CN114216980A - Method for establishing HPLC-ELSD (high Performance liquid chromatography-evaporative light scattering) fingerprint spectrum of starwort root - Google Patents
Method for establishing HPLC-ELSD (high Performance liquid chromatography-evaporative light scattering) fingerprint spectrum of starwort root Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 47
- 238000001228 spectrum Methods 0.000 title claims description 23
- 238000001474 liquid chromatography-evaporative light scattering detection Methods 0.000 title description 2
- 240000007641 Spergula rubra Species 0.000 title 1
- 244000018687 Stellaria dichotoma Species 0.000 claims abstract description 30
- 240000006694 Stellaria media Species 0.000 claims abstract description 30
- 238000000105 evaporative light scattering detection Methods 0.000 claims abstract description 22
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- NJKOMDUNNDKEAI-UHFFFAOYSA-N beta-sitosterol Natural products CCC(CCC(C)C1CCC2(C)C3CC=C4CC(O)CCC4C3CCC12C)C(C)C NJKOMDUNNDKEAI-UHFFFAOYSA-N 0.000 claims description 32
- KZJWDPNRJALLNS-VJSFXXLFSA-N sitosterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CC[C@@H](CC)C(C)C)[C@@]1(C)CC2 KZJWDPNRJALLNS-VJSFXXLFSA-N 0.000 claims description 32
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention discloses a starwort root HPLC-ELSD fingerprint establishing method, which belongs to the technical field of traditional Chinese medicine fingerprints, and the fingerprint establishing method comprises the step of analyzing and measuring the high performance liquid chromatography fingerprint of the starwort root by adopting an HPLC-ELSD chromatography, wherein the chromatographic conditions of the HPLC-ELSD chromatography comprise: the chromatographic column is C18; mobile phase: performing gradient elution with the mobile phase A being methanol and the mobile phase B being water; a detector: an evaporative light scattering detector. According to the method, the problem that sterol which is a main active ingredient of Stellaria dichotoma is difficult to detect due to ultraviolet terminal absorption is solved, the obtained characteristic peak of the fingerprint can reflect the content of chemical substances in Stellaria dichotoma more directly and accurately through the relative peak area, the quality of the Stellaria dichotoma medicinal material is further better evaluated, a convenient extraction method of the Stellaria dichotoma ethyl acetate extract and a fingerprint establishment method are established, and the obtained chromatographic characteristic peak separation effect of the Stellaria dichotoma is better.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine fingerprints, and particularly relates to a starwort root HPLC-ELSD fingerprint establishing method.
Background
The dichotoma is a genuine medicinal material for Ningxia and is a traditional good medicine for clearing deficiency heat and removing infantile malnutrition heat in China. With the deepening of modern medical research, the good efficacies of the starwort root such as clearing heat, cooling blood, resisting inflammation, resisting allergy, resisting cancer and the like are gradually discovered, the medicinal value is continuously improved, and the social demand is continuously increased. In recent years, under the condition that the wild bupleurum chinense resources tend to be deficient, the cultivated bupleurum chinense becomes the mainstream of the market. However, in the process of changing from wild cultivation to cultivation, the medicinal material quality of Stellaria dichotoma is uneven due to factors such as different cultivation production areas, field management, processing modes and the like. However, related scientific research of the starwort root as a small-variety medicinal material is still imperfect, so that an effective method for evaluating the quality of the starwort root medicinal material still does not exist at present, and the development of the starwort root industry and the progress of basic research are influenced to a certain extent.
The fingerprint spectrum is based on the knowledge of the whole action of the Chinese medicine substance group, and the spectrum or chromatogram of the chemical components of the Chinese medicine is obtained by means of the technologies of spectrum, chromatogram and the like, so that the fingerprint spectrum is a feasible mode for realizing the identification of the authenticity of the Chinese medicine, the evaluation of quality consistency and the stability of products. Chromatography is one of the fastest developing and most widely applied analysis methods in the field of analytical chemistry, is also the most basic technology of traditional Chinese medicine fingerprints and the key technology of modern traditional Chinese medicine industry, and High Performance Liquid Chromatography (HPLC) fingerprints are one of the more mature technologies of traditional Chinese medicine quality control at present. The detector in high performance liquid analysis is an important hardware component and a qualitative and quantitative basis of substances. In the effective components of Stellaria dichotoma in the prior research and report, sterol is always considered as the main component for clearing deficiency heat, but the sterol component generally has ultraviolet terminal absorption, so that the detection effect of the commonly used optical detector on the main effective components of Stellaria dichotoma is poor.
