CN115598229A - High performance liquid chromatography identification method for gastrodia elata - Google Patents
High performance liquid chromatography identification method for gastrodia elata Download PDFInfo
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Abstract
The invention provides a high performance liquid chromatography identification method of gastrodia elata, which comprises the following steps: extracting Gastrodia elata Blume with a first solvent, and preparing a standard substance; extracting at least one of rhizoma Gastrodiae and radix Aconiti Sungpanensis with a second solvent to obtain a reference substance; extracting rhizoma Gastrodiae to be tested with a third solvent to obtain a sample; respectively performing high performance liquid chromatography determination on the standard substance, the reference substance and the test substance; wherein, the mobile phase A adopted by the high performance liquid chromatography is formic acid aqueous solution, and the mobile phase B is acetonitrile; gradient elution mode is adopted. The high performance liquid chromatography identification method can express the difference components between the gastrodia elata and the gastrodia elata, and between the gastrodia elata and the gastrodia elata on the map, so that the aim of quickly, standardly and effectively identifying the gastrodia elata is fulfilled.
Description
Technical Field
The invention relates to the field of traditional Chinese medicine variety analysis, in particular to a high performance liquid chromatography identification method for gastrodia elata.
Background
Gastrodia elata is one of the commonly used famous and precious traditional Chinese medicines, also known as red arrow, sedge, shencao and the like, is a dry tuber of Gastrodia elata Bl. Tianma is neutral in nature and sweet in taste, and enters liver meridian; has effects in calming endogenous wind, relieving spasm, suppressing liver yang, dispelling pathogenic wind, and dredging collaterals; can be used for treating hyperactivity of liver-yang, hyperactivity of liver-wind, apoplexy, and arthralgia due to wind-dampness. Gastrodia elata is a perennial parasitic plant, and takes its host Armillaria mellea as a nutrient source. The chemical components of gastrodia elata are various, and mainly comprise phenol components represented by gastrodine (gastrodine) and p-hydroxybenzyl alcohol, barcinoside components formed by condensing gastrodine and citric acid, sterols such as beta-sitosterol, amino acids such as lysine and polysaccharides, and various chemical components. Rhizoma gastrodiae is known as a wind-treating psychotropic drug, is commonly used for treating diseases such as infantile convulsion, epilepsy and convulsion, tetanus, headache and dizziness, limb paralysis, limb numbness, rheumatism and arthralgia and the like in clinical traditional Chinese medicine, has wide pharmacological action and relates to a plurality of systems such as a central nervous system, a cardiovascular system, an immune system and the like. Modern pharmacological research shows that the gastrodia elata also has pharmacological activities of resisting cerebral ischemia, AD, oxidation, aging, inflammation, neuroprotection, immunity enhancement and the like.
The gastrodia elata is used as a traditional rare Chinese medicinal material in China, is widely applied in the clinical field of traditional Chinese medicine, and is an important raw material medicine of more than ten common Chinese patent medicines such as gastrodia elata and polygonum multiflorum tablets, gastrodia elata and uncaria granules, large ligusticum wallichii tablets and the like. Common gastrodia elata medicinal materials mainly comprise gastrodia elata, red gastrodia elata and Wuhong hybrid gastrodia elata, the character difference of underground tubers of different varieties of gastrodia elata is small, after the gastrodia elata medicinal material commodities are processed, the character difference of different varieties of gastrodia elata medicinal materials is not obvious and difficult to distinguish, and great difficulty is brought to distinguishing and identifying different varieties of gastrodia elata medicinal materials. Among various gastrodia elata medicinal materials, the genuine medicinal material gastrodia elata is expensive and has obvious price difference with the red gastrodia elata and the black red gastrodia elata, so that the phenomenon that the red gastrodia elata and the black red gastrodia elata are used for being sold as the black gastrodia elata is caused, market circulation is influenced, and the quality of related products is reduced.
