: the relative retention time is 1.80-1.84, and the relative peak area is 0.02-0.12;
peak No. 19: the relative retention time is 1.85-1.89, and the relative peak area is 0.27-1.93;
peak No. 20: the relative retention time is 1.89-1.93, and the relative peak area is 0.12-0.47;
peak No. 21: the relative retention time is 1.90-1.94, and the relative peak area is 0.05-0.90;
peak No. 22: the relative retention time is 1.98-2.02, and the relative peak area is 0.03-0.20.
In a specific embodiment of the present invention, in the fingerprint, 6 # Peak is saikosaponin c, 7 # Peak is saikosaponin f, 8 # The peak is saikosaponin a, 11 # Peak is saikosaponin e, 13 # The peak is saikosaponin d.
The invention compares 22 common characteristic peaks in the fingerprint with the reference substance, and identifies chemical components corresponding to 5 common characteristic peaks in the fingerprint.
In the specific embodiment of the invention, 20 batches of bupleurum medicinal materials are detected according to the HPLC-CAD method, chromatograms of the bupleurum medicinal materials are obtained, and the chromatograms are processed by traditional Chinese medicine fingerprint similarity evaluation software to obtain a control map.
In actual operation, the chromatogram of the sample to be detected can be guided into traditional Chinese medicine fingerprint similarity evaluation software, similarity with a control chromatogram is calculated, and quality control is performed on medicinal materials through the similarity.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention establishes an HPLC-CAD fingerprint spectrum measuring method of bupleurum medicinal materials, extracts and single preparations, and realizes good separation of chromatographic peaks;
(2) The fingerprint obtained by the method contains 22 common characteristic peaks, not only contains ultraviolet-absorbing components (such as flavonoids and the like), but also contains weak ultraviolet-absorbing components (such as saponins and the like) and partial non-ultraviolet-absorbing components, and can more comprehensively represent the material composition and relative content of bupleurum medicinal materials, extracts and single preparations.
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 fingerprint of Bupleurum root medicinal material measured in example 1 of the present invention;
FIG. 2 is a chromatogram of a mixed control solution measured in example 1 of the present invention;
FIG. 3 shows chromatograms measured under different conditions according to example 2 of the present invention;
FIG. 4 shows chromatograms measured under different conditions of example 3 of the present invention;
FIG. 5 shows chromatograms measured under different conditions according to example 4 of the present invention;
FIG. 6 is a chromatogram measured under the conditions of example 5 of the present invention;
FIG. 7 is a chromatogram measured under the conditions of example 6 of the present invention;
FIG. 8 is a chromatogram measured under the conditions of example 7 of the present invention;
FIG. 9 is a chromatogram obtained under different conditions of examples 8 and 9 of the present invention;
FIG. 10 is a chromatogram measured in example 10 of the present invention;
FIG. 11 is a graph of 20 batches of bupleurum medicinal material of example 11 of the present invention;
FIG. 12 is a comparative map of Bupleurum root medicinal material of example 11 of the present invention;
FIG. 13 is a chromatogram obtained in comparative example 1;
FIG. 14 is a chromatogram obtained in comparative example 2 at a detection wavelength of 254 nm;
FIG. 15 is a chromatogram obtained in comparative example 2 at a detection wavelength of 280 nm;
fig. 16 is a chromatogram of a hollow white solvent in a specificity test.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and detailed description, but it will be understood by those skilled in the art that the examples described below are some, but not all, examples of the present invention, and are intended to be illustrative of the present invention only and should not be construed as limiting the scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In the specific embodiment, the verification of the items such as specificity, precision, repeatability, stability and the like is carried out according to the guideline related to the annex of the current edition ' pharmacopoeia of the people's republic of China '.
The information of materials, instruments, reagents and the like adopted in the specific embodiment of the invention can be as follows:
materials and reagents: acetonitrile (HPLC, fepmrue); methanol (HPLC, fepmrue); formic acid (AR, MREDA); pure water (MiLLi-Q preparation);
saikosaponin a (110777-201912, national food and drug verification institute); saikosaponin c (PS 000193, dupusi biotechnology Co., ltd.); saikosaponin d (110779-201912, national food and drug verification institute); saikosaponin e (PS 200702-15, dupusi biotechnology Co., ltd.); saikosaponin f (6131, shanghai Shiadad standard technical service Co., ltd.);
the bupleurum medicinal material is purchased in the medicinal material market, and the specific bupleurum medicinal material sample source information is shown in the following table 1;
TABLE 1 bupleurum root medicinal material sample Source information Table
Instrument: high performance liquid chromatograph Uitimate3000 Thermo (CAD detector); agilent ZORBAX SB-C18 (150 mm. Times.4.6 mm,3.5 μm); electronic balance (ME 204/02,METTLER TOLEDO).