Disclosure of Invention
The invention aims to: in order to solve the problem that the detection effect of a common optical detector on the main active ingredients of Stellaria dichotoma is poor, the method for establishing the HPLC-ELSD fingerprint spectrum of Stellaria dichotoma is provided.
In order to achieve the purpose, the invention adopts the following technical scheme:
a starwort root HPLC-ELSD fingerprint spectrum establishment method comprises the step of analyzing and measuring a high performance liquid chromatography fingerprint spectrum of the starwort root by adopting an HPLC-ELSD chromatography, wherein the HPLC-ELSD chromatography conditions comprise that:
the chromatographic column is C18;
mobile phase: performing gradient elution with the mobile phase A being methanol and the mobile phase B being water;
a detector: an evaporative light scattering detector.
As a further description of the above technical solution:
the standard control solution for HPLC-ELSD chromatography was prepared according to the following steps: taking a proper amount of standard reference substances of the alpha-bromosterol and the beta-sitosterol, precisely weighing, dissolving by using ethyl acetate, fixing the volume, preparing a standard reference substance solution of the alpha-bromsterol and the beta-sitosterol, and storing at a dark place at 4 ℃ for later use.
As a further description of the above technical solution:
the test solution for HPLC-ELSD chromatography was prepared according to the following procedure: precisely weighing 2-5g of starwort root powder which is sieved by a 40-mesh sieve, placing the powder in a 50ml conical flask with a plug, pouring an extraction solvent, soaking for 25-30min, carrying out ultrasonic extraction for 30min, standing for 3min, taking out supernatant, pouring residues in the extraction solvent, carrying out secondary extraction, combining extracting solutions, rapidly filtering, placing filtrate in a rotary evaporator, concentrating to 2-5ml at 60 ℃, transferring concentrated solution to a 10ml volumetric flask, and precisely fixing the volume by using the extraction solvent to obtain the medicine.
As a further description of the above technical solution:
the extraction solvent is ethyl acetate.
As a further description of the above technical solution:
the gradient elution procedure was performed in the following volume concentration configuration:
at 0 minute, the volume percent of mobile phase a was 30.0%, and the volume percent of mobile phase B was 70%;
at 5 minutes, the volume percent of the mobile phase A is 30.0 percent, and the volume percent of the mobile phase B is 70 percent;
at 10 minutes, the volume percent of the mobile phase A is 70.0 percent, and the volume percent of the mobile phase B is 30 percent;
at 15 minutes, the volume percent of the mobile phase A is 70.0 percent, and the volume percent of the mobile phase B is 30 percent;
at 18 minutes, the volume percent of mobile phase a was 86.0%, and the volume percent of mobile phase B was 14%;
at 32 minutes, the volume percent of mobile phase a was 86.0%, and the volume percent of mobile phase B was 14%;
at 37 minutes, the volume percent of the mobile phase A is 91.0 percent, and the volume percent of the mobile phase B is 9 percent;
at 57 minutes, the volume percent of mobile phase a was 91.0%, and the volume percent of mobile phase B was 9%;
at 60 minutes, the volume percent of the mobile phase A is 95.0 percent, and the volume percent of the mobile phase B is 5 percent;
at 95 minutes, the volume percent of the mobile phase A is 95.0 percent, and the volume percent of the mobile phase B is 5 percent;
at 100 minutes, the volume percent of the mobile phase A is 30.0 percent, and the volume percent of the mobile phase B is 70 percent;
at 110 minutes, the volume percent of mobile phase a was 30.0% and the volume percent of mobile phase B was 70%.
As a further description of the above technical solution:
the chromatographic column is a Waters Sun Fire C18 chromatographic column, and the specification is as follows: 4.6X 250mm,5 μm.
As a further description of the above technical solution:
the chromatographic conditions further comprise:
the drift tube temperature of the evaporative light scattering detector was 75 ℃;
the flow rate of the carrier gas is 1.6L/min;
the column temperature was 40 ℃;
the injection amount is 20 mul each time;
the flow rate was 0.8 ml/min.
