CN111929378A - Method for measuring content of 6 index components of gastrodia elata in Qingda granules - Google Patents
Method for measuring content of 6 index components of gastrodia elata in Qingda granules Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
Abstract
The invention provides a method for measuring the content of 6 index components of gastrodia elata in Qingda particles, which improves the preparation method of a sample solution, takes Qingda particles with different loading amounts, grinds the Qingda particles, precisely scales the Qingda particles, adds a phosphoric acid aqueous solution, scales the weight, carries out ultrasonic treatment, cools the Qingda particles, complements the lost weight with the phosphoric acid aqueous solution, carries out centrifugal treatment, takes supernatant liquid to pass through a polyamide column, adds an elution solvent to carry out elution, collects eluent, adds the phosphoric acid aqueous solution to fix the volume, shakes the mixture evenly, and takes a subsequent filtrate. The method is rapid and accurate, can effectively control the quality of the rhizoma gastrodiae, realizes qualitative research on the rhizoma gastrodiae and the scutellaria baicalensis in the Qingda granules, and provides a basis for the development of the Qingda granules and other preparations. The invention can reduce the interference of other 3 medicinal materials in the Qingdao granules on the gastrodia elata, can greatly reduce other impurity components in the gastrodia elata, enriches 6 index components, improves the purity of the index components and reduces the interference of impurity peaks.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical analysis, and relates to a method for determining the content of 6 index components of gastrodia elata in Qingda granules.
Background
Hypertension is a main risk factor of various cardiovascular diseases, such as cerebral apoplexy, heart failure, atherosclerosis, vascular hypertrophy and the like, and about one third of adults in China suffer from hypertension and show a continuously rising trend. The Chinese guidelines for hypertension control in 2018 show that the treatment rate and the control rate of adult hypertension in China are 46.1% and 16.9% respectively. Still at a lower level compared to developed countries. Therefore, hypertension prevention and treatment are still key problems to be solved urgently in the research field at present.
The ancient books of traditional Chinese medicine do not have the names of hypertension, and medical doctors at all ages classify the ancient books of traditional Chinese medicine into diseases such as vertigo, headache, liver wind and the like according to other symptoms and physical signs. The traditional Chinese medicine university Chenke Ji hospital thinks that the onset of hypertension has an evolution process from yang exuberance to yin-yang deficiency, and the primary clinical hypertension is mainly characterized by hyperactivity of liver yang or flaring up of liver fire, so the Chenke Ji hospital establishes a decoction for clearing and reducing blood pressure and dizziness on the basis of the decoction of gastrodia and uncaria so as to treat the hypertension patients with the symptoms. Clinical studies have shown that: the dizziness-clearing and blood pressure-reducing soup can better control blood pressure and effectively improve clinical symptoms and life quality of hypertension patients (Yujun, Xufengqi, the dizziness-clearing and blood pressure-reducing soup treats hypertension with liver-kidney yin deficiency and liver-yang hyperactivity type [ J ] the journal of cardiovascular and cerebrovascular diseases combining traditional Chinese and western medicine, 2010,8(1): 1-3). The Qingda granules are prepared by condensing the Qingxuan Jiangyu decoction according to the concept of early treatment and prevention. The QINGDA granule is prepared from rhizoma Gastrodiae, ramulus Uncariae cum uncis, plumula Nelumbinis, and Scutellariae radix (CN 106421447A). The rhizoma Gastrodiae mainly contains phenolic compounds containing benzene ring structure, and exists in the form of glycoside and aglycone, and typical glycoside component gastrodine, and aglycone thereof p-hydroxybenzol, and also contains polysaccharide and other phenolic substances. Modern pharmacological research shows that the gastrodia elata has the effects of reducing blood pressure, protecting cardiac muscle, resisting blood coagulation, resisting thrombus, regulating immunity and the like. The gastrodia elata contains gastrodin, gastrodin and the like, the blood pressure reducing parts are positioned in the nerve center, so that the vascular resistance can be reduced, arterioles and capillaries can be dilated, the blood pressure can be reduced for more than 3 hours, the sympathetic nerve is prevented from being activated, and the blood pressure is stably and effectively controlled. The total amount of gastrodin, parahydroxybenzyl alcohol and barbalin A, B, C, E is used as the quality control index of rhizoma Gastrodiae because the conversion relationship exists between the Barrison glycoside substance and gastrodin in rhizoma Gastrodiae.
At present, the content determination method of the gastrodia elata mainly comprises high performance liquid chromatography (HPLC method), reverse high performance liquid chromatography, near infrared spectroscopy, fingerprint spectrum and the like, wherein the HPLC method is most widely applied, and the content of the gastrodin is determined by the HPLC method according to the specification under the content determination item of the gastrodia elata in Chinese pharmacopoeia (2015 edition). However, the method is more suitable for measuring the content of gastrodin in the gastrodia elata medicinal material, and the report on the content measurement of the gastrodia elata in the compound prescription is less. Simultaneously, for other 5 effective chemical components in the gastrodia elata: the determination of the content of p-hydroxybenzyl alcohol, balsamoside A, B, C, E, also did not specify whether it is suitable. Meanwhile, the related determination methods for determining the content of 6 components of the gastrodia elata are few in reports, and in addition, the HPLC method is long in analysis time and complicated in operation process. How to realize the method for rapidly, accurately and suitably and simultaneously measuring 6 index components in the gastrodia elata, wherein the method comprises the step of simultaneously measuring 6 effective components in the gastrodia elata in the compound preparation such as Qingda granules, and further research and discussion are needed.
Disclosure of Invention
Problems to be solved by the invention
In order to overcome the technical problems in the prior art, the invention provides a method for measuring the content of 6 index components of gastrodia elata in Qingda granules, and the method can also realize qualitative analysis of gastrodia elata and scutellaria baicalensis in Qingda granules.
Means for solving the problems
In one technical scheme, the invention provides a method for determining the content of 6 index components of gastrodia elata in Qingda granules, which comprises the following steps: preparing test solution, preparing reference solution, measuring with ultra-high performance liquid chromatograph,
wherein, the 6 index components comprise gastrodin, barban A, barban B, barban C, barban E and p-hydroxybenzyl alcohol;
the preparation of the test solution comprises the steps of taking QINGDA particles under the condition of different loading amounts, grinding, precisely weighing, adding a phosphoric acid aqueous solution, weighing, carrying out ultrasonic treatment, cooling, complementing the weight loss by using the phosphoric acid aqueous solution, carrying out centrifugal treatment, taking supernate, passing through a polyamide column, adding an elution solvent for elution, collecting eluent, adding the phosphoric acid aqueous solution for constant volume, shaking uniformly, and taking a subsequent filtrate.