The traditional method distinguishes and determines the varieties of the gastrodia elata through the color of the rachis, and the method is strong in experience, low in standardization degree and difficult to master. The traditional method for identifying gastrodia elata, gastrodia elata and gastrodia elata mostly adopts a capillary electrophoresis technology, a nuclear magnetic resonance spectroscopy technology and an electron bombardment ion source-mass spectrometry combined technology, and the detection technologies are complex and complicated to operate. The traditional high performance liquid chromatography technology is adopted to detect the gastrodia elata, the gastrodia elata and the gastrodia elata, although the detection is quick and convenient, the different components of the gastrodia elata and other gastrodia elata cannot be found, and the gastrodia elata cannot be identified through index chemical components (such as gastrodin, balansin A, balansin B, balansin C, balansin E, hydroxybenzaldehyde and the like) of the gastrodia elata.
Disclosure of Invention
Based on the method, the invention provides a high performance liquid chromatography identification method for gastrodia elata, which can express the difference components between gastrodia elata and gastrodia elata, and between gastrodia elata and gastrodia elata on a map, so that the aim of quickly, normatively and effectively identifying gastrodia elata is fulfilled.
The invention is realized by the following technical scheme.
A high performance liquid chromatography identification method of gastrodia elata comprises the following steps:
extracting Gastrodia elata Blume with a first solvent, and preparing a standard substance;
extracting at least one of rhizoma Gastrodiae and radix Aconiti Sungpanensis with a second solvent to obtain a reference substance;
extracting the gastrodia elata to be detected by a third solvent to prepare a test sample;
respectively carrying out high performance liquid chromatography determination on the standard substance, the reference substance and the test substance;
wherein, the mobile phase A adopted by the high performance liquid chromatography is formic acid aqueous solution, and the mobile phase B is acetonitrile; adopting a gradient elution mode;
the gradient elution mode is as follows: 0 min-5 min, wherein the volume percentage of the mobile phase B is changed from 0% to 2%;5 min-10 min, wherein the volume percentage of the mobile phase B is changed from 2% to 10%;10 min-25 min, wherein the volume percentage of the mobile phase B is changed from 10% to 23%;25 min-30 min, wherein the volume percentage of the mobile phase B is changed from 23% to 65%; 30-35 min, wherein the volume percentage of the mobile phase B is changed from 65% to 90%; 35-37 min, wherein the volume percentage of the mobile phase B is changed from 90% to 100%; 37-40 min, and the volume percentage of the mobile phase B is kept as 100%.
In one embodiment, the volume fraction of formic acid in the aqueous formic acid solution is 0.05% to 0.15%.
In one embodiment, the detection wavelength for the HPLC assay is 280nm to 300nm.
In one embodiment, the HPLC assay uses a column C 18 A chromatographic column.
In one embodiment, the HPLC assay uses a column temperature of 30 ℃ to 40 ℃.
In one embodiment, the HPLC assay uses a flow rate of 0.25mL/min to 0.45mL/min.
In one embodiment, the sample injection volume for HPLC is 5-15 μ L.
In one embodiment, the first solvent, the second solvent, and the third solvent are each independently selected from: at least one of methanol, ethanol, isopropanol, acetonitrile, formic acid, and water.
In one embodiment, the extraction of gastrodia elata with a first solvent comprises the following steps: performing ultrasonic treatment and filtering; and/or
The extraction of at least one of gastrodia elata and gastrodia elata with the second solvent includes the following steps: performing ultrasonic treatment and filtering; and/or
The method for extracting the gastrodia elata to be detected by adding the third solvent comprises the following steps: and (4) performing ultrasonic treatment and filtering.
In one embodiment, the gastrodia elata to be tested is gastrodia elata, gastrodia elata or gastrodia elata.
In one embodiment, the high performance liquid chromatogram of the standard does not include the chromatographic peaks of the compound a, the compound b and the compound c, and the high performance liquid chromatogram of the control includes the chromatographic peaks of the compound a, the compound b and the compound c;
in the high performance liquid chromatogram of the reference substance, the retention time of the compound a is 16.94-17.01 min; the retention time of the compound b is 19.79min-19.86 min; the retention time of the compound c is 20.83min-20.88 min.