As a further description of the above technical solution:
and the chromatogram analysis adopts computer data processing software to subtract a base line from the obtained chromatogram, calculate retention time, peak height and peak area and carry out characteristic peak identification.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. in the invention, a high performance liquid evaporation light scattering combined technology (HPLC-ELSD) is applied to the radix Stellariae fingerprint technology, so that the problem that sterol which is a main active ingredient of radix Stellariae is difficult to detect due to ultraviolet terminal absorption is solved, the evaporation light scattering gets rid of the dependence of a sample on optical characteristics, the samples respond to all substances, and the response value of the substances can better reflect the content of chemical substances in traditional Chinese medicines. Therefore, the established radix Stellariae fingerprint characteristic peak can more directly and accurately reflect the content of chemical substances in the radix Stellariae through the relative peak area, and further better evaluate the quality of the radix Stellariae medicinal material. Meanwhile, the method for extracting the bupleurum falcatum ethyl acetate extract and the method for establishing the fingerprint spectrum are convenient and fast, the separation effect of the chromatographic characteristic peak of the obtained bupleurum falcatum is good, and the used reagent materials are simple and low in toxicity, are suitable for popularization and have high practicability.
2. In the invention, the reference standard substance is added, chromatographic peaks of alpha-bromosterol and beta-sitosterol are identified in the fingerprint, and accurate, qualitative and quantitative determination of the alpha-bromsterol and the beta-sitosterol can be simultaneously realized while the starwort root fingerprint information is obtained.
3. The process and conditions of the gradient elution procedure are important parts of the method, and by adopting the gradient elution procedure and conditions, the characteristic peaks of the starwort root fingerprint can be better separated, the separation effect is improved, the starwort root fingerprint characteristics are better reflected, and the accuracy of the starwort root quality identification result is greatly improved.
Drawings
FIG. 1 is ELSD-HPLC chromatogram fingerprint of Bupleurum scorzonerifolium Linn sample provided by the invention;
FIG. 2 is an ELSD-HPLC chromatogram of standard reference samples of alpha-bromsterol and beta-sitosterol provided by the invention;
FIG. 3 is the chromatogram fingerprint of Bupleurum scorzonerifolium Linn.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The invention provides a technical scheme that: a starwort root HPLC-ELSD fingerprint spectrum establishment method comprises the steps of analyzing and measuring the high performance liquid phase fingerprint spectrum of the starwort root by adopting an HPLC-ELSD chromatography, and the method comprises the following 4 steps:
s1, preparation of standard control solution for HPLC-ELSD chromatography:
taking a proper amount of standard reference substances of alpha-bromosterol and beta-sitosterol, precisely weighing, dissolving with ethyl acetate, fixing the volume, preparing a solution of the standard reference substances of alpha-bromsterol and beta-sitosterol, and storing at a dark place at 4 ℃ for later use;
s2, preparation of test solution for HPLC-ELSD chromatography:
precisely weighing 2-5g of starwort root powder which is sieved by a 40-mesh sieve, placing the powder in a 50ml conical flask with a plug, pouring an ethyl acetate solution, soaking for 25-30min, carrying out ultrasonic extraction for 30min, standing for 3min, taking out supernatant, pouring residues in the ethyl acetate solution, carrying out secondary extraction, combining extracting solutions, rapidly filtering, placing filtrate in a rotary evaporator, concentrating to 2-5ml at 60 ℃, transferring the concentrated solution to a 10ml volumetric flask, and precisely fixing the volume of the ethyl acetate solution to obtain the medicine;
s3, HPLC-ELSD chromatography determination:
analyzing the prepared reference solution and test solution, wherein the chromatographic conditions of HPLC-ELSD chromatography comprise:
the chromatographic column is C18;
mobile phase: performing gradient elution with the mobile phase A being methanol and the mobile phase B being water;
a detector: an evaporative light scattering detector;
wherein the gradient elution conditions are detailed in table 1:
TABLE 1 gradient elution conditions
| Time (min) | A(％) | B(％) |
| 0.00 | 30.0 | 70.0 |
| 5.00 | 30.0 | 70.0 |
| 10.00 | 70.0 | 30.0 |
| 15.00 | 70.0 | 30.0 |
| 18.00 | 86.0 | 14.0 |
| 32.00 | 86.0 | 14.0 |
| 37.00 | 91.0 | 9.0 |
| 57.00 | 91.0 | 9.0 |
| 60.00 | 95.0 | 5.0 |
| 95.00 | 95.0 | 5.0 |
| 100.00 | 30.0 | 70.0 |
S4, spectrum analysis:
and generating chromatographic maps of the standard reference substance solution and the test substance solution by using computer software.