Example 1
The embodiment provides a detection method of a fingerprint spectrum of bupleurum medicinal materials, which comprises the following steps:
(1) Preparation of control solution
Taking reference substances of saikosaponin a, saikosaponin c, saikosaponin d, saikosaponin e and saikosaponin f, respectively precisely weighing, and adding a methanol aqueous solution with the volume fraction of 80% to prepare a mixed reference substance solution containing 0.10mg of saikosaponin a, 50 mug of saikosaponin c, 0.15mg of saikosaponin d, 20 mug of saikosaponin e and 50 mug of saikosaponin f in each 1 mL.
(2) Preparation of test solutions
Taking bupleurum medicinal material powder (sieving with a third sieve), precisely weighing about 1.0g, adding 25mL of methanol water solution with the volume fraction of 80%, weighing, performing ultrasonic treatment (500W, 40 KHZ) for 30min, taking out, cooling, supplementing the lost weight with extraction solvent (methanol water solution with the volume fraction of 80%), shaking, filtering, and collecting subsequent filtrate.
(3) Detection conditions
Chromatographic conditions: chromatographic column Agilent ZORBAX SB-C18 (150 mm. Times.4.6 mm,3.5 μm); mobile phase A is formic acid water solution with volume fraction of 0.1%, mobile phase B is acetonitrile, gradient elution is carried out, and specific gradient elution program is shown in table 2; column temperature 40 ℃; the flow rate is 1.4mL/min; the sample injection amount is 20 mu L; the CAD drift tube temperature was 50deg.C and the gas flow rate was 2.4L/min.
TABLE 2 gradient elution procedure (volume fraction)
| Time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0～20 | 90→70 | 10→30 |
| 20～35 | 70→65 | 30→35 |
| 35～60 | 65→60 | 35→40 |
| 60～65 | 60→55 | 40→45 |
| 65～70 | 55→55 | 45→45 |
| 70～85 | 55→35 | 45→65 |
| 85～90 | 35→15 | 65→85 |
| 90～100 | 15→10 | 85→90 |
| 100～105 | 10→5 | 90→95 |
(4) Detection step
And (3) respectively precisely sucking 20 mu L of the mixed reference substance solution in the step (1) and 20 mu L of the sample solution in the step (2), injecting into a liquid chromatograph, measuring, and recording chromatograms. The fingerprint spectrum measured in this example is shown in fig. 1, and the chromatogram of the mixed reference substance is shown in fig. 2. Wherein Ssa, ssc, ssd, sse and Ssf marked in the figure correspond to saikosaponin a, saikosaponin c, saikosaponin d, saikosaponin e and saikosaponin f, respectively.
Example 2
The present embodiment differs from the detection method of embodiment 1 only in that: in the preparation of the sample solution in the step (2), the extraction solvents are different, and specific information of the extraction solvents is shown in table 3. The corresponding chromatograms under different extraction conditions are shown in fig. 3.
TABLE 3 extraction solvents and corresponding numbering
Example 3
The present embodiment differs from the detection method of embodiment 1 only in that: in the preparation of the sample solution in the step (2), the ratio of the extracted liquid is different, and specific information of the extracted liquid is shown in table 4. The corresponding chromatograms under different extraction conditions are shown in fig. 4.
TABLE 4 extract to feed ratio and corresponding numbering
| Numbering device | 3-1 | 3-2 |
| Feed-to-liquid ratio | 1.5g﹕25mL | 0.5g﹕25mL |
Example 4
The present embodiment differs from the detection method of embodiment 1 only in that: in the preparation of the sample solution in the step (2), the extraction time is different, and specific information of the extraction time is shown in table 5. The corresponding chromatograms under different extraction conditions are shown in fig. 5.
TABLE 5 extraction time and corresponding numbering
| Numbering device | 4-1 | 4-2 |
| Extraction time | 60min | 90min |
Example 5
The present embodiment differs from the detection method of embodiment 1 only in that: in the preparation of the sample solution in the step (2), the extraction modes are different, and the extraction is performed for 30min by adopting a heating reflux extraction mode. The corresponding chromatogram is shown in fig. 6.