Compared with the prior art, the high performance liquid chromatography identification method for gastrodia elata has the following beneficial effects:
the identification method can show the difference components between the gastrodia elata and between the gastrodia elata and the gastrodia elata on a high performance liquid chromatogram map by limiting the types of the mobile phases and a gradient elution mode, so that the gastrodia elata can be effectively identified, and the accuracy is high. In addition, the high performance liquid chromatography identification method of gastrodia elata is simple and rapid to operate, economic and environment-friendly, saves detection time and cost to a great extent, and can be popularized and applied.
Drawings
FIG. 1 is a high performance liquid chromatogram of a Gastrodia elata Blume provided by the invention;
FIG. 2 is a high performance liquid chromatogram of 15 batches of Gastrodia elata Blume provided by the invention;
FIG. 3 is a high performance liquid chromatogram of the Gastrodia elata Blume provided by the present invention;
FIG. 4 is a high performance liquid chromatogram of 15 batches of Gastrodia elata Blume provided by the present invention;
FIG. 5 is a high performance liquid chromatogram of the tall gastrodia tuber;
FIG. 6 is a high performance liquid chromatogram of 14 batches of tall gastrodia tuber;
FIG. 7 is a comparison graph of different HPLC differences of different varieties of Gastrodia elata Blume provided by the present invention;
FIG. 8 is EIC, UV and TIC chromatograms of a Gastrodia elata sample provided in the present invention;
fig. 9 is a high performance liquid chromatogram of rhizoma gastrodiae under different mobile phases.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the accompanying examples. The examples set forth preferred embodiments of the invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all 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. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or to implicitly indicate the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the invention, "a plurality" means at least two, e.g., two, three, etc., unless explicitly specified otherwise. In the description of the present invention, "a plurality" means at least one, e.g., one, two, etc., unless explicitly specified otherwise.
The words "preferably," "more preferably," and variations thereof herein mean embodiments of the invention that provide certain benefits, under certain circumstances. However, other embodiments may be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.
When a range of values is disclosed herein, the range is considered to be continuous and includes both the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range describing features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein.
Unless defined otherwise, all 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. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The invention provides a high performance liquid chromatography identification method of gastrodia elata, which comprises the following steps:
extracting Gastrodia elata Blume with a first solvent, and preparing a standard substance;
extracting at least one of rhizoma Gastrodiae and rhizoma Gastrodiae with a second solvent to obtain a reference substance;
extracting rhizoma Gastrodiae to be tested with a third solvent to obtain a sample;
respectively performing high performance liquid chromatography determination on the standard substance, the reference substance and the test substance;
wherein, the mobile phase A adopted by the high performance liquid chromatography is formic acid aqueous solution, and the mobile phase B is acetonitrile; adopting a gradient elution mode;
the gradient elution mode is as follows: the volume percentage of the mobile phase B is changed from 0 percent to 2 percent within 0-5 min; 5-10 min, and changing the volume percentage of the mobile phase B from 2% to 10%; 10-25 min, and changing the volume percentage of the mobile phase B from 10% to 23%; the volume percent of the mobile phase B is changed from 23 percent to 65 percent within 25-30 min; 30-35 min, and the volume percentage of the mobile phase B is changed from 65% to 90%; 35-37 min, wherein the volume percentage of the mobile phase B is changed from 90% to 100%; the volume percentage of the mobile phase B is kept at 100 percent for 37 min-40 min.
In a specific example, the volume fraction of formic acid in the aqueous formic acid solution is 0.05% to 0.15%. It is understood that, in the present invention, the volume fraction of formic acid in the aqueous formic acid solution includes, but is not limited to, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%.