Example 2
The invention provides another specific implementation scheme, a starwort root HPLC-ELSD fingerprint spectrum establishing method, which comprises the steps of analyzing and measuring the high performance liquid phase fingerprint spectrum of the starwort root by adopting an HPLC-ELSD chromatography, and the method comprises the following 4 steps:
s1, preparation of standard control solution for HPLC-ELSD chromatography:
taking a proper amount of standard reference substances of alpha-bromosterol and beta-sitosterol, precisely weighing, dissolving with ethyl acetate, fixing the volume, preparing 0.5696mg/ml of alpha-bromsterol and 0.4024mg/ml of beta-sitosterol standard reference substance solution, and storing at a dark place at 4 ℃ for later use;
s2, preparation of test solution for HPLC-ELSD chromatography:
precisely weighing 2g of starwort root powder which is sieved by a 40-mesh sieve, placing the powder in a 50ml conical flask with a plug, pouring an ethyl acetate solution, soaking for 30min, ultrasonically extracting for 30min, standing for 3min, taking out supernatant, pouring residues into 20ml of the ethyl acetate solution, carrying out secondary extraction, combining extracting solutions, rapidly filtering, placing filtrate in a rotary evaporator, concentrating to 2-5ml at 60 ℃, transferring the concentrated solution into a 10ml volumetric flask, and precisely fixing the volume of the ethyl acetate solution to obtain the product;
wherein, the ethyl acetate adopts chromatographic grade, and the concentration is required to be more than 98 percent;
s3, HPLC-ELSD chromatography determination:
analyzing the prepared reference solution and test solution, wherein the chromatographic conditions of HPLC-ELSD chromatography comprise:
the chromatographic column is C18;
mobile phase: performing gradient elution with the mobile phase A being methanol and the mobile phase B being water;
a detector: an evaporative light scattering detector;
wherein Agilent 1260 high performance liquid chromatograph, Agilent 1260Infinity evaporation light scattering detector and Waters Sun Fire C18 chromatographic column (5 μm, 4.6 × 250mm) are selected, the set parameters are drift tube temperature of 75 ℃, carrier gas flow rate of 1.6L/min, column temperature of 40 ℃, 20 μ L of sample injection each time, flow rate of 0.8ml/min, mobile phase A is methanol, B is water, gradient elution is carried out, and the gradient elution conditions are the same as those of embodiment 1;
s4, spectrum analysis:
adopting data processing software such as Origin 2018 and the like to subtract a base line from chromatographic spectrums of the obtained standard reference substance solution and the obtained test substance solution, calculating retention time, peak height, peak area and the like, and performing characteristic peak identification; and comprehensively evaluating the similarity of the chromatogram by adopting 'traditional Chinese medicine chromatogram fingerprint similarity evaluation system software' 2012A edition.
Example 3
The present invention further provides an embodiment, which can quantitatively determine the content of α -bromsterol and β -sitosterol in a test sample, and the embodiment is different from the embodiment 2 in that the embodiment further comprises:
taking 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0ml to 10ml volumetric flasks of the standard reference product mother liquor prepared in the step S1 of example 2 respectively, accurately fixing the volume with ethyl acetate solution, preparing standard reference product solutions with different concentrations, detecting the obtained solution according to the method, drawing a standard curve by taking the concentration of the reference product as a horizontal coordinate (X) and the peak area of the peak of the reference product as a vertical coordinate (Y), and calculating a regression equation and a correlation coefficient (namely R)2Correlation coefficient greater than 0.99 is required);
substituting the peak areas of the alpha-bromsterol peak and the beta-sitosterol peak of the test sample measured in the step S4 in example 2 into the regression equation by taking the obtained regression equation as a standard, and calculating the contents of the alpha-bromsterol and the beta-sitosterol in the test sample.