Conclusion: the best preparation conditions of the sample solution are those in example 1 by examining the extraction solvent, the extraction feed liquid ratio, the extraction time and the extraction mode of the sample solution to comprehensively characterize the material composition of the bupleurum medicinal material: the extraction solvent is aqueous solution of methanol with volume fraction of 80%, the extraction liquid-to-liquid ratio is 1 g/25 mL, the extraction time is 30min, and the extraction mode is ultrasonic extraction.
Example 6
The present embodiment differs from the detection method of embodiment 1 only in that: in the detection condition of the step (3), gradient elution procedures are different.
The gradient elution procedure of this example is shown in table 6 below. The corresponding chromatogram is shown in FIG. 7.
TABLE 6 gradient elution procedure (volume fraction)
| Time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0～20 | 85→70 | 15→30 |
| 20～60 | 70→50 | 30→50 |
| 60～70 | 50→40 | 50→60 |
| 70～80 | 40→35 | 60→65 |
Example 7
The present embodiment differs from the detection method of embodiment 1 only in that: in the detection condition of the step (3), the mobile phase system is different, and the gradient elution program is different.
The mobile phase system of this example is: mobile phase a-water, mobile phase B-acetonitrile. The gradient elution procedure of this example is shown in table 7 below. The corresponding chromatogram is shown in FIG. 8.
TABLE 7 gradient elution procedure (volume fraction)
Conclusion: by examining the mobile phase system and the gradient elution condition of the mobile phase, the mobile phase system and the gradient elution condition in the detection condition of the embodiment 1 are obtained by taking good separation degree and stable baseline as principles.
Example 8
The present embodiment differs from the detection method of embodiment 1 only in that: in the detection condition of the step (3), the column temperatures are different, and specific information of the column temperatures is shown in Table 8. The corresponding chromatograms at different column temperatures are shown in fig. 9.
Table 8 column temperatures and corresponding numbers
| Numbering device | 8-1 | 8-2 |
| |
30 |
35℃ |
Example 9
The present embodiment differs from the detection method of embodiment 1 only in that: in the detection condition of the step (3), the flow rates are different, and specific information of the flow rates is shown in Table 9. The chromatograms corresponding to the different flow rates are shown in fig. 9.
TABLE 9 flow rates and corresponding numbering
| Numbering device | 9-1 | 9-2 |
| Flow rate | 1.0mL/min | 1.2mL/min |
Conclusion: the chromatograms obtained under each condition are respectively shown in figure 3 through investigation of column temperature and flow rate.
Example 10
The present embodiment differs from the detection method of embodiment 1 only in that: in the detection condition of the step (3), the detection time is different.
This example extends the detection time to 210min, and after 105min, elutes with 5vol.% mobile phase a, 95vol.% mobile phase B following the gradient elution according to table 1. The chromatogram is shown in FIG. 10.
As can be seen from fig. 10, substantially no chromatographic peak is detected after 105min, and the detection and acquisition time is determined to be 105min, so that the integrity of the spectrum can be ensured.
Example 11
The embodiment provides a method for establishing a comparison map, which comprises the following steps:
according to the detection method in the embodiment 1, 20 batches of bupleurum medicinal materials in the regions of origin in table 1, namely Shanxi, gansu and Hebei, are detected to obtain 20 fingerprint patterns; the fingerprint of the specific 20 batches of bupleurum medicinal materials is shown in figure 11;
20 fingerprint chromatograms are introduced into the traditional Chinese medicine fingerprint similarity evaluation software to synthesize a control spectrum, see figure 12.
As can be seen from the chromatogram of the mixed control of example 1, 6 # Peak is saikosaponin c, 7 # Peak is saikosaponin f, 8 # The peak is saikosaponin a, 11 # Peak is saikosaponin e, 13 # Peak is saikosaponin d, option 13 # The peak was used as a reference peak.
The fingerprints of 20 batches of bupleurum medicinal materials in table 1 are imported into traditional Chinese medicine fingerprint similarity evaluation software, the similarity with the control fingerprints is calculated, the result is shown in table 10, and the similarity between 20 batches of bupleurum medicinal materials and the control fingerprints is larger than 0.90.
TABLE 10 similarity of 20 batches of bupleurum medicinal materials
Comparative example 1
Comparative example 1A test solution prepared according to the method of example 1 was tested by HPLC-ELSD and a chromatogram was recorded as shown in FIG. 13.
Wherein, the conditions of the HPLC-ELSD method are as follows: ELSD atomizing tube temperature 70 ℃, drift tube temperature 70 ℃, nitrogen flow rate 1.7L/min. Other chromatographic conditions refer to the procedure of example 1.
Comparative example 2
Comparative example 2 the test solution prepared according to the method of example 1 was tested by HPLC-UV and chromatograms were recorded as shown in fig. 14 and 15.