In one specific example, the detection wavelength for the HPLC assay is 280nm to 300nm. It is understood that in the present invention, the detection wavelengths used in the HPLC assay include, but are not limited to, 280nm, 285nm, 286nm, 287nm, 288nm, 289nm, 290nm, 295nm, 300nm.
In one specific example, the HPLC assay employs a column C 18 A chromatographic column. More specifically, the high performance liquid chromatography adopts HSS T3C as chromatographic column 18 (100 mm. Times.2.10 mm,1.8 μm) chromatography column.
In one specific example, the HPLC assay employs a column temperature of 30 ℃ to 40 ℃. As will be appreciated, in the present invention, the HPLC assay employs column temperatures including, but not limited to, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃.
In one specific example, the HPLC assay uses a flow rate of 0.25mL/min to 0.45mL/min. It is understood that, in the present invention, the high performance liquid chromatography assay employs flow rates including, but not limited to, 0.25mL/min, 0.3mL/min, 0.31mL/min, 0.32mL/min, 0.33mL/min, 0.34mL/min, 0.35mL/min, 0.36mL/min, 0.37mL/min, 0.38mL/min, 0.39mL/min, 0.4mL/min, 0.45mL/min.
In a specific example, the HPLC assay uses a sample volume of 5 μ L to 15 μ L. It is understood that in the present invention, the sample volumes used for HPLC assay include, but are not limited to, 5. Mu.L, 6. Mu.L, 7. Mu.L, 8. Mu.L, 9. Mu.L, 10. Mu.L, 11. Mu.L, 12. Mu.L, 13. Mu.L, 14. Mu.L, and 15. Mu.L.
In a particular example, the first solvent, the second solvent, and the third solvent are each independently selected from: at least one of methanol, ethanol, isopropanol, acetonitrile, formic acid, and water.
In a specific example, the extraction of gastrodia elata with a first solvent comprises the following steps: performing ultrasonic treatment and filtering; and/or
The extraction of at least one of the red gastrodia tuber and the black red gastrodia tuber by adding a second solvent comprises the following steps: performing ultrasonic treatment and filtering; and/or
The method for extracting the gastrodia elata to be detected by adding the third solvent comprises the following steps: and (4) performing ultrasonic treatment and filtering.
In one specific example, the filtration is through a 0.45 μm microfiltration membrane.
In a specific example, the time of the ultrasound is 20min to 40min.
In a specific example, the gastrodia elata to be tested is gastrodia elata, gastrodia elata or gastrodia elata.
In a specific example, the high performance liquid chromatogram of the standard does not include the chromatographic peaks of the compound a, the compound b and the compound c, and the high performance liquid chromatogram of the reference includes the chromatographic peaks of the compound a, the compound b and the compound c;
in the high performance liquid chromatogram of the reference substance, the retention time of the compound a is 16.94-17.01 min; the retention time of the compound b is 19.79min-19.86 min; the retention time of the compound c is 20.83min-20.88 min.
The high performance liquid chromatography identification method for gastrodia elata according to the invention is further described in detail with reference to specific examples. The starting materials used in the following examples are all commercially available products unless otherwise specified.
Example 1
The embodiment provides a high performance liquid chromatography identification method for gastrodia elata, which comprises the following specific steps:
1. medicinal material sample information
The sample information of 44 batches of gastrodia elata medicinal materials in different producing areas adopted in the present embodiment is shown in table 1.
