CN113514576A - Establishing method of fingerprint of bupleurum medicinal material, extract and single preparation - Google Patents
Establishing method of fingerprint of bupleurum medicinal material, extract and single preparation Download PDFInfo
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Abstract
The invention relates to the technical field of quality analysis of bupleurum medicinal materials, in particular to a method for establishing a fingerprint of bupleurum medicinal materials, an extract and a single preparation. 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 containing 22 common characteristic peaks; the detection conditions of the HPLC-CAD method comprise: and performing gradient elution by using aqueous solution of formic acid as a mobile phase A and acetonitrile as a mobile phase B. The invention establishes an HPLC-CAD fingerprint spectrum measuring method of bupleurum medicinal materials, extracts and single preparations, realizes good separation of chromatographic peaks, contains components with ultraviolet absorption, also contains components with weak ultraviolet absorption and components without part of ultraviolet absorption in 22 common characteristic peaks, and can more comprehensively represent the material compositions and relative contents of the bupleurum medicinal materials, the extracts and the single preparations.
Description
Technical Field
The invention relates to the technical field of quality analysis of bupleurum medicinal materials, in particular to a method for establishing a fingerprint of bupleurum medicinal materials, an extract and a single preparation.
Background
The bupleuri radix is dried root of Bupleurum chinense DC or Bupleurum scorzonerifolium Willd of Umbelliferae, collected in spring and autumn, removed stem and leaf, and dried. The bupleurum medicinal material is mainly produced in Shanxi, Shaanxi, Gansu, Hebei and other places, and has the effects of dispelling and abating fever, soothing liver and relieving depression, lifting yang qi and the like.
The bupleurum root mainly contains saponin, flavone, volatile oil, polysaccharide and other components, the quality of the bupleurum root is generally evaluated by the saikoside at present, under the item of bupleurum root medicinal materials in the section of Chinese pharmacopoeia, the saikoside a and the saikoside d are used as content control indexes, and a fingerprint method is not available. The traditional Chinese medicine has complex components, the clinical efficacy is the result of the combined action of the contained chemical components, the contained chemical components directly influence the quality of the medicinal materials, and the content measurement of one or two components hardly reflects the quality of the traditional Chinese medicine really, so a fingerprint method needs to be established to analyze the whole chemical components of the traditional Chinese medicine, scientifically and comprehensively evaluate the quality of the medicinal materials, and provide scientific basis for the clinical application of the traditional Chinese medicine.
At present, the research on the fingerprint of bupleurum root in the literature mostly adopts an Ultraviolet (UV) or Evaporative Light Scattering (ELSD) detector. The UV detector can only detect components with ultraviolet absorption, and materials without chromophore do not respond and cannot be detected; although the ELSD is a general detector, it is difficult to achieve a good detection effect for some low-content components in the traditional Chinese medicine due to its low detection sensitivity and poor repeatability. Therefore, a fingerprint detection method of a universal detector with high sensitivity and good reproducibility needs to be established.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for establishing a fingerprint of a bupleurum medicinal material, an extract and a single preparation, so as to solve the technical problem that the quality of the bupleurum medicinal material can not be comprehensively characterized in the prior art.
In order to achieve the above purpose of the present invention, the following technical solutions are 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 containing 22 common characteristic peaks;
the detection conditions of the HPLC-CAD method comprise:
taking a formic acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B to carry out gradient elution; the gradient elution comprises: changing the volume fraction of the mobile phase A from 88-92% to 68-72% in 0-20 min; changing the volume fraction of the mobile phase A from 68-72% to 63-67% in 20-35 min; changing the volume fraction of the mobile phase A from 63-67% to 58-62% in 35-60 min; changing the volume fraction of the mobile phase A from 58-62% to 53-57% in 60-65 min; 65-70 min, wherein the volume fraction of the mobile phase A is 53-57%; changing the volume fraction of the mobile phase A from 53-57% to 33-37% in 70-85 min; 85-90 min, changing the volume fraction of the mobile phase A from 33-37% to 13-17%; changing the volume fraction of the mobile phase A from 13-17% to 8-12% for 90-100 min; changing the volume fraction of the mobile phase A from 8-12% to 3-7% in 100-105 min.