Wherein, the conditions of the HPLC-UV method are as follows: the ultraviolet detection wavelengths were 254nm and 280nm, respectively. Other chromatographic conditions refer to the procedure of example 1.
Experimental example 1
As can be seen from the chromatograms of example 1, comparative example 1 and comparative example 2, the HPLC-CAD method of the present invention has the most abundant information of chromatographic peaks in the chromatograms, stable base line, high sensitivity and good reproducibility.
Experimental example 2
Methodology investigation
(1) Specificity test
The blank solvent (80% methanol aqueous solution by volume fraction) was taken and detected under the detection conditions of example 1, the chromatogram is shown in fig. 16, and the result shows that the blank solvent has no interference, and the method has good specificity.
(2) Precision test
Taking radix bupleuri (S1), preparing a sample solution according to the method of example 1, detecting according to the detection conditions of example 1, continuously injecting 6 needles, and calculating the peak-to-peak ratio of each chromatographic peak to 13 # Relative retention time of peak and relative peak area. The results show that the relative retention time and the RSD of the relative peak area of each chromatographic peak are less than 5.0 percent, and the method has good precision. The specific data are shown in Table 11.
(3) Repeatability test
Taking bupleurum medicinal material (S1), preparing 6 parts of sample solution in parallel according to the method of example 1, sampling and detecting according to the method of example 1, and calculating each chromatographic peak relative to 13 # The relative retention time and relative peak area of the peaks show that the relative retention time and relative peak area of each chromatographic peak have RSD less than 5.0 percent, and the method has good repeatability. The specific data are shown in Table 11.
(4) Stability test
Taking bupleurum medicinal material (S1), preparing a sample solution according to the method of example 1, sampling and detecting at 0 hour, 4 hours, 8 hours, 12 hours, 20 hours, 30 hours and 48 hours according to the method of example 1, and calculating the peak-to-peak ratio of each chromatographic peak to 13 # Relative retention time of peak and relative peak surfaceAnd (3) accumulation. The results showed that the relative retention time of each chromatographic peak and RSD of the relative peak area were less than 5.0% for 48 hours, and the test sample solution was stable for 48 hours at room temperature. The specific data are shown in Table 11.
Table 11 methodology verifies the relative retention times of 22 common peaks
Table 12 methodological verification of the relative peak areas of 22 common peaks
The method has high sensitivity, and the obtained fingerprint has more abundant information of common characteristic peaks, thereby meeting the requirement of the integrity of the fingerprint.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (11)
1. The method for establishing the fingerprint of the bupleurum medicinal material, the extract and the single preparation is characterized by comprising the following steps:
detecting the sample solution by adopting an HPLC-CAD method to obtain a fingerprint spectrum containing 22 common characteristic peaks;
the detection conditions of the HPLC-CAD method comprise:
carrying out gradient elution by taking formic acid aqueous solution with the volume fraction of 0.05% -0.15% as a mobile phase A and acetonitrile as a mobile phase B; the gradient elution includes: 0-20 min, wherein the volume fraction of the mobile phase A is changed from 90% to 70%; 20-35 min, wherein the volume fraction of the mobile phase A is changed from 70% to 65%; 35-60 min, wherein the volume fraction of the mobile phase A is changed from 65% to 60%; 60-65 min, wherein the volume fraction of the mobile phase A is changed from 60% to 55%; 65-70 min, wherein the volume fraction of the mobile phase A is 55%; 70-85 min, wherein the volume fraction of the mobile phase A is changed from 55% to 35%; 85-90 min, wherein the volume fraction of the mobile phase A is changed from 35% to 15%; 90-100 min, wherein the volume fraction of the mobile phase A is changed from 15% to 10%; 100-105 min, wherein the volume fraction of the mobile phase A is changed from 10% to 5%;
the chromatographic column is C18 chromatographic column, 3.5 μm;
the detection flow rate is 1.0-1.5 mL/min; the column temperature of the chromatographic column is 30-40 ℃;
detecting the reference substance solution by adopting the HPLC-CAD method; the reference substance comprises any one or more of saikosaponin a, saikosaponin c, saikosaponin d, saikosaponin e and saikosaponin f;
the preparation method of the bupleurum medicinal material, the bupleurum extract in solid or semisolid dosage form and the test solution of the bupleurum single preparation in solid or semisolid dosage form comprises the following steps: performing ultrasonic extraction or reflux extraction on the bupleurum medicinal material, bupleurum extract or bupleurum single preparation by taking a methanol aqueous solution as an extraction solvent, supplementing the reduced weight by the extraction solvent, filtering, and taking a subsequent filtrate as a sample solution;
for the bupleurum extract in liquid dosage form and bupleurum single preparation in liquid dosage form, corresponding liquid is directly obtained as the test solution.