Of course, this embodiment is only for the convenience of understanding of those skilled in the relevant art, and is not limited to the preconditions such as the mother liquor concentration of the standard reference substance set in embodiment 2, and may satisfy that the correlation coefficient is greater than 0.99.
Test examples
The experimental example passes through the verification method feasibility and carries out the example experiment, and the main scheme is as follows:
1. methodology investigation
(1) And (3) linear relation investigation: taking 0.5 ml to 1.0 ml, 1.5 ml to 2.0 ml, 2.5 ml to 3.0ml volumetric flasks of the standard reference substance solution in the example 2 respectively, accurately fixing the volume by using ethyl acetate solution, preparing standard reference substance solutions with different concentrations, detecting the obtained solution according to the method, taking the concentration of the reference substance as an abscissa (X),the peak area of the reference peak is an ordinate (Y), a standard curve is drawn, the linear regression equation of the alpha-borsterol is obtained, namely Y is 7047X-0.8942, and R is20.9912, the linear regression equation of beta-sitosterol is that Y is 1480X-27.441, R20.9952, indicating that the linear relationship is good, and can be used for subsequent content determination.
(2) Precision investigation: taking the same Bupleurum scorzonerifolium medicinal material, preparing a test solution according to the method in the embodiment 2, setting chromatographic conditions and the like, continuously injecting a sample of 6 needles, recording a chromatogram, taking alpha-bromsterol and beta-sitosterol as reference peaks, and calculating the relative retention time and the relative peak area of the two chromatographic peaks.
The result shows that the RSD values of the relative retention time of the alpha-bromsterol and the beta-sitosterol in the fingerprint are 0.45 percent and 0.39 percent respectively, and the relative standard deviation (RSD value) of the relative peak area is 3.58 percent and 3.04 percent respectively, which indicates that the precision of the instrument is good.
(3) And (3) repeatability inspection: taking 6 parts of the same Stellaria dichotoma medicinal material, preparing 6 parts of test solution in parallel according to the method in example 2, recording a chromatogram under the same chromatographic condition, taking alpha-bromsterol and beta-sitosterol as reference peaks, and calculating the relative retention time and the relative peak area of the two chromatographic peaks.
The result shows that the RSD values of the relative retention time of the alpha-bromsterol and the beta-sitosterol in the fingerprint are 0.42 percent and 0.46 percent respectively, and the RSD values of the relative peak areas are 3.78 percent and 3.95 percent respectively, which indicates that the method has good repeatability.
(4) And (3) stability investigation: taking the same Bupleurum scorzonerifolium medicinal material, preparing a test solution according to the method in the embodiment 2, setting chromatographic conditions, carrying out sample injection analysis on the prepared test solution for 0, 2, 4, 8, 12 and 24 hours respectively, recording a chromatogram, and calculating relative retention time and relative peak area of two chromatographic peaks by taking alpha-bromsterol and beta-sitosterol as reference peaks.
The result shows that the RSD values of the relative retention time of the alpha-bromsterol and the beta-sitosterol in the fingerprint are 0.37 percent and 0.42 percent respectively, and the RSD values of the relative peak areas are 3.57 percent and 3.45 percent respectively, which indicates that the test solution has good stability within 24 hours at normal temperature.
(5) And (3) sample recovery rate investigation: precisely weighing 2g and 6 parts of starwort root powder which is sieved by a 40-mesh sieve and has known contents of alpha-bromsterol and beta-sitosterol, precisely adding 1ml of standard reference substance solution prepared in linear relation investigation and having different concentrations respectively, preparing a sample solution by referring to the method in example 2, measuring peak areas of an alpha-bromsterol peak and a beta-sitosterol peak of a sample, calculating the contents of the alpha-bromsterol and the beta-sitosterol according to a regression equation in the linear relation investigation, and calculating to obtain an average recovery rate and an RSD value.
The result shows that the average recovery rate of the alpha-bromsterol is between 95.46 and 103.23 percent, the RSD is less than 5 percent, and the method has good recovery rate.
2. Example test:
(1) sample collection
Collecting 17 parts of Stellaria dichotoma including wild and cultivated, different producing areas and different drying methods, sun-drying the collected Stellaria dichotoma until the weight is constant, wherein the detailed information of the sample is shown in Table 2, pulverizing the dried Stellaria dichotoma sample, sieving with a 40-mesh sieve, and storing at low temperature in a dark place in a sealed manner for later use.