In another embodiment, the polyamide column is 80-100 mesh or 100-200 mesh polyamide column, preferably 80-100 mesh polyamide column.
In another embodiment, the elution solvent comprises one or more of an aqueous phosphoric acid solution, water or an ethanol solution, preferably the elution solvent is an aqueous phosphoric acid solution.
In another embodiment, the aqueous phosphoric acid solution is a 0.05% aqueous phosphoric acid solution, the percentage being volume percent.
In another embodiment, the hplc adopts a binary mobile phase system for gradient elution, wherein the mobile phase a is a 60-80% methanol solution, preferably a 70% methanol solution, and the mobile phase B is a phosphoric acid aqueous solution, preferably a 0.02-0.1% phosphoric acid aqueous solution, more preferably a 0.05% phosphoric acid aqueous solution, where the percentages are by volume.
In another embodiment, the methanol solution comprises a sodium dodecyl sulfate solution. Further, the concentration of the sodium dodecyl sulfate solution is 1-15 mmol/L, more preferably 3-8 mmol/L, and still more preferably 5 mmol/L.
In another embodiment, the preparation of the reference solution comprises taking a proper amount of gastrodin reference, precisely weighing, adding acetonitrile-water solution to prepare (preferably to prepare 1mL of mixed solution containing 80 μ g of gastrodin), and shaking up.
In another embodiment, the gradient elution conditions of the present invention are as follows:
| time/min | Mobile phase A/%) | Mobile phase B/%) |
| 0 | 8 | 92 |
| 5 | 8 | 92 |
| 6 | 26 | 74 |
| 9 | 28 | 72 |
| 19 | 55 | 45 |
| 20 | 100 | 0 |
Further preferably, the flow rate of the gradient elution is 0.2-0.4 mL/min, and more preferably, the flow rate of the gradient elution is 0.3 mL/min.
In another embodiment, the chromatographic column of the ultra high performance liquid chromatograph uses dodecyl silane bonded silica gel as a filler, and the chromatographic column is further preferably selected from an Agilent Eclipse Plus C18 chromatographic column.
In another embodiment, the column temperature of the ultra-high performance liquid chromatograph is 30 to 40 ℃, and further the column temperature is 35 ℃.
Further, the invention also provides a method for qualitatively analyzing the gastrodia elata and the scutellaria baicalensis in the Qingda granules, and the method comprises any one of the methods.
ADVANTAGEOUS EFFECTS OF INVENTION
The method disclosed by the invention realizes simultaneous determination of the contents of 6 components including gastrodin, barbadensin A, B, C, E and p-hydroxybenzene methanol in Gastrodia elata Blume particles by using UPLC and a one-test-multiple evaluation method, and also realizes qualitative research on Gastrodia elata Blume and Scutellaria baicalensis in the Qingda particles. By improving the preparation method of the rhizoma gastrodiae test solution, the interference of other 3 medicinal materials in the Qingda granules on the rhizoma gastrodiae can be further reduced, particularly the interference of scutellaria baicalensis on the determination of the rhizoma gastrodiae is reduced, 6 index components are enriched, the purity of the index components is improved, and the interference of impurity peaks is reduced. The method is simple, good in reproducibility, accurate and reliable, convenient to operate, and less in time, solvent consumption and environmental pollution compared with HPLC.
Drawings
Fig. 1 shows the ultra-high performance liquid chromatogram of gastrodin control.
Figure 2 shows an ultra high performance liquid chromatogram of a dada particle sample.
Figure 3 shows a qingda particle ultra high performance liquid chromatogram with mobile phase a of 70% methanol.
FIG. 4 shows an ultra high performance liquid chromatogram of Qingdao particles with mobile phase A of 70% methanol (containing 1mmol/L sodium dodecyl sulfate).
FIG. 5 shows an ultra high performance liquid chromatogram of Qingdao particles with mobile phase A of 70% methanol (containing 10mmol/L sodium dodecyl sulfate).
FIG. 6 shows an ultra high performance liquid chromatogram of Qingdao particles with mobile phase A of 70% methanol (containing 5mmol/L dipotassium hydrogen phosphate).
Fig. 7 shows the ultra-high performance liquid chromatogram of the gastrodia elata sample which is not processed by the polyamide column.
FIG. 8 shows an ultra-high performance liquid chromatogram of a sample of Gastrodia elata Blume processed through a polyamide column (100-200 mesh).
Fig. 9 shows ultra-high performance liquid chromatogram and mixed standard chromatogram of rhizoma Gastrodiae sample treated with different elution solvents.
Fig. 10 shows ultra high performance liquid chromatograms for different column temperature studies.
Figure 11 shows an ultra high performance liquid chromatogram for different flow rate studies.
Figure 12 shows ultra high performance liquid chromatograms for different chromatographic column studies.
Fig. 13 shows a gastrodin linear relationship diagram.
FIG. 14 shows a linear relationship of p-hydroxybenzyl alcohol.
Figure 15 shows a linear plot of the balisonide E.
Figure 16 shows a linear plot of the bacitracin B.
Figure 17 shows a linear plot of the bacitracin C.
Figure 18 shows a linear plot of the bacitracin a.
Detailed Description
In the following detailed description, numerous specific details are set forth in order to provide a better understanding of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other instances, methods, means, devices and steps which are well known to those skilled in the art have not been described in detail so as not to obscure the invention.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
In the present specification, the numerical range represented by "numerical value a to numerical value B" means a range including the end point numerical value A, B.
In the present specification, the meaning of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.
It should be understood that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
In the present specification, reference to "one or some particular/preferred embodiments", "another or some other particular/preferred embodiments", "one or another embodiment", or the like, means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments.
The terms "comprises" and "comprising," and any variations thereof, in the description and claims of this invention and the above-described drawings are intended to cover non-exclusive inclusions. For example, a process, method, or system, article, or apparatus that comprises a list of steps or elements is not limited to only those steps or elements listed, but may alternatively include other steps or elements not listed, or inherent to such process, method, article, or apparatus.