TABLE 1
2. High performance liquid chromatography conditions
Adopting Agilent 1260HPLC high performance liquid chromatography system equipped with HSS T3C 18 (100 mm. Times.2.10 mm,1.8 μm) column, column temperature set at 35 ℃, mobile phase consisting of 0.1% FA (A) and acetonitrile (B), flow rate of 0.35mL/min, sample size of 10 μ L, detection wavelength of 290nm, elution gradient set as shown in Table 2:
TABLE 2
Time (min) | Mobile phase B: acetonitrile (%) |
0-5 | 0-2 |
5-10 | 2-10 |
10-25 | 10-23 |
25-30 | 23-65 |
30-35 | 65-90 |
35-37 | 90-100 |
37-40 | 100 |
3. The concrete operation steps
(1) Testing of a standard gastrodia elata sample: taking 1.0g of gastrodia elata medicinal material powder, precisely weighing, adding 25mL of methanol, carrying out ultrasonic treatment for 30min, filtering an extracting solution through a 0.45-micron microporous filter membrane, taking a subsequent filtrate as a test solution, precisely absorbing 10 mu L of the test solution, and injecting the test solution into a high performance liquid chromatograph to obtain an HPLC fingerprint of the gastrodia elata medicinal material;
(2) Testing of a rhizoma gastrodiae sample or a black rhizoma gastrodiae sample: taking 1.0g of powder of the red gastrodia tuber or the black red gastrodia tuber medicinal material, precisely weighing, adding 25mL of methanol, carrying out ultrasonic treatment for 30min, filtering an extracting solution through a 0.45 mu m microporous filter membrane, taking a subsequent filtrate as a test solution, precisely absorbing 10 mu L of the test solution, and injecting the test solution into a high performance liquid chromatograph to obtain an HPLC fingerprint of the red gastrodia tuber or the black red gastrodia tuber medicinal material;
(3) Testing a gastrodia elata sample to be tested: taking 1.0g of rhizoma gastrodiae medicinal material powder, precisely weighing, adding 25mL of methanol, carrying out ultrasonic treatment for 30min, filtering an extracting solution through a 0.45 mu m microporous filter membrane, taking a subsequent filtrate as a test solution, precisely sucking 10 mu L of the test solution, and injecting the sample into a high performance liquid chromatograph to obtain a rhizoma gastrodiae medicinal material sample HPLC (high performance liquid chromatography) spectrum.
4. Results
15 batches of gastrodia elata, 15 batches of gastrodia elata and 14 batches of gastrodia elata are subjected to variety identification by gastrodia elata research institute in showtong city of Yunnan province.
The HPLC-UV characteristic spectrum of Gastrodia elata Blume is shown in figure 1, wherein P3 is gastrodin; p6 is the balsamoside E; p9 is the balsamoside B; p11 is balaneboside a. The HPLC-UV characteristic spectrum of 15 batches of gastrodia elata medicinal materials is shown in figure 2.
The HPLC-UV characteristic spectrum of rhizoma Gastrodiae is shown in figure 3, wherein P3 is gastrodine; p6 is the Balison glycoside E; p9 is the balsamoside B; p11 is balaneboside a. The HPLC-UV characteristic spectrum of 15 batches of the gastrodia elata medicinal material is shown in figure 4.
The HPLC-UV characteristic spectrum of rhizoma Gastrodiae is shown in figure 5, wherein P3 is gastrodine; p6 is the Balison glycoside E; p9 is the Balison B; p11 is basilixoside a. The HPLC-UV characteristic spectrum of 14 batches of tall gastrodia tuber medicinal materials is shown in figure 6.
As shown in fig. 7, by comparing the HPLC fingerprints of gastrodia elata, gastrodia elata and gastrodia elata, we found 3 different components, which are labeled as a, b and c, respectively. The chemical components a, b and c are absent in the gastrodia elata, and the gastrodia elata both contain the 3 components, wherein the content of the 3 components in the gastrodia elata is slightly higher than that of the gastrodia elata.
To further characterize these 3 differential components, the experiment used a tandem Agilent MSD-iQ single quadrupole mass spectrometer to acquire LC-MS data in positive ion mode in SCAN mode, and the results are shown in fig. 8, where the chromatographic peak a (m/z 848, c) was extracted by comparing the Total Ion Current (TIC) chromatogram and the 290nm Ultraviolet (UV) chromatogram to obtain a chromatographic peak a (m/z 848, c) 41 H 53 NO 18 ),b(m/z 802,C 40 H 51 NO 16 ) And c (m/z 772,C) 39 H 49 NO 15 ). Comparing the retention time and the relative retention time of the 3 chromatographic peaks with obvious missing characteristics, wherein the distribution ranges of the retention time of chromatographic peaks a, b and c are respectively 16.94-17.01 min, 19.79-19.86 min and 20.83-20.88 min; by P9 chromatographyThe peak (the balaneboside B) is used as a reference, and the relative retention time distribution ranges of chromatographic peaks a, B and c are 1.18-1.21, 1.39-1.42 and 1.47-1.49 respectively.