TABLE 2 sample Collection information
(2)17 parts of radix Stellariae sample fingerprint
Preparing a test solution of 17 parts of Stellaria dichotoma medicinal material according to the method in example 2, setting chromatographic conditions and the like, and detecting to obtain 17 parts of synthetic Stellaria dichotoma fingerprint, as shown in figure 1;
17 portions of Bupleurum scorzonerifolium High Performance Liquid Chromatography (HPLC) spectra were overlaid by Origin 2018 software to determine 15 common peaks (retention time of common peaks is shown in Table 3). The fingerprint spectrums and characteristic peaks of the starwort root which are displayed by the spectrums and have different producing areas, wild areas, cultivation methods and different drying methods show different differences, which shows that the content of chemical substances of the starwort root samples has different differences, and further the quality of medicinal materials has larger difference, so that the quality of the starwort root is judged according to the difference of the content of specific chemical substances.
ELSD-HPLC chromatogram fingerprint peak positions of 315 Stellaria dichotoma Maxim
(3) Identification of characteristic peaks
The standard alpha-bromsterol and beta-sitosterol control prepared in example 2 were used for detection under the same conditions, 15 common characteristic peaks were identified by the same retention time and peak shape characteristics, and finally C13 and C15 in table 3 were identified as the characteristic peaks of alpha-bromsterol and beta-sitosterol, respectively (the chromatographic peaks and starwort fingerprint of the standard control are shown in fig. 2 and fig. 3).
(4) Determination of contents of starwort alpha-borsterol and beta-sitosterol
According to the method in example 2 and the regression equation in linear relationship investigation, the contents of alpha-bromsterol and beta-sitosterol in 17 parts of Stellaria dichotoma are measured, peak areas corresponding to alpha-bromsterol and beta-sitosterol in different samples are substituted into the regression equation, and X is obtained to obtain the contents of alpha-bromsterol and beta-sitosterol in the samples, and the results are shown in Table 4.
The content of alpha-bromsterol and beta-sitosterol of 17 parts of Stellaria dichotoma is different, which shows that the content of effective components of Stellaria dichotoma medicinal materials from different sources is different, and further reflects that the quality of the medicinal materials is different, so that the quality of the Stellaria dichotoma is identified and graded according to the content of the effective components.
TABLE 417 weight Stellaria root samples fingerprint alpha-bromsterol and beta-sitosterol content (mg/g)
(5) Evaluation of similarity
The method comprises the following steps of comprehensively evaluating the similarity of 17 Stellaria dichotoma samples by adopting ' traditional Chinese medicine chromatogram fingerprint similarity evaluation system software ' 2012A version ', wherein the result is shown in Table 5, the fingerprint similarity of all samples is over 0.90, but certain difference exists, which shows that the fingerprints of the 17 Stellaria dichotoma samples have certain consistency, the similarity of all Stellaria dichotoma medicinal materials reaches over 0.900, and certain specificity exists at the same time, such as: YCH 1-YCH 4 are all wild Stellaria delavayi Diels medicinal materials, the similarity is between 0.91-0.93, YCH 6-YCH 11 are cultivated medicinal materials, and the similarity is over 0.93; YCH 12-YCH 17 are used as the same batch of Stellaria dichotoma and are treated by adopting different drying methods, wherein the YCH12 and YCH13 of the naturally dried Stellaria dichotoma sample are both lower than 0.91, and the similarity of the samples treated by hot air drying is more than 0.91, so the fingerprint has certain application effect on distinguishing Stellaria dichotoma medicinal materials from different sources, has good Stellaria dichotoma quality evaluation application prospect, and can be applied on identifying the Stellaria dichotoma medicinal materials with different qualities and sources.
TABLE 517 fingerprint similarity of Bupleurum scorzonerifolium samples
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (8)
1. The dichotoma HPLC-ELSD fingerprint spectrum establishing method is characterized in that the spectrum establishing method comprises the step of analyzing and measuring the dichotoma by adopting an HPLC-ELSD chromatography method to obtain a high performance liquid fingerprint spectrum, wherein the HPLC-ELSD chromatography condition comprises the following steps:
the chromatographic column is C18;
mobile phase: performing gradient elution with the mobile phase A being methanol and the mobile phase B being water;
a detector: an evaporative light scattering detector.