The term "control", as used herein, unless otherwise indicated, refers to a standard substance used for identification, inspection, assay and calibration of the performance of assay instruments, generally approved by the national drug certification authority for examination, which standard should not be less than the quality standard for the product.
The term "reference drug substance" (or "reference drug substance") as used herein refers to a standard drug substance that has been species-identified for identifying a test drug substance, unless otherwise specified.
The term "test article" as used herein, unless otherwise specified, refers to an experimental sample used for detection or identification.
As used herein, unless otherwise specified, the term "precisely weigh" means that the weight is weighed to the nearest thousandth of the weight taken, the term "weigh" means that the weight is weighed to the nearest hundredth of the weight taken, the term "precisely measure" means that the volume is measured to the nearest thousandth of the volume taken, and the term "precisely aspirate" means that the sample is accurately measured by the microsyringe.
As used herein, unless otherwise indicated, the term "secondary filtrate" refers to the filtrate collected on filtration after discarding the primary filtrate. Compared with the primary filtrate, the secondary filtrate is closer to the real concentration of the sample, because the filtering medium (such as a filter membrane, filter paper and the like) can possibly adsorb the solute, the concentration of the sample in the primary filtrate is lower; in addition, the subsequent filtrate is cleaner, and because the solute adsorbed by the filter medium can form a filter cake, the filter pore size is reduced, and therefore, more tiny particles can be trapped.
The term "reconstitution solvent" as used herein, unless otherwise specified, refers to a solvent used to dissolve a residue after drying treatment or an extract after concentration treatment to obtain a corresponding solution.
As used herein, unless otherwise indicated, the term "filler" refers to a substance used as a stationary phase in chromatography, the term "stationary phase" refers to a phase that is immobilized in position in chromatography and exerts an adsorptive effect on a sample, and the term "mobile phase" refers to a phase that is not immobilized in position in chromatography and can freely flow and exert a desorption effect on a sample.
As used herein, unless otherwise indicated, the term "binary mobile phase system" refers to a mobile phase consisting of two components, one of which (preferably the former) is generally designated as "mobile phase a" and the other (preferably the latter) is designated as "mobile phase B". For example, in a binary mobile phase system of acetonitrile-aqueous ammonium acetate, mobile phase a is acetonitrile and mobile phase B is aqueous ammonium acetate. However, the above labeling method is not fixed, and the aqueous ammonium acetate solution may be referred to as mobile phase A and the acetonitrile as mobile phase B.
The term "isocratic elution" as used herein, unless otherwise specified, refers to a manner of elution in which the composition, proportion, and flow rate of the mobile phase are constant throughout the chromatographic analysis cycle of a sample.
Unless otherwise indicated, the term "ultra performance liquid chromatography" (or "UPLC") used herein refers to a novel technique developed on the basis of High Performance Liquid Chromatography (HPLC), and has the characteristics of small filler particles, fast detection speed, large analysis flux, high sensitivity, and the like.
The term "retention time" as used herein, unless otherwise specified, refers to the time elapsed from the start of the sample introduction to the time when the maximum concentration of the component occurs after the column, i.e., the time elapsed from the start of the sample introduction to the time when the peak of the chromatographic peak of the separated component occurs; the term "relative retention time" refers to the ratio of the corrected retention time of the separated component to the corrected retention time of the standard; the term "corrected retention time" refers to the retention time of the separated component minus the retention time of air. For example, if the retention time of air is 3s, the retention time of the separated component is 9s, and the retention time of the standard sample is 15s, then the corrected retention time of the separated component is 6s, the corrected retention time of the standard sample is 12s, and the relative retention time of the separated component with respect to the standard sample is 0.5.
As used herein, unless otherwise indicated, the term "reference peak" (or "control peak") refers to the chromatographic peak to which the standard corresponds when calculating the relative retention time.
The term "corresponding peak" as used herein, unless otherwise indicated, refers to the chromatographic peak in the chromatogram of the control of the component corresponding to the standard in the control.
References herein to concentration percentages of solutions "%" refer to volume percentages, unless otherwise indicated. For example, the 0.05% phosphoric acid aqueous solution means that 0.5mL of phosphoric acid is added to 1000mL of pure water, and since a measuring cylinder is not recommended as a container for the mixed solution, the 0.05% phosphoric acid aqueous solution is also approximately equal to 1000mL of pure water mixed with 0.5mL of phosphoric acid.
Specifically, the invention provides a method for measuring the content of 6 index components of gastrodia elata in Qingda granules, which comprises the following steps: preparing a test solution, preparing a reference solution and measuring by adopting an ultra-high performance liquid chromatograph.
< preparation of test sample solution >
The Qingda granules are prepared from Qingxuan Jiangya decoction by melting and cutting, and are mainly prepared from the following raw material medicines in parts by weight: 10-30 parts of gastrodia elata, 10-20 parts of uncaria, 5-15 parts of lotus plumule and 5-15 parts of scutellaria baicalensis. The Qingda granules are mainly used for treating hypertension in the early stage or 1-grade hypertension, gastrodia elata is used as a monarch drug for calming the liver and suppressing yang, uncaria is used as an auxiliary drug for assisting the gastrodia elata in clearing heat, calming the liver and stopping wind, scutellaria baicalensis is used for clearing liver heat, and lotus plumule discharges heart fire, so that the liver floats and yang, is hidden, the heart and liver fire heat is reduced, the purposes of suppressing yang and calming the liver, inhibiting stirring wind, reducing blood pressure and recovering heart, brain and kidney injuries are achieved. The gastrodia elata in the formula is a traditional Chinese medicine in China, has the functions of calming wind, relieving spasm, calming liver yang and the like, is used for treating infantile convulsion, epilepsy, convulsion and the like, is rich in active ingredients such as phenols, gastrodin and the like, has the functions of relieving pain, resisting depression, reducing blood pressure, resisting thrombus, resisting aging, improving learning memory and microcirculation and also has the functions of enhancing cellular immunity and organism nonspecific immunity. The 6 index components in the Qingda granule rhizoma Gastrodiae include gastrodine, Balison glycoside A, B, C, E, and p-hydroxybenzyl alcohol. The qingda granules of the present invention are self-made by the applicant.