In a specific embodiment of the invention, the gradient elution comprises: changing the volume fraction of the mobile phase A from 90% to 70% in 0-20 min; changing the volume fraction of the mobile phase A from 70% to 65% in 20-35 min; changing the volume fraction of the mobile phase A from 65% to 60% in 35-60 min; changing the volume fraction of the mobile phase A from 60% to 55% in 60-65 min; 65-70 min, wherein the volume fraction of the mobile phase A is 55%; the volume fraction of the mobile phase A is changed from 55% to 35% in 70-85 min; 85-90 min, and changing the volume fraction of the mobile phase A from 35% to 15%; changing the volume fraction of the mobile phase A from 15% to 10% for 90-100 min; the volume fraction of the mobile phase A is changed from 10% to 5% in 100-105 min.
The fingerprint spectrum of the bupleurum medicinal material, which is established by adopting a certain detection method, chromatographic conditions and the like, has 22 common characteristic peaks, the common characteristic peaks not only contain components with ultraviolet absorption (such as flavonoids and the like), but also comprise components with weak ultraviolet absorption (such as saponins and the like) and partial components without ultraviolet absorption, the quality of the bupleurum medicinal material can be comprehensively represented and evaluated through various components, scientific basis is provided for the full utilization of bupleurum resources, and the problems of single quality evaluation index of the bupleurum medicinal material and the like are solved. The method of the invention also has the advantages of high sensitivity, good reproducibility and the like.
In a specific embodiment of the present invention, the method further comprises: detecting the reference substance solution by adopting the HPLC-CAD method; the reference substance comprises one or more of saikosaponin a, saikosaponin c, saikosaponin d, saikosaponin e and saikosaponin f. Preferably, the reference substance comprises 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 aqueous formic acid solution is 0.05% to 0.15%. Further, the mobile phase A is 0.1% by volume of formic acid aqueous solution.
In the specific embodiment of the invention, the detection flow rate is 1.0-1.5 mL/min, preferably 1.4 mL/min.
As in various embodiments, the detection flow rate can be 1.0mL/min, 1.1mL/min, 1.2mL/min, 1.3mL/min, 1.4mL/min, 1.5mL/min, and the like.
By adopting the flow velocity detection method, the chromatographic peak separation degree and the detection efficiency can be further improved.
In the specific embodiment of the invention, the temperature of the chromatographic column is 30-40 ℃, and preferably 40 ℃.
As in the different embodiments, the column temperature of the chromatographic column can be 30 ℃, 32 ℃, 34 ℃, 35 ℃, 36 ℃, 38 ℃, 40 ℃ and so on.
In a specific embodiment of the invention, the HPLC-CAD method uses a C18 column, preferably an 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 a formic acid aqueous solution with the volume fraction of 0.1%, and the mobile phase B is acetonitrile;
the column was Agilent ZORBAX SB-C18 (150X 4.6mm, 3.5 μm);
the column temperature was 40 ℃;
the flow rate is 1.4 mL/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 various embodiments, the CAD parameters may include a gas flow rate of 2.2L/min, 2.3L/min, 2.4L/min, 2.5L/min, 2.6L/min, etc., such as 2.4L/min.
In the embodiment of the invention, the sample amount is 10-20 μ L, preferably 20 μ L.
In a specific embodiment of the present invention, the method for preparing the test solution comprises:
when the substance to be detected is radix bupleuri, performing ultrasonic extraction or reflux extraction on the radix bupleuri by using methanol water solution as an extraction solvent, supplementing the loss weight with the extraction solvent, filtering, and taking the subsequent filtrate as a sample solution.
For solid or semisolid bupleuri radix extract and bupleuri radix single preparation, the preparation method of the sample solution with fingerprint spectrum is the same as that of bupleuri radix medicinal material. The method specifically comprises the following steps: ultrasonic extracting or reflux extracting the bupleuri radix extract or single preparation with methanol water solution as extraction solvent, adding the weight loss of the extraction solvent, filtering, and collecting the filtrate as sample solution.
For bupleurum extract and single bupleurum preparation in liquid dosage form, the liquid extract or liquid preparation can be directly taken as a test solution for analysis.