In summary, when the high performance liquid chromatography identification method for gastrodia elata is adopted to analyze a gastrodia elata sample, if chromatographic peaks of compounds a, b and c are found in an obtained liquid chromatogram, and within the range of the retention time and the relative retention time, the gastrodia elata sample is red gastrodia elata or black red gastrodia elata, and if the chromatographic peaks of a, b and c are not found in the liquid chromatogram, the gastrodia elata is black.
Example 2
The embodiment provides a high performance liquid chromatography identification method for gastrodia elata, which comprises the following specific steps:
1. information on samples of medicinal materials
10 commercially available batches of gastrodia elata samples.
2. High performance liquid chromatography conditions
Adopting Agilent 1260HPLC high performance liquid chromatography system equipped with HSS T3C 18 (100 mm. Times.2.10 mm,1.8 μm) column, column temperature was set at 35 ℃ and mobile phase was composed of 0.1% FA (A) and acetonitrile (B), flow rate was 0.35mL/min, sample size was 10 μ L, detection wavelength was 290nm, and elution gradient was set as shown in Table 2 above.
3. The concrete operation steps
Taking 1.0g of commercially available rhizoma Gastrodiae powder, precisely weighing, adding 25mL of methanol, performing ultrasonic treatment for 30min, filtering the extractive solution with 0.45 μm microporous membrane, taking the subsequent filtrate as a sample solution, precisely absorbing 10 μ L of the sample solution, and injecting into a high performance liquid chromatograph to obtain HPLC chromatogram of rhizoma Gastrodiae sample.
4. Results
The results of testing and identifying 10 commercially available gastrodia elata samples are shown in table 3. By carrying out liquid phase detection and rhizoma gastrodiae bolting variety identification on 10 batches of commercially available rhizoma gastrodiae samples (completed by rhizoma gastrodiae research institute in showtong, yunnan province), the high performance liquid chromatography identification method for the gastrodia elata glauca provided by the invention can distinguish whether the samples are gastrodia elata glauca or gastrodia elata glauca, the accuracy reaches 100%, wherein one batch of samples are gastrodia elata glauca, the high performance liquid chromatography identification method for the gastrodia glauca provided by the invention is identified as gastrodia elata glauca or gastrodia elata glauca, the samples are identified as gastrodia elata glauca by bolting, and the high performance liquid chromatography identification method for the gastrodia glauca provided by the invention can be used for identifying whether the gastrodia elata glauca or gastrodia glauca is used for simulating the gastrodia glauca in the market.
TABLE 3
In order to compare the influence of different mobile phase additives on the chromatogram, 0.1% formic acid water, water and 0.1% phosphoric acid water are respectively considered as the aqueous phase mobile phase on the premise that acetonitrile is used as the organic phase mobile phase. As a result, as shown in FIG. 9, 0.1% formic acid in water as the aqueous mobile phase can effectively improve the peak shape of the chromatographic peak and increase the peak capacity, and 0.1% formic acid and 0.1% phosphoric acid have similar effects on the chromatographic peak, but 0.1% formic acid is finally selected as the aqueous mobile phase in consideration of the potential requirement for subsequent LC-MS analysis.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, which is convenient for specific and detailed understanding of the technical solutions of the present invention, but the present invention should not be construed as being limited to the scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. It should be understood that the technical solutions provided by the present invention, which are obtained by logical analysis, reasoning or limited experiments, are within the scope of the appended claims. Therefore, the protection scope of the present invention should be subject to the content of the appended claims, and the description and the drawings can be used for explaining the content of the claims.