The preparation of the test solution comprises the steps of taking QINGDA particles under the condition of different loading amounts, grinding, precisely weighing, adding a phosphoric acid aqueous solution, weighing, carrying out ultrasonic treatment, cooling, complementing the weight loss by using the phosphoric acid aqueous solution, carrying out centrifugal treatment, taking supernate, passing through a polyamide column, adding an elution solvent for elution, collecting eluent, adding the phosphoric acid aqueous solution for constant volume, shaking up, and taking a subsequent filtrate.
In consideration of the solubility of gastrodia elata, the invention adopts phosphoric acid aqueous solution for extraction. In some embodiments of the present invention, the phosphoric acid aqueous solution is 0.02 to 0.1% by volume phosphoric acid aqueous solution, and further, preferably, the phosphoric acid aqueous solution is 0.05% by volume phosphoric acid aqueous solution.
In some embodiments of the present invention, the ratio of the amount of the test sample after the Qingda particles are ground to the amount of the phosphoric acid aqueous solution added for the first time is: 40-70mL of 1g, preferably 1g, 50mL, to achieve sufficient extraction and meet the concentration requirements for detection.
In other embodiments of the present invention, the time of the ultrasonic treatment is 10 to 70min, preferably 10 to 40min, and more preferably 15 to 35min, and the factors such as the inspection period and the sufficient extraction are considered comprehensively, and more preferably the ultrasonic time is 30 min. The ultrasonic power is 100-400W, preferably 200-300W, further the ultrasonic power is 250W, and the ultrasonic frequency is 10-100 KHz, preferably 30-50 KHz, and more preferably 40 KHz.
After the ultrasonic treatment, the mixture is cooled, the weight loss is compensated by phosphoric acid solution, and the mixture is shaken up and then centrifuged, the equipment and parameters used for centrifugation are not particularly limited, and in one specific embodiment of the invention, the centrifugation rotating speed of 3000r/min is adopted.
In order to reduce the interference of other 3 medicinal materials of the QINGDA granules on the gastrodia elata, especially the interference of scutellaria baicalensis on the gastrodia elata, the inventor finds that the centrifuged supernatant of the QINGDA granules (such as 10mL of the supernatant precisely measured) passes through a polyamide column, an elution solvent is added for elution, the eluent is collected, the phosphoric acid aqueous solution is added for constant volume and shaking up, and a sample solution is obtained by taking a subsequent filtrate, so that other impurity components in the gastrodia elata can be greatly reduced, 6 index components of the gastrodia elata are enriched, the purity of the index components is improved, and the interference of impurity peaks is reduced. In addition, the inventor finds that by adopting the preparation method of the test solution, the chromatographic peaks of 2 index components (baicalin and wogonoside) of the scutellaria baicalensis can be obviously obtained during the subsequent chromatographic analysis. Therefore, qualitative analysis of the gastrodia elata and the scutellaria baicalensis in the Qingda granules can be realized.
In some embodiments of the present invention, the polyamide column is 80-100 mesh or 100-200 mesh polyamide column, preferably 80-100 mesh polyamide column, and the gastrodia elata processed by the polyamide column (80-100 mesh) has good peak shape and less interference of journal peak, and the chromatographic peaks of baicalin and wogonoside are more obvious than those of 100-200 mesh polyamide column. Further, the polyamide column is 80-100 meshes, 2g and 1cm in inner diameter, and the column is packed by a dry method.
After passing through the polyamide column, the sample is eluted with an elution solvent, which in some embodiments of the invention comprises one or more of an aqueous phosphoric acid solution, water, or an ethanol solution. The elution effect is close to that described above, and it is preferable that the elution solvent is an aqueous phosphoric acid solution in view of the amount of impurities to be eluted.
After elution, collecting the effluent liquid and the eluent in a volumetric flask, adding a phosphoric acid aqueous solution to a constant volume, shaking up, and taking a subsequent filtrate to obtain a test solution.
The preparation method of the test solution can effectively and fully extract 6 index components of the gastrodia elata, and can simultaneously obtain chromatographic peaks of 2 index components (baicalin and wogonoside) of the scutellaria baicalensis when the index components of the gastrodia elata are detected by using ultra-high performance liquid chromatography.
< preparation of control solution >
The preparation of the reference substance solution comprises the steps of taking a proper amount of gastrodin reference substance, precisely weighing, adding acetonitrile-water solution for preparation, and shaking up. Further, 1mL of a mixed solution containing 80. mu.g of gastrodin is preferably prepared.
< measurement Using ultra high Performance liquid chromatography >
Compared with the conventional HPLC method, the UPLC method has the advantages of short analysis period, higher separation degree and more environmental protection, the detection period of the UPLC is about one third of that of the HPLC, the precision and the efficiency of research and detection are greatly improved, and in addition, certain components of HPLC peak are separated by the UPLC.
Confirmation of chromatographic conditions
The inventor researches a mobile phase in order to reduce interference of other chemical components (especially scutellaria baicalensis) in the qingda particles, and finds that the separation of a chromatographic peak can be realized and the peak shape is good if gradient elution is carried out by adopting a binary mobile phase system with a mobile phase A of 60-80% methanol solution and a mobile phase B of phosphoric acid aqueous solution. If the concentration of the methanol solution is too high or too low, the chromatographic separation and the peak pattern are affected and the impurities may interfere significantly. Further, a 70% methanol solution (i.e., 100mL of methanol solution containing 70mL of high grade pure methanol) is preferred, i.e., 100mL of water containing 70mL of methanol. Further, the phosphoric acid aqueous solution is 0.02-0.1% phosphoric acid aqueous solution, and preferably the phosphoric acid aqueous solution is 0.05% phosphoric acid aqueous solution.
In some embodiments of the invention, the methanol solution comprises a sodium dodecyl sulfate solution. Further, in some embodiments of the present invention, the concentration of the sodium dodecyl sulfate solution is 1 to 15mmol/L, preferably 3 to 8mmol/L, and further preferably 5mmol/L from the viewpoint of reducing impurity interference.