In a specific embodiment of the present invention, the single preparation of bupleurum includes any one of bupleurum injection, bupleurum oral liquid, bupleurum dripping pill and bupleurum cough-relieving tablet. Wherein the bupleuri radix dripping pill and bupleuri radix cough relieving tablet are solid preparations, and the bupleuri radix injection and bupleuri radix oral liquid are liquid preparations.
The preparation conditions of the test solution are described below by taking bupleurum medicinal materials as an example:
in a particular embodiment of the invention, the ratio of the bupleurum root material to the extraction solvent is from 0.5 g: 25mL to 1.5 g: 25mL, preferably (0.8 to 1.2) g: 25mL, e.g. 1.0 g: 25 mL.
In various embodiments, the ratio of the bupleuri radix to the extraction solvent can be 0.5 g: 25mL, 0.6 g: 25mL, 0.7 g: 25mL, 0.8 g: 25mL, 0.9 g: 25mL, 1.0 g: 25mL, 1.1 g: 25mL, 1.2 g: 25mL, 1.3 g: 25mL, 1.4 g: 25mL, 1.5 g: 25mL, or the like.
In a specific embodiment of the present invention, the extraction is ultrasonic extraction. Furthermore, the power of the ultrasound is 300-500W, and the frequency of the ultrasound is 40 kHz.
In a specific embodiment of the present invention, the extraction time is 30-90 min, preferably 30-60 min, such as 30 min.
As in the different embodiments, the time of the extraction may be 30min, 45min, 60min, 75min, 90min, and the like.
In a specific embodiment of the present invention, the volume fraction of methanol in the methanol aqueous solution is 70% to 90%, preferably 80%.
In actual operation, the extraction is carried out by adopting a powdery bupleurum medicinal material. Further, the powdered bupleurum medicinal material is powder sieved by a third sieve.
In a specific embodiment of the present invention, the method for preparing the control solution comprises: precisely weighing saikosaponin a, saikosaponin c, saikosaponin d, saikosaponin e and saikosaponin f, and dissolving by 70-90% volume fraction of methanol water solution; in the reference solution, the concentrations of saikosaponin a, saikosaponin c, saikosaponin d, saikosaponin e and saikosaponin f are 0.1mg/mL, 50 μ g/mL, 0.15mg/mL, 20 μ g/mL and 50 μ g/mL respectively.
In the specific embodiment of the invention, 13 is used#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 is 1.00, and the relative peak area is 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 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#The peak is saikosaponin c, 7#The peak is saikosaponin f, 8#The peak is saikosaponin a, 11#The peak is saikosaponin e, 13#The peak is saikosaponin d.
The invention compares 22 common characteristic peaks in the fingerprint with a reference substance, and identifies the chemical components corresponding to 5 common characteristic peaks in the fingerprint.
In the specific implementation mode of the invention, 20 batches of bupleurum medicinal material sample solution are detected according to the HPLC-CAD method to obtain the chromatogram of each batch of bupleurum medicinal material, and the chromatogram is processed by traditional Chinese medicine fingerprint similarity evaluation software to obtain a reference spectrum.
In actual operation, the chromatogram of the sample to be detected can be introduced into traditional Chinese medicine fingerprint similarity evaluation software, the similarity between the traditional Chinese medicine fingerprint similarity evaluation software and the reference chromatogram can be calculated, and the quality of the medicinal materials can be controlled 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 spectrum obtained by the method contains 22 common characteristic peaks, wherein the fingerprint spectrum not only contains components with ultraviolet absorption (such as flavonoids and the like), but also contains components with weak ultraviolet absorption (such as saponins and the like) and parts of components without ultraviolet absorption, and can more comprehensively represent the material compositions and relative contents of the bupleurum medicinal material, the extract and a single preparation.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a finger-print of bupleuri radix 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 is a chromatogram measured under different conditions according to example 2 of the present invention;
FIG. 4 is a chromatogram measured under different conditions according to example 3 of the present invention;
FIG. 5 is a chromatogram measured under different conditions according to example 4 of the present invention;
FIG. 6 is a chromatogram obtained under the conditions of example 5 according to the present invention;
FIG. 7 is a chromatogram obtained under the conditions of example 6 according to the present invention;
FIG. 8 is a chromatogram obtained under the conditions of example 7 according to the present invention;
FIG. 9 is a chromatogram obtained under different conditions in examples 8 and 9 according to the present invention;
FIG. 10 is a chromatogram obtained in example 10 of the present invention;
FIG. 11 is a map of 20 batches of bupleuri radix herbs in example 11 of the present invention;
FIG. 12 is a reference map of the bupleuri radix herb established in example 11 of the present invention;
FIG. 13 is a chromatogram obtained in comparative example 1;
FIG. 14 is a chromatogram obtained at a detection wavelength of 254nm in comparative example 2;
FIG. 15 is a chromatogram obtained at a detection wavelength of 280nm in comparative example 2;
FIG. 16 is a chromatogram of a blank solvent in a specificity test.