Claims (11)
1. A high performance liquid chromatography identification method of gastrodia elata is characterized by comprising the following steps:
extracting Gastrodia elata Blume with a first solvent, and preparing a standard substance;
extracting at least one of rhizoma Gastrodiae and rhizoma Gastrodiae with a second solvent to obtain a reference substance;
extracting rhizoma Gastrodiae to be tested with a third solvent to obtain a sample;
respectively carrying out high performance liquid chromatography determination on the standard substance, the reference substance and the test substance;
wherein, the mobile phase A adopted by the high performance liquid chromatography is formic acid aqueous solution, and the mobile phase B is acetonitrile; adopting a gradient elution mode;
the gradient elution mode is as follows: 0 min-5 min, wherein the volume percentage of the mobile phase B is changed from 0% to 2%;5 min-10 min, wherein the volume percentage of the mobile phase B is changed from 2% to 10%;10 min-25 min, wherein the volume percentage of the mobile phase B is changed from 10% to 23%;25 min-30 min, wherein the volume percentage of the mobile phase B is changed from 23% to 65%; 30-35 min, wherein the volume percentage of the mobile phase B is changed from 65% to 90%; 35-37 min, wherein the volume percentage of the mobile phase B is changed from 90% to 100%; 37-40 min, and the volume percentage of the mobile phase B is kept to be 100%.
2. The Gastrodia elata Blume high performance liquid chromatography identification method according to claim 1, wherein formic acid in said aqueous formic acid solution is in a volume fraction of 0.05-0.15%.
3. The Gastrodia elata Blume high performance liquid chromatography identification method of claim 1, wherein the detection wavelength used for high performance liquid chromatography is 280 nm-300 nm.
4. The HPLC identification method of Gastrodia elata as claimed in claim 1, wherein HPLC determination uses column C 18 A chromatographic column.
5. The method for identifying Gastrodia elata Blume of claim 1, wherein the column temperature for HPLC determination is 30-40 ℃.
6. The Gastrodia elata Blume high performance liquid chromatography identification method of claim 1, wherein the flow rate for high performance liquid chromatography is 0.25-0.45 mL/min.
7. The method for identifying Gastrodia elata Blume by high performance liquid chromatography according to claim 1, wherein the sample injection volume for high performance liquid chromatography is 5-15 μ L.
8. A Gastrodia elata Blume high performance liquid chromatography identification method as claimed in any one of claims 1-7, wherein said first solvent, said second solvent and said third solvent are each independently selected from the group consisting of: at least one of methanol, ethanol, isopropanol, acetonitrile, formic acid, and water.
9. The high performance liquid chromatography identification method of gastrodia elata according to any one of claims 1 to 7, wherein the step of extracting gastrodia elata with a first solvent comprises the steps of: performing ultrasonic treatment and filtering; and/or
The extraction of at least one of the red gastrodia tuber and the black red gastrodia tuber by adding a second solvent comprises the following steps: performing ultrasonic treatment and filtering; and/or
The method for extracting the gastrodia elata to be detected by adding the third solvent comprises the following steps: and (4) performing ultrasonic treatment and filtering.
10. The HPLC identification method of Gastrodia elata Blume of any one of claims 1-7, wherein said Gastrodia elata Blume to be tested is Gastrodia elata Blume, gastrodia elata Blume or Gastrodia elata Blume.
11. The method for identifying Gastrodia elata Blume according to any one of claims 1-7, wherein the high performance liquid chromatogram of said standard does not include the chromatographic peaks of compound a, compound b and compound c, and the high performance liquid chromatogram of said reference does include the chromatographic peaks of compound a, compound b and compound c;
in the high performance liquid chromatogram of the reference substance, the retention time of the compound a is 16.94-17.01 min; the retention time of the compound b is 19.79min-19.86 min; the retention time of the compound c is 20.83min-20.88 min.
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