In some embodiments of the invention, the gradient elution conditions are as set forth in table 1:
TABLE 1 gradient elution conditions
| Time/min | Mobile phase A/%) | Mobile phase B/%) |
| 0 | 8 | 92 |
| 5 | 8 | 92 |
| 6 | 26 | 74 |
| 9 | 28 | 72 |
| 19 | 55 | 45 |
| 20 | 100 | 0 |
The inventor researches and discovers that chromatographic peaks have different impurity interferences under the conditions of different column temperatures and different flow rates, and in some specific embodiments of the invention, the flow rate of gradient elution is 0.2-0.4 mL/min, and more preferably, the flow rate of gradient elution is 0.3 mL/min. Further, the column temperature is 30 to 40 ℃, and further preferably 35 ℃.
The chromatographic column of the invention takes octadecylsilane chemically bonded silica as a filler, and different chromatographic columns have slight difference in separation degree, and the inventor finds that when Agilent Eclipse Plus C18 (the column length is 100mm, the inner diameter is 2.1mm, and the particle size is 1.8 mu m) is used as the chromatographic column, the separation effect is better, and the retention time is moderate.
Full-wavelength scanning is carried out on 6 index components of the gastrodia elata, and the result shows that the maximum absorption occurs at 220nm, so that 220nm is selected as the detection wavelength.
In some embodiments of the present invention, the chromatographic conditions of the hplc are as follows: octadecylsilane chemically bonded silica is used as a filler (the column length is 100mm, the inner diameter is 2.1mm, and the particle size is 1.8 mu m); gradient elution is carried out by taking 70% methanol as phase A and 0.05% phosphoric acid as phase B; the detection wavelength is 220 nm; the column temperature is 35 ℃; the flow rate is 0.3 mL/min; the number of theoretical plates is not less than 5000 calculated according to gastrodin. The gradient elution conditions are shown in table 1. Further preferably, the chromatographic conditions of the ultra high performance liquid chromatograph are as follows: octadecylsilane chemically bonded silica is used as a filling agent; gradient elution is carried out by taking 70% methanol (containing 5mmol/L sodium dodecyl sulfate) as phase A and 0.05% phosphoric acid as phase B; the detection wavelength is 220 nm; the column temperature is 35 ℃; the flow rate is 0.3 mL/min; the number of theoretical plates is not less than 5000 calculated according to gastrodin. The gradient elution conditions are shown in table 1.
Determination and analysis
In some embodiments of the invention, the present invention precisely sucks 2 μ L of each of the reference solution and the test solution, injects the sucked solutions into the ultra-high performance liquid chromatograph, and measures the absorbed solutions.
The invention adopts an analysis method of one test and multiple comments. The one-measurement-multiple-evaluation method is based on the fact that the quantity (quality/concentration) of a certain substance is in direct proportion to the response value of an instrument in a certain linear range, namely, under the same sample quantity and chromatographic conditions in the certain linear range, the correction factor (peak area/concentration) of the certain substance is a determined value, and the correction factors of different substances are possibly different, so that one-measurement-multiple-evaluation can be realized through the determined function (proportion) relation of the internal reference substance to the correction factor (Fsi relative correction factor) of the substance to be detected, and the purposes of quickly detecting the content of multiple components, simplifying operation and saving cost are achieved.
The formula for calculating the relative correction factor is as follows:
Fsi＝(As/Cs)/(Ai/Ci)
in the formula, Fsi is a relative correction factor, As is the peak area of an internal reference substance s, Cs is the concentration of the internal reference substance s, Ai is the peak area of a component to be detected reference substance i, and Ci is the concentration of the component to be detected reference substance i.
The formula for calculating the relative retention time is as follows:
R＝ti/ts
wherein R is relative retention time, ts is retention time of s peak of internal reference substance, and ti is retention time of s peak of internal reference substance.
In the invention, the peak area of a gastrodin reference substance is used as a reference, the contents of p-hydroxybenzyl alcohol, barban E, barban B, barban C and barban A are respectively calculated by using relative correction factors, the relative retention time of a chromatographic peak of a component to be detected and a gastrodin (S1) peak and a barban A (S2) peak is determined, and the relative retention time is within +/-5% of a specified value. The relative retention times and correction factors are shown in table 2 below.
TABLE 2 relative retention time and correction factor
Based on dry product, each bag contains gastrodin (C)13H18O7) Hydroxybenzyl alcohol (C)7H8O2) Balison glycoside E (C)19H24O13) Balison B (C)32H40O19) Balison C (C)32H40O19) Balison A (C)45H56O25) The total amount should not be less than 54.0 mg.
The multi-evaluation method can quickly and accurately measure the content of 6 index components of the gastrodia elata. The determination method has high precision, good reproducibility, good stability, and high accuracy of determination result, and can effectively control the quality of rhizoma Gastrodiae, thereby ensuring the safety and effectiveness of clinical application.
In another technical scheme, the invention further provides a method for qualitatively analyzing the gastrodia elata and the scutellaria baicalensis in the qingda particles, which comprises any one of the methods for content determination through liquid chromatography.
The technical solution of the present invention will be further described with reference to specific examples. It will be readily understood by those skilled in the art that the specific experimental conditions and results thereof described in the following examples are illustrative of the present invention only and should not be, and should not be construed as, limiting the invention. Changes and substitutions in detail and form may be made without departing from the spirit and scope of the invention, but it is intended that such changes and substitutions fall within the scope of the invention. In addition, unless otherwise specified, instruments, materials, reagents and the like used in the examples can be obtained by conventional commercial means.
Examples
Experimental materials:
control (for content determination, no treatment before use):
gastrodin (batch number: 110807-;
p-hydroxybenzyl alcohol (batch number: 111970) -201702, purity 99.4%) was purchased from China institute for food and drug testing;
baricin E (Shidande 6942, 98% purity) was purchased from Shanghai Shidande Biotech Co., Ltd;
baricin B (Shidande 6251, purity 97%) was purchased from Shanghai Shidande Biotech Co., Ltd;
baricin C (Shidande 6229, purity 98%) was purchased from Shanghai Shidande Biotech Co., Ltd;
balison A (Shidande, 6250, purity 98%) was purchased from Shanghai Shidande Biotechnology Co., Ltd.
And (3) testing the sample:
qingda granule batch number: 1905310, 1905001, 1905002 and 1905003, provided by Jiangyin Tianjiang pharmaceutical Co.