Detailed Description
The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and the detailed description, but those skilled in the art will understand that the following described embodiments are some, not all, of the embodiments of the present invention, and are only used for illustrating the present invention, and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In the specific implementation mode, the verification of the items such as specificity, precision, repeatability, stability and the like is carried out according to the relevant guidance principle in the appendix of the pharmacopoeia of the people's republic of China in the current edition.
The information of materials, instruments, reagents and the like adopted in the specific embodiment of the invention can be as follows:
materials, reagents: acetonitrile (HPLC, fepue); methanol (HPLC, fepue); formic acid (AR, MREDA); pure water (manufactured by MiLLI-Q);
saikosaponin a (110777-; saikosaponin c (PS000193, Kyoto Pusi Biotech Co., Ltd.); saikosaponin d (110779-; saikosaponin e (PS200702-15, Chengdu Pusi Biotechnology GmbH); saikosaponin f (6131, shanghai shidan standard technical service ltd);
the bupleurum medicinal material is purchased in the medicinal material market, and the source information of the specific bupleurum medicinal material sample is shown in the following table 1;
TABLE 1 bupleuri radix sample source information Table
The instrument comprises the following steps: high performance liquid chromatograph ulitimate 3000 Thermo (CAD detector); agilent ZORBAX SB-C18(150 mm. times.4.6 mm, 3.5 μm); electronic balance (ME204/02, METTLER TOLEDO).
Example 1
The embodiment provides a method for detecting a fingerprint of a radix bupleuri medicinal material, which comprises the following steps:
(1) preparation of control solutions
Taking saikosaponin a, saikosaponin c, saikosaponin d, saikosaponin e and saikosaponin f as reference substances, respectively precisely weighing, and adding 80% methanol aqueous solution by volume fraction to prepare a mixed reference substance solution containing 0.10mg of saikosaponin a, 50 μ g of saikosaponin c, 0.15mg of saikosaponin d, 20 μ g of saikosaponin e and 50 μ g of saikosaponin f per 1 mL.
(2) Preparation of test solution
Taking bupleuri radix powder (sieved by a third sieve), about 1.0g, precisely weighing, adding 25mL of 80% methanol aqueous solution by volume fraction, weighing, performing ultrasonic treatment (500W, 40KHZ) for 30min, taking out, cooling, supplementing the weight loss with an extraction solvent (80% methanol aqueous solution by volume fraction), shaking uniformly, filtering, and taking the subsequent filtrate.
(3) Detection conditions
Chromatographic conditions are as follows: column Agilent ZORBAX SB-C18(150 mm. times.4.6 mm, 3.5 μm); the mobile phase A is formic acid aqueous solution with volume fraction of 0.1%, the mobile phase B is acetonitrile, and the gradient elution is carried out, wherein the specific gradient elution procedure is shown in table 2; the column temperature is 40 ℃; the flow rate is 1.4 mL/min; the sample volume is 20 mu L; the temperature of the CAD drift tube is 50 ℃, and the gas flow rate is 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) precisely sucking 20 mu L of the mixed reference solution in the step (1) and the test solution in the step (2) respectively, injecting into a liquid chromatograph, measuring, and recording a chromatogram. The fingerprint obtained in this example is shown in FIG. 1, and the chromatogram of the mixed control 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
This example refers to the detection method of example 1, with the only difference that: in the step (2) of preparing the test solution, the extraction solvents are different, and the 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
This example refers to the detection method of example 1, with the only difference that: in the step (2) of preparing the test solution, the extraction material-liquid ratios are different, and the specific information of the extraction material-liquid ratios is shown in Table 4. The corresponding chromatograms under different extraction conditions are shown in fig. 4.