2. The method for establishing HPLC-ELSD fingerprint spectrum of Bupleurum scorzonerifolium Linn according to claim 1, wherein the standard reference solution for HPLC-ELSD chromatography is prepared by the following steps: taking a proper amount of standard reference substances of the alpha-bromosterol and the beta-sitosterol, precisely weighing, dissolving by using ethyl acetate, fixing the volume, preparing a standard reference substance solution of the alpha-bromsterol and the beta-sitosterol, and storing at a dark place at 4 ℃ for later use.
3. The method for establishing HPLC-ELSD fingerprint spectrum of Bupleurum scorzonerifolium Linn according to claim 1, wherein the sample solution of HPLC-ELSD chromatography is prepared by the following steps: precisely weighing 2-5g of starwort root powder which is sieved by a 40-mesh sieve, placing the powder in a 50ml conical flask with a plug, pouring an extraction solvent, soaking for 25-30min, carrying out ultrasonic extraction for 30min, standing for 3min, taking out supernatant, pouring residues in the extraction solvent, carrying out secondary extraction, combining extracting solutions, rapidly filtering, placing filtrate in a rotary evaporator, concentrating to 2-5ml at 60 ℃, transferring concentrated solution to a 10ml volumetric flask, and precisely fixing the volume by using the extraction solvent to obtain the medicine.
4. The HPLC-ELSD fingerprint establishment method of Bupleurum scorzonerifolium Linn as claimed in claim 3, wherein the extraction solvent is ethyl acetate.
5. The HPLC-ELSD fingerprint establishment method of Bupleurum scorzonerifolium Linn as claimed in claim 1, wherein the gradient elution procedure is performed according to the following volume concentration configuration:
at 0 minute, the volume percent of mobile phase a was 30.0%, and the volume percent of mobile phase B was 70%;
at 5 minutes, the volume percent of the mobile phase A is 30.0 percent, and the volume percent of the mobile phase B is 70 percent;
at 10 minutes, the volume percent of the mobile phase A is 70.0 percent, and the volume percent of the mobile phase B is 30 percent;
at 15 minutes, the volume percent of the mobile phase A is 70.0 percent, and the volume percent of the mobile phase B is 30 percent;
at 18 minutes, the volume percent of mobile phase a was 86.0%, and the volume percent of mobile phase B was 14%;
at 32 minutes, the volume percent of mobile phase a was 86.0%, and the volume percent of mobile phase B was 14%;
at 37 minutes, the volume percent of the mobile phase A is 91.0 percent, and the volume percent of the mobile phase B is 9 percent;
at 57 minutes, the volume percent of mobile phase a was 91.0%, and the volume percent of mobile phase B was 9%;
at 60 minutes, the volume percent of the mobile phase A is 95.0 percent, and the volume percent of the mobile phase B is 5 percent;
at 95 minutes, the volume percent of the mobile phase A is 95.0 percent, and the volume percent of the mobile phase B is 5 percent;
at 100 minutes, the volume percent of the mobile phase A is 30.0 percent, and the volume percent of the mobile phase B is 70 percent;
at 110 minutes, the volume percent of mobile phase a was 30.0% and the volume percent of mobile phase B was 70%.
6. The HPLC-ELSD fingerprint establishment method of Bupleurum scorzonerifolium Linn according to claim 1, wherein the chromatographic column is Waters Sun Fire C18 chromatographic column with specification: 4.6X 250mm,5 μm.
7. The HPLC-ELSD fingerprint establishment method of Bupleurum scorzonerifolium Linn according to claim 1, wherein the chromatographic conditions further include:
the drift tube temperature of the evaporative light scattering detector was 75 ℃;
the flow rate of the carrier gas is 1.6L/min;
the column temperature was 40 ℃;
the injection amount is 20 mul each time;
the flow rate was 0.8 ml/min.
8. The HPLC-ELSD fingerprint establishment method for Stellaria dichotoma according to claim 1, wherein the chromatogram analysis adopts computer data processing software to subtract a baseline from the obtained chromatogram, calculate retention time, peak height and peak area, and perform characteristic peak identification.
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