Other reagents:
sodium dodecyl sulfate, available from Shanghai Aladdin Biotechnology GmbH;
methanol, chromatographically pure;
methanol, superior pure;
acetonitrile, chromatographic purity;
phosphoric acid, chromatographic purity;
ultrapure water, self-made.
Instruments and consumables:
Thermo-UPLC ultra-high performance liquid chromatograph, model/specification: thermo VANQUISH, available in Cheng Guangzhou City
Stand out and import and export Limited;
KQ-250E ultrasonic cleaning machine, available from Kunshan ultrasonic instruments Inc.;
electronic analytical balance, available from mettler-toledo instruments (shanghai) ltd;
a temperature-controlled water bath, purchased from south China Mount Tai laboratory instruments, Inc.;
pure water system, available from Sartori μ s corporation;
Agilent Eclipse Plμs C18chromatography column (2.1X 100mm, 1.8 μm);
Thermo Acclaim C18chromatography column (2.1X 100mm, 2.2 μm);
Wates BEH C18column (2.1X 100mm, 1.7 μm).
Chromatographic conditions are as follows:
with Agilent Eclipse Plus C18The chromatographic column (2.1X 100mm, 1.8 μm) is the stationary phase; gradient elution is carried out by taking 70% methanol (containing 5mmol/L sodium dodecyl sulfate) as phase A and 0.05% phosphoric acid as phase B; the detection wavelength is 220 nm; the column temperature is 35 ℃; the flow rate is 0.3 ml/min; the number of theoretical plates is not less than 5000 calculated according to gastrodin. The gradient elution conditions are shown in table 1. Preparation of control solutions:
accurately weighing appropriate amount of gastrodine reference substance, adding acetonitrile-water (3:97) to obtain solution containing gastrodine 80 μ g per 1ml, and shaking.
First, example 1
Preparation of test solution
Grinding the QINGDA particles with different loading amounts, uniformly mixing, taking about 0.5g, precisely weighing, precisely adding 25mL of 0.05% phosphoric acid aqueous solution, weighing, carrying out ultrasonic treatment (power 250W and frequency 40kHz) for 30 minutes, cooling, supplementing the lost weight with 0.05% phosphoric acid solution, shaking uniformly, centrifuging (3000r/min), precisely weighing 10mL of supernate, passing through a polyamide column (80-100 meshes, 2g, the inner diameter of the column is 1cm, loading the column by a dry method), adding 0.05% phosphoric acid for elution, collecting eluent to a near-scale line, adding 0.05% phosphoric acid for fixing the volume to the scale line, shaking uniformly, taking a subsequent filtrate, and obtaining a test solution.
The sample solution and the control solution were each 2. mu.L each, precisely aspirated, and UPLC was injected and measured under the above-described chromatographic conditions.
Fig. 1 is an ultra-high performance liquid chromatogram of a gastrodin reference substance, and fig. 2 is an ultra-high performance liquid chromatogram of a Qingda granule sample. The abscissa is time, unit: minute (min), the ordinate is milliabsorbance value (mAu), peak 1 is gastrodin, peak 2 is p-hydroxybenzyl alcohol, peak 3 is balansin E, peak 4 is balansin B, peak 5 is balansin C, peak 6 is balansin a, peak 7 is baicalin, and peak 8 is wogonoside. Therefore, the method can realize qualitative research on the gastrodia elata and the scutellaria baicalensis.
The content of 6 index components of gastrodia elata is measured on different Qingda granules, and the results obtained by combining the relative retention time and the relative correction factor determined in the table 2 and utilizing one-test-multiple evaluation are shown in the table 3. The test result shows that: the 4 batches of Qingdao granules meet the standard limit of the quality standard Gastrodia elata items of the Qingdao granules. The product contains gastrodine (C)13H18O7) Hydroxybenzyl alcohol (C)7H8O2) Balison glycoside E (C)19H24O13) Balison B (C)32H40O19) Balison C (C)32H40O19) Balison A (C)45H56O25) The total amount should not be less than 54.0 mg.
TABLE 3 measurement results of different lot numbers of Qingda granule
Therefore, the method can be practically applied to the quality control of the Qingda granules, the content and the total amount of 6 index components of the gastrodia elata in different batches of Qingda granules can be measured, and the method has operability.
Second, investigation of mobile phase
2.1 sample test solutions were prepared by the test solution preparation method of example 1 by taking about 0.5g of the Qingda particles in the loading difference term and grinding them in parallel 3 parts, and gradient elution was performed according to Table 1 using a.70% methanol, b.70% methanol (containing 1mmol/L sodium dodecylsulfonate solution) and c.70% methanol (containing 10mmol/L sodium dodecylsulfonate solution) as mobile phase A instead of 70% methanol (containing 5mmol/L sodium dodecylsulfonate) of example 1 and 0.05% phosphoric acid as mobile phase B, respectively. The results are shown in FIGS. 3-5. The test result shows that: sodium dodecyl sulfate with different concentrations is added into 70 percent methanol, and chromatographic peaks have impurity interference with different degrees. In general, 70% methanol (containing 5mmol/L sodium dodecyl sulfate) is preferably selected as the mobile phase A in the invention.
2.2 sample test solutions were prepared by the test solution preparation method of example 1 by taking about 0.5g of fine powder of QINGDA particles in terms of the difference in loading amount, taking 2 portions in parallel, and gradient elution was performed according to Table 1 using 70% methanol (containing 5mmol/L dipotassium hydrogen phosphate solution) as mobile phase A instead of 70% methanol (containing 5mmol/L sodium dodecylsulfate) as example 1 and 0.05% phosphoric acid as mobile phase B. The results are shown in FIG. 6. The test result shows that: 5mmol/L dipotassium hydrogen phosphate is added into 70% methanol, the interference of impurity peaks is serious, and the result peak separation is low. In comprehensive consideration, the invention selects 70% methanol (containing 5mmol/L sodium dodecyl sulfate solution) as the mobile phase A.
Study on extraction process of rhizoma Gastrodiae
3.1 compared with the example 1, the preparation method of the test solution is changed: grinding the QINGDA granule, mixing, collecting about 0.5g, precisely weighing, precisely adding 0.05% phosphoric acid water solution 25mL, weighing, ultrasonic treating (power 250W, frequency 40kHz) for 30min, cooling, supplementing with 0.05% phosphoric acid solution, shaking, and collecting filtrate to obtain test solution. The results are shown in FIG. 7. The test result shows that: when the sample which did not pass through the polyamide column was subjected to chromatography, the number of impurity peaks was large and the degree of separation was low.