TABLE 4 feed and extract ratios and corresponding numbering
| Numbering | 3-1 | 3-2 |
| Ratio of material to liquid | 1.5g﹕25mL | 0.5g﹕25mL |
Example 4
This example refers to the detection method of example 1, with the only difference that: in the step (2) of preparing the test solution, the extraction time is different, and the 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 | 4-1 | 4-2 |
| Extraction time | 60min | 90min |
Example 5
This example refers to the detection method of example 1, with the only difference that: in the step (2), the preparation of the test solution is carried out in different extraction modes, and the extraction is carried out for 30min by adopting a heating reflux extraction mode. The corresponding chromatogram is shown in FIG. 6.
And (4) conclusion: through the investigation of the extraction solvent, the extraction material-liquid ratio, the extraction time and the extraction mode of the test solution, the substance composition of the bupleurum medicinal material is comprehensively represented, and the optimal preparation conditions of the test solution are obtained as the preparation conditions in the embodiment 1: the extraction solvent is 80% methanol water solution, the extraction ratio of material to liquid is 1 g: 25mL, the extraction time is 30min, and the extraction method is ultrasonic extraction.
Example 6
This example refers to the detection method of example 1, with the only difference that: and (4) in the detection conditions of the step (3), gradient elution programs are different.
The gradient elution procedure for this example is shown in table 6 below. The corresponding chromatogram obtained 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
This example refers to the detection method of example 1, with the only difference that: and (3) in the detection conditions of the step (3), the mobile phase systems are different, and the gradient elution procedures are different.
The mobile phase system of the present embodiment is: mobile phase a-water, mobile phase B-acetonitrile. The gradient elution procedure for this example is shown in table 7 below. The corresponding chromatogram obtained is shown in FIG. 8.
TABLE 7 gradient elution procedure (volume fraction)
And (4) conclusion: the mobile phase system and the gradient elution conditions in the detection conditions of example 1 are obtained by examining the mobile phase system and the gradient elution conditions of the mobile phase and taking good separation degree and stable baseline as the principles.
Example 8
This example refers to the detection method of example 1, with the only difference that: in the detection conditions in the step (3), the column temperatures are different, and the 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 numbering
| Numbering | 8-1 | 8-2 |
| |
30 |
35℃ |
Example 9
This example refers to the detection method of example 1, with the only difference that: in the detection conditions in the step (3), the flow rates are different, and the specific information of the flow rates is shown in a table 9. The chromatograms for the different flow rates are shown in fig. 9.
TABLE 9 flow rates and corresponding numbering
| Numbering | 9-1 | 9-2 |
| Flow rate of flow | 1.0mL/min | 1.2mL/min |
And (4) conclusion: the chromatogram obtained under each condition is shown in FIG. 3 by examining the column temperature and the flow rate.
Example 10
This example refers to the detection method of example 1, with the only difference that: and (4) in the detection condition of the step (3), the detection time is different.
This example extended the detection time to 210min, after gradient elution according to table 1, and after 105min, elution was with 5 vol.% mobile phase a, 95 vol.% mobile phase B. The chromatogram is shown in FIG. 10.
As can be seen from FIG. 10, basically no chromatographic peak is detected after 105min, and the detection acquisition time is determined to be 105min, so that the integrity of the chromatogram can be ensured.
Example 11
This example provides a method for establishing a control map, comprising the steps of:
according to the detection method in the embodiment 1, 20 batches of radix bupleuri medicinal materials with the production places of Shanxi, Shaanxi, Gansu and Hebei in Table 1 are detected to obtain 20 fingerprint spectrums; the fingerprint of 20 batches of bupleuri radix materials is shown in figure 11;
introducing 20 finger prints into the Chinese medicinal finger print similarity evaluation software, and synthesizing reference prints (see FIG. 12).
From the chromatogram of the mixed control in example 1, 6#The peak is saikosaponin c, 7#The peak is saikosaponin f, 8#The peak is saikosaponin a, 11#The peak is saikosaponin e, 13#The peak is saikosaponin d, and 13 is selected#The peak was used as a reference peak.