2. A method according to claim 1, wherein the control comprises saikosaponin a, saikosaponin c, saikosaponin d, saikosaponin e and saikosaponin f.
3. The method of claim 1, wherein the mobile phase a is an aqueous formic acid solution having a volume fraction of 0.1%.
4. A method according to any one of claims 1 to 3, wherein the detection conditions of the HPLC-CAD method comprise:
the chromatographic column is Agilent ZORBAX SB-C18, 150X4.6mm, 3.5 μm;
column temperature is 40 ℃;
the flow rate is 1.4mL/min;
the CAD drift tube temperature was 35 ℃ or 50 ℃.
5. The method of claim 1, wherein the single bupleurum formulation comprises any one of bupleurum injection, bupleurum oral liquid, bupleurum dripping pill and bupleurum cough relieving tablet.
6. The method according to claim 1, wherein the extraction is by ultrasonic extraction;
the power of the ultrasonic wave is 300-500W, and the frequency of the ultrasonic wave is 40kHz;
the extraction time is 30-90 min.
7. The method according to claim 1, wherein the ratio of the bupleurum medicinal material to the extraction solvent is 0.5 g/25 mL to 1.5 g/25 mL.
8. The method according to claim 1, wherein the volume fraction of methanol in the extraction solvent is 70% -90%.
9. A method according to any one of claims 1-3, characterized in that the method is carried out at 13 # The peak is a reference peak, and the relative retention time and the relative peak area of the 22 common characteristic peaks are respectively as follows:
peak No. 1: the relative retention time is 0.04-0.05, and the relative peak area is 0.01-0.15;
peak No. 2: the relative retention time is 0.21-0.23, and the relative peak area is 0.01-0.26;
peak No. 3: the relative retention time is 0.23-0.24, and the relative peak area is 0.01-0.13;
peak No. 4: the relative retention time is 0.30-0.31, and the relative peak area is 0.04-0.26;
peak No. 5: the relative retention time is 0.49-0.50, and the relative peak area is 0.08-0.53;
peak No. 6: the relative retention time is 0.51-0.52, and the relative peak area is 0.21-0.60;
peak No. 7: the relative retention time is 0.54-0.55, and the relative peak area is 0.18-0.95;
peak No. 8: the relative retention time is 0.68-0.69, and the relative peak area is 0.92-1.51;
peak No. 9: the relative retention time is 0.76-0.77, and the relative peak area is 0.10-0.42;
peak No. 10: the relative retention time is 0.78-0.79, and the relative peak area is 0.13-0.59;
peak No. 11: the relative retention time is 0.86-0.87, and the relative peak area is 0.07-0.23;
peak No. 12: the relative retention time is 0.93-0.94, and the relative peak area is 0.03-0.33;
peak No. 13: the relative retention time was 1.00 and the relative peak area was 1.00;
peak No. 14: the relative retention time is 1.14-1.16, and the relative peak area is 0.22-0.62;
peak No. 15: the relative retention time is 1.17-1.19, and the relative peak area is 0.32-1.18;
peak No. 16: the relative retention time is 1.32-1.34, and the relative peak area is 0.27-1.35;
peak No. 17: the relative retention time is 1.67-1.70, and the relative peak area is 0.02-0.16;
peak No. 18: the relative retention time is 1.80-1.84, and the relative peak area is 0.02-0.12;
peak No. 19: the relative retention time is 1.85-1.89, and the relative peak area is 0.27-1.93;
peak No. 20: the relative retention time is 1.89-1.93, and the relative peak area is 0.12-0.47;
peak No. 21: the relative retention time is 1.90-1.94, and the relative peak area is 0.05-0.90;
peak No. 22: the relative retention time is 1.98-2.02, and the relative peak area is 0.03-0.20.
10. The method according to claim 9, wherein 6 in the fingerprint # Peak is firewoodHu Zaogan c, 7 # Peak is saikosaponin f, 8 # The peak is saikosaponin a, 11 # Peak is saikosaponin e, 13 # The peak is saikosaponin d.
11. A method according to any one of claims 1 to 3, wherein 20 batches of the test solution of bupleurum medicinal material are detected by the HPLC-CAD method to obtain chromatograms of the bupleurum medicinal material batches, and the chromatograms are processed by the traditional Chinese medicine fingerprint similarity evaluation software to obtain a control spectrum.
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