3.2 compared with example 1, the test solution was prepared by changing the mesh number of the filler in the polyamide column, i.e., by using a polyamide column (100 to 200 mesh, 2g, inner diameter of 1cm, dry-packed column) instead of the polyamide column of example 1. The results are shown in FIG. 8. Comparing fig. 2 and fig. 8, the results show that: the gastrodia elata processed by a polyamide column (100-200 meshes) is short and sharp in peak shape, and the peak area is small; the gastrodia elata processed by the polyamide column (80-100 meshes) is good in peak shape, less in impurity peak interference and capable of obviously seeing chromatographic peaks of baicalin and wogonoside. Therefore, the invention preferably uses polyamide columns (80-100 meshes) as the column type for preparing the rhizoma gastrodiae test sample.
3.3 compared with example 1, only after passing through the polyamide column elution solvent difference, with water instead of 0.05% phosphoric acid elution, other the same, get the sample solution.
3.4 compared with example 1, only after passing through the polyamide column elution solvent difference, with 10% ethanol instead of 0.05% phosphoric acid elution, other the same, get the sample solution.
The single standard of example 1 was changed to a mixed standard (i.e., a mixed standard), and the mixed standard solution was prepared by: taking a proper amount of a gastrodin reference substance, a p-hydroxybenzyl alcohol reference substance, a barban glycoside E reference substance, a barban glycoside B, a barban glycoside C and a barban glycoside A reference substance, precisely weighing, adding acetonitrile-water (3:97) to prepare a mixed solution containing 70 mu g of gastrodin, 10 mu g of p-hydroxybenzyl alcohol, 40 mu g of barban glycoside E, 20 mu g of barban glycoside B, 20 mu g of barban glycoside C and 10 mu g of barban glycoside A in each 1mL, and shaking up to obtain the gastrodin-B-C-A mixed solution. The sample solution obtained in example 1, and the sample solution and the mixed standard solution obtained in example 3.3 and 3.4 were each 2. mu.L each, and then UPLC was injected thereinto to measure the concentration under the above-mentioned chromatographic conditions. The results are shown in FIG. 9. In addition, the contents of 6 index components in the gastrodia elata are calculated, and the results are shown in table 4. Taken together, the results show that: the elution with 10% ethanol shows few peaks, and water and 0.05% phosphoric acid have substantially the same elution effect, but 0.05% phosphoric acid elutes less impurities, so that the present invention preferably uses 0.05% phosphoric acid as the elution solvent.
Table 4 elution solvent investigation
Fourth, investigation of column temperature, flow rate and chromatographic column
The product particles with different loading amounts are ground into fine powder (batch number: 1905001), 2 portions of about 0.5g of the product particles are taken in parallel, sample test solution is prepared according to the text test solution preparation method, 2 mu L of the sample test solution is injected respectively, and different column temperatures, different flow rates and different chromatographic columns are examined. The results are shown in FIGS. 10, 11, 12, Table 5, 6 and 7. The test result shows that: under the conditions of different column temperatures and different flow rates, the chromatographic peak of the barbaloin A has different impurity interferences, so that the flow rate of gradient elution is determined to be 0.3mL/min, and the column temperature is 35 ℃. Different columns differ slightly in the degree of separation. In the one-test multi-evaluation of Gastrodia elata, in order to determine a chromatographic peak, a chromatographic column was defined by Agilent Eclipse Plus C18 (column length 100mm, inner diameter 2.1mm, particle size 1.8 μm).
TABLE 5 different column temperatures
TABLE 6 different flow rates
TABLE 7 different chromatography columns
Fifth, methodology verification
5.1 relative correction factor validation test
The contents of gastrodin, p-hydroxybenzyl alcohol, barbalin E, barbalin B, barbalin C and barbalin A in the gastrodia elata are determined by adopting a one-test-multiple-evaluation method. The relative retention time and relative correction factor need to be re-established due to the mobile phase change. Meanwhile, the difference between instruments and between chromatographic columns is that the peaks are positioned by taking gastrodine and balsamioside A as S peaks, and as can be seen from the data in the following table, the relative retention time (calculated by gastrodine) of p-hydroxybenzyl alcohol is 1.69, and the retention time of the balsamioside E, B, C (calculated by the balsamioside A) is 0.57, 0.81 and 0.87 respectively.
2 portions of QINGDA granules (batch No. 1905001) are taken, sample test solution is prepared according to the preparation method of the test solution in the embodiment 1 respectively, 2 mu L of sample is injected respectively, the peak area values of gastrodin, p-hydroxybenzyl alcohol and balaneboside E, B, C, A are measured on different instruments, different sample injection amounts are examined by taking linear reference substances, and the relative retention time, the relative correction factor and the RSD are calculated. The results are shown in tables 8 and 9. The test result shows that: the correction factors are consistent with those in a medicinal material determination methodology, so that the relative correction factors (calculated by gastrodin) of the correction factors are respectively 0.57, 1.55, 1.31, 1.35 and 1.27 when the correction factors are determined by the medicinal materials, namely, the parahydroxybenzyl alcohol, the barbanoside E, the barbanoside B, the barbanoside C and the barbanoside A.
TABLE 8 relative retention time determination
TABLE 9 relative correction factor determination
5.2 Linear relationship investigation test
Respectively and precisely sucking a gastrodin reference solution (138.0683 mu g/mL), a p-hydroxybenzyl alcohol reference solution (9.83 mu g/mL), a barban E (92.276 mu g/mL), a barban B (84.5258 mu g/mL), a barban C (48.746 mu g/mL) and a barban A (77.94 mu g/mL) reference solution of 0.1, 0.2, 0.5, 1.0, 1.5, 2.0 and 3.0 mu L, injecting the solution into a liquid chromatograph, measuring according to the chromatographic conditions of example 1, drawing a standard curve by taking a peak area integral value as a vertical coordinate and a sample injection amount (mu g) as a horizontal coordinate, obtaining a regression equation, wherein the result is shown in tables 10-16, and the graph is shown in tables 13-18.