The respective fingerprints of 20 batches of bupleurum medicinal materials in the table 1 are led into traditional Chinese medicine fingerprint similarity evaluation software, and the similarity with the reference map is calculated, the result is shown in the table 10, and the similarity between the 20 batches of bupleurum medicinal materials and the reference map is more than 0.90.
TABLE 1020 batch radix bupleuri medicinal material similarity
Comparative example 1
Comparative example 1 the sample solution prepared according to the method of example 1 was examined by HPLC-ELSD and a chromatogram was recorded, as shown in FIG. 13.
Wherein, the conditions of the HPLC-ELSD method are as follows: the ELSD atomizing tube temperature is 70 ℃, the drift tube temperature is 70 ℃, and the nitrogen flow rate is 1.7L/min. Other chromatographic conditions refer to the procedure of example 1.
Comparative example 2
Comparative example 2 the sample solution prepared according to the method of example 1 was subjected to HPLC-UV detection and a chromatogram was recorded, as shown in FIGS. 14 and 15.
Wherein, the conditions of the HPLC-UV method are as follows: the ultraviolet detection wavelength is 254nm and 280nm respectively. Other chromatographic conditions refer to the procedure of example 1.
Experimental example 1
According to the chromatograms of the embodiment 1, the comparative example 1 and the comparative example 2, the HPLC-CAD method of the invention has the advantages of most abundant chromatographic peak information, stable base line, high sensitivity and better reproducibility.
Experimental example 2
Methodology investigation
(1) Specificity test
A blank solvent (80 volume percent methanol aqueous solution) is taken and subjected to sample injection detection according to the detection conditions of the example 1, a chromatogram is shown in figure 16, and the result shows that the blank solvent has no interference, so that the method has good specificity.
(2) Precision test
Taking bupleuri radix (S1), preparing test solution according to the method of example 1, detecting according to the detection conditions of example 1, continuously injecting sample 6 needles, calculating each chromatographic peak relative to 13#Relative retention time of peaks and relative peak area. The results show the phase of the individual chromatographic peaksThe RSD of the retention time and the relative peak area are both less than 5.0 percent, and the method has good precision. The data are shown in Table 11.
(3) Repeatability test
Taking bupleuri radix (S1), preparing 6 parts of test solution in parallel according to the method of example 1, injecting sample according to the method of example 1, calculating each chromatographic peak relative to 13#The relative retention time of the peak and the relative peak area show that the RSD of the relative retention time and the relative peak area of each chromatographic peak are less than 5.0 percent, and the method has good repeatability. The data are shown in Table 11.
(4) Stability test
Taking bupleuri radix (S1), preparing test solution according to the method of example 1, injecting sample for 0 hr, 4 hr, 8 hr, 12 hr, 20 hr, 30 hr, and 48 hr according to the method of example 1, detecting, and calculating each chromatographic peak relative to 13#Relative retention time of peaks and relative peak area. The results show that the relative retention time of each chromatographic peak and the RSD of the relative peak area are less than 5.0% in 48 hours, and the test solution is stable in 48 hours at room temperature. The data are shown in Table 11.
Table 11 methodology verifies the relative retention time of 22 common peaks
Table 12 methodology verifies the relative peak area of the 22 common peaks
The method has high sensitivity, and the obtained fingerprint has richer information of common characteristic peaks and better meets the integral requirement of the fingerprint.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
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 containing 22 common characteristic peaks;
the detection conditions of the HPLC-CAD method comprise:
taking a formic acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B to carry out gradient elution; the gradient elution comprises: changing the volume fraction of the mobile phase A from 88-92% to 68-72% in 0-20 min; changing the volume fraction of the mobile phase A from 68-72% to 63-67% in 20-35 min; changing the volume fraction of the mobile phase A from 63-67% to 58-62% in 35-60 min; changing the volume fraction of the mobile phase A from 58-62% to 53-57% in 60-65 min; 65-70 min, wherein the volume fraction of the mobile phase A is 53-57%; changing the volume fraction of the mobile phase A from 53-57% to 33-37% in 70-85 min; 85-90 min, changing the volume fraction of the mobile phase A from 33-37% to 13-17%; changing the volume fraction of the mobile phase A from 13-17% to 8-12% for 90-100 min; changing the volume fraction of the mobile phase A from 8-12% to 3-7% in 100-105 min.
2. The method of claim 1, further comprising: detecting the reference substance solution by adopting the HPLC-CAD method; the reference substance comprises one or more of saikosaponin a, saikosaponin c, saikosaponin d, saikosaponin e and saikosaponin f;
preferably, the reference substance comprises saikosaponin a, saikosaponin c, saikosaponin d, saikosaponin e and saikosaponin f.
3. The method of claim 1, wherein the gradient elution comprises: changing the volume fraction of the mobile phase A from 90% to 70% in 0-20 min; changing the volume fraction of the mobile phase A from 70% to 65% in 20-35 min; changing the volume fraction of the mobile phase A from 65% to 60% in 35-60 min; changing the volume fraction of the mobile phase A from 60% to 55% in 60-65 min; 65-70 min, wherein the volume fraction of the mobile phase A is 55%; the volume fraction of the mobile phase A is changed from 55% to 35% in 70-85 min; 85-90 min, and changing the volume fraction of the mobile phase A from 35% to 15%; changing the volume fraction of the mobile phase A from 15% to 10% for 90-100 min; changing the volume fraction of the mobile phase A from 10% to 5% in 100-105 min;
preferably, in the formic acid aqueous solution, the volume fraction of formic acid is 0.05-0.15%;
preferably, the mobile phase A is 0.1% by volume of formic acid aqueous solution.
4. The method according to any one of claims 1 to 3, wherein in the HPLC-CAD method, the detection flow rate is 1.0 to 1.5 mL/min;
and/or, the chromatographic column adopted is a C18 chromatographic column;
and/or the column temperature of the chromatographic column is 30-40 ℃.
5. The method according to any one of claims 1 to 3, wherein the detection conditions of the HPLC-CAD method comprise:
the mobile phase A is a formic acid aqueous solution with the volume fraction of 0.1%, and the mobile phase B is acetonitrile;
the chromatographic column is Agilent ZORBAX SB-C18, 150X 4.6mm, 3.5 μm;
the column temperature was 40 ℃;
the flow rate is 1.4 mL/min;
the CAD drift tube temperature was 35 ℃ or 50 ℃.
6. The method according to any one of claims 1 to 3, wherein the method for preparing the test solution of Bupleurum root crude drug, Bupleurum extract in solid or semisolid form, Bupleurum single preparation in solid or semisolid form comprises: performing ultrasonic extraction or reflux extraction on the bupleurum medicinal material, the bupleurum extract or the single preparation of bupleurum by taking methanol aqueous solution as an extraction solvent, supplementing the weight loss by the extraction solvent, filtering, and taking the subsequent filtrate as a test solution;
directly obtaining corresponding liquid as a test solution for a liquid type bupleurum extract and a liquid type bupleurum single preparation;
preferably, the single preparation of bupleurum comprises any one of bupleurum injection, bupleurum oral liquid, bupleurum dripping pill and bupleurum cough-relieving tablet;
preferably, the extraction mode is ultrasonic extraction;
preferably, the power of the ultrasound is 300-500W, and the frequency of the ultrasound is 40 kHz;
preferably, the extraction time is 30-90 min.
7. The method of claim 6, wherein the ratio of the Bupleurum chinense to the extraction solvent is 0.5 g: 25mL to 1.5 g: 25 mL;
preferably, the volume fraction of methanol in the extraction solvent is 70-90%.
8. A method according to any one of claims 1 to 3, characterised in that 13 is added#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 is 1.00, and the relative peak area is 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 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.
9. The method of claim 8,in the fingerprint, 6#The peak is saikosaponin c, 7#The peak is saikosaponin f, 8#The peak is saikosaponin a, 11#The peak is saikosaponin e, 13#The peak is saikosaponin d.
10. The method according to any one of claims 1-3, wherein 20 batches of the test solution of bupleuri radix medicinal materials are detected by HPLC-CAD method to obtain chromatogram of each batch of bupleuri radix medicinal materials, and processed by traditional Chinese medicine fingerprint similarity evaluation software to obtain reference chromatogram.
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