TABLE 10 Linear relationship examination results of 6 index components of rhizoma Gastrodiae reference sample
TABLE 11 Gastrodin sample size and Peak area
TABLE 12 p-hydroxybenzol sample introduction and Peak area
TABLE 13 Balisin E sample amount and Peak area plot
TABLE 14 Balisin B sample size and Peak area
TABLE 15 Balisin C sample introduction amount and Peak area
TABLE 16 Balisin A sample size and Peak area
5.2 precision test
Preparing a mixed reference solution: taking a proper amount of a gastrodin reference substance, a p-hydroxybenzyl alcohol reference substance, a barban glycoside E reference substance, a barban glycoside B, a barban glycoside C and a barban glycoside A reference substance, precisely weighing, adding acetonitrile-water (3:97) to prepare a mixed solution containing 70 mu g of gastrodin, 10 mu g of p-hydroxybenzyl alcohol, 40 mu g of barban glycoside E, 20 mu g of barban glycoside B, 20 mu g of barban glycoside C and 10 mu g of barban glycoside A in each 1mL, and shaking up to obtain the gastrodin-B-C-A mixed solution.
Precisely sucking the mixed reference solution, injecting into a liquid chromatograph, measuring according to the chromatographic conditions, injecting 2 μ L of the sample, continuously injecting for 6 times, recording the peak area measurement value, and calculating the relative standard deviation. The results are shown in Table 17. As can be seen from the table, the precision of the instrument is good and can be used for measuring the content.
TABLE 17 precision test
5.3 stability test
Collecting QINGDA granule (batch number: 1905001), preparing sample test solution according to the text preparation method, injecting 2 μ L sample at 0 hr (following Table WD0h, and so on), 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 10 hr, and 12 hr, measuring peak area value, and calculating RSD%. The results are shown in Table 18. As can be seen from the table, 6 components to be tested in the test solution were substantially stable within 12 hours at room temperature.
TABLE 18 stability test
5.3 recovery test
Taking about 0.25g of sample with known content, precisely weighing, adding gastrodin control solution (690.3413 μ g/mL) respectively, p-hydroxybenzyl alcohol reference substance solution (49.15 mu g/mL), barban E (461.38 mu g/mL), barban B (422.629 mu g/mL), barban C (243.73 mu g/mL), and barban A (389.7 mu g/mL) total 6 parts, 0.05% phosphoric acid aqueous solution is added to 25mL, the weight is weighed, a plug is sealed, ultrasonic treatment (power 250W and frequency 40kHz) is carried out for 30 minutes, weight loss is compensated, centrifugation (3000r/min) is carried out, 10mL of supernatant is precisely taken, the supernatant passes through a polyamide column (80-100 meshes, 2g, inner diameter is 1cm, the column is dried and filled with 0.05% phosphoric acid for elution, effluent liquid and 24.5mL of eluent are collected in a 25mL volumetric flask, water is added to fix the volume, and continuous filtrate is obtained after shaking. According to the above chromatographic conditions, 2. mu.L of each sample was introduced, and the recovery rate, RSD%, was calculated according to the following formula, and the results are shown in Table 19. The experimental results show that: the recovery rate of each index component of the gastrodia elata is 92-105%, and the requirement of accuracy of a related content limit (1%) of 9101 in the three general rules of the 'Chinese pharmacopoeia' 2015 edition is met.
Recovery (%). yield (%). measured amount (mg) -content in sample (mg))/addition of control amount (mg). times.100%
TABLE 19 sample recovery test
The above examples are intended only to illustrate several embodiments of the present invention, which are described in more detail and detail, but are not to be construed as imposing any limitation on the scope of the present invention. It should be clear that a person skilled in the art can make several variations and modifications without departing from the inventive concept, which fall within the scope of protection of the present invention.
Claims (10)
1. A method for measuring the content of 6 index components of gastrodia elata in Qingda granules is characterized by comprising the following steps: preparing test solution, preparing reference solution, measuring with ultra-high performance liquid chromatograph,
wherein, the 6 index components comprise gastrodin, barban A, barban B, barban C, barban E and p-hydroxybenzyl alcohol;
the preparation of the test solution comprises the steps of taking QINGDA particles under the condition of different loading amounts, grinding, precisely weighing, adding a phosphoric acid aqueous solution, weighing, carrying out ultrasonic treatment, cooling, complementing the weight loss by using the phosphoric acid aqueous solution, carrying out centrifugal treatment, taking supernate, passing through a polyamide column, adding an elution solvent for elution, collecting eluent, adding the phosphoric acid aqueous solution for constant volume, shaking uniformly, and taking a subsequent filtrate.
2. The method according to claim 1, wherein the polyamide column is 80 to 100 mesh or 100 to 200 mesh, preferably 80 to 100 mesh.
3. The method according to claim 1 or 2, wherein the elution solvent comprises one or more of an aqueous phosphoric acid solution, water or an ethanol solution, preferably the elution solvent is an aqueous phosphoric acid solution.
4. The method according to any one of claims 1 to 3, wherein the aqueous phosphoric acid solution is a 0.05% aqueous phosphoric acid solution, and the percentage is a volume percentage.
5. The method according to any one of claims 1 to 4, wherein the ultra high performance liquid chromatograph performs gradient elution by using a binary mobile phase system, wherein the mobile phase A is 60 to 80% methanol solution, and the mobile phase B is phosphoric acid aqueous solution, preferably 0.02 to 0.1% phosphoric acid aqueous solution.
6. The method of claim 5, wherein the methanol solution comprises a sodium dodecyl sulfate solution.
7. The method according to claim 6, wherein the concentration of the sodium dodecyl sulfate solution is 1 to 15mmol/L, preferably 3 to 8mmol/L, and more preferably 5 mmol/L.
8. The method according to any one of claims 1 to 7,
the preparation of the reference solution comprises precisely weighing appropriate amount of gastrodin reference, adding acetonitrile-water solution, and shaking.
9. The method according to claim 5, wherein the gradient elution conditions are as follows:
preferably, the flow rate of the gradient elution is 0.2-0.4 mL/min, and more preferably, the flow rate of the gradient elution is 0.3 mL/min.
10. A method of qualitatively analysing gastrodia elata and scutellaria baicalensis in qingda granules, comprising the method of any one of claims 1-9.
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