Preparation method and quality detection method of high-purity Hyuganin D

Abstract

The invention discloses a preparation method and a detection method of high-purity Hyuganin D by taking umbelliferae radix peucedani plant radix peucedani sphenanthera as a raw material. The method comprises the following steps: drying and crushing umbelliferae peucedanum plant peucedanum sphenanthera, weighing powder, carrying out ultrasonic extraction by using methanol, carrying out vacuum concentration and drying on a methanol extract, carrying out silica gel column chromatography on an extract, and carrying out gradient elution by using a petroleum ether (60-90 DEG C)-ethyl acetate mixed solution to obtain a Hyuganin D coarse crystal; the method comprises the following steps: collecting a Hyuganin D component with the purity of 90-95% and a Hyuganin D component with the purity of more than 95%, separating by using a reverse phase silica gel C-18 column preparation high performance liquid chromatography, detecting each collected eluent by using analytical high performance liquid chromatography, merging the Hyuganin D component with the same retention time to obtain the Hyuganin D component with the purity of 90-95% and the Hyuganin D component with the purity of more than 95%, and carrying out quality detection by using thin-layer chromatography and liquid chromatography. And the production cost can be reduced.

Description

Technical Field

[0001] The present invention relates to the field of compound monomer preparation, and in particular to a preparation method of high-purity Hyuganin D and a quality detection method thereof. Background Art

[0002] Chemical reference substances, also known as standard substances, are the physical comparisons for quality standard research, quality testing and quality inspection of traditional Chinese medicine. The research on chemical reference substances of traditional Chinese medicine is an important part of the standardization research of traditional Chinese medicine. It plays an extremely important role in the quality evaluation of products, especially in the quality inspection of drug production. It is the foundation and core of the quality inspection of traditional Chinese medicine.

[0003] Hyuganin D is an angular pyranocoumarin, one of the active ingredients of plants, and an indicator ingredient for the quality inspection of many plants and medicines. At present, there is no report on the systematic study of Hyuganin D at home and abroad. Referring to the technical requirements of Chinese medicine chemical reference substances (for content determination), the Hyuganin D chemical reference substance was studied, and the batch extraction process, purity and content of the Hyuganin D reference substance, and the analytical determination method for impurity inspection were established, so as to establish the technical standards of Hyuganin D chemical reference substances, and provide a scientific basis and guarantee for its use as a Chinese medicine chemical reference substance and the quality standard research of medicinal materials and preparations.

[0004] Hyuganin D is a chemical reference substance for plants, medicinal materials and their products, and is a technical key to quality detection. Many enterprises, scientific research and inspection departments need high-purity Hyuganin D reference substances. Due to the low content of Hyuganin D in medicinal materials, the extraction and separation technology requirements are very high and the difficulty is very large. The present invention conducts research on the preparation of Hyuganin D Chinese medicine chemical standard substances and its quality detection technology, solves the problem of high-purity Hyuganin D chemical reference substances, and has an obvious effect on the modernization of Chinese medicine, and has great practical significance and academic value.

[0005] The existing technology extracts and separates Hyuganin D from different botanicals. The content of Hyuganin D in the herbs is relatively low, and the extraction cost is high. There is no method for controlling the purity and quality of Hyuganin D, and the need for high-purity Hyuganin D chemical reference substances cannot be met. Summary of the invention

[0006] The invention is the first to extract and separate Hyuganin D from Peucedanum sphenanthera of the Umbelliferae family. The Hyuganin D prepared by the method has high purity, high content and high quality, can be used as a chemical reference substance or standard substance to ensure quality detection, and overcomes the shortcomings of low yield and high cost of Hyuganin D.

[0007] The present invention is a Hyuganin D obtained by extraction, separation, refinement and purification from the dried root of PeucedanummedicumDunn of the genus Peucedanum of the Apiaceae family. Its chemical name is propionic acid, 2-methyl-, (9R, 10R)-9-(acetyloxy)-9,10-dihydro-8,8-dimethyl-2-oxo-2H,8H-benzo[1,2-b:3,4-b']bipyran-10-ester. Its structural formula is as follows:

[0008]

[0009] The object of the present invention is achieved through the following solutions:

[0010] A method for preparing high-purity hyuganin D, comprising the following steps:

[0011] (1) The dried root of Peucedanum sphenantherum of the Umbelliferae family was crushed, extracted with methanol, filtered, the filtrate was combined, concentrated under reduced pressure, and dried to obtain an extract, which was subjected to silica gel column chromatography and gradient elution with petroleum ether-ethyl acetate to collect the fraction containing hyuganin D, and concentrated to obtain crude hyuganin D crystals;

[0012] (2) The crude hyuganin D crystals obtained in step (1) are separated and purified by preparative high performance liquid chromatography, and the hyuganin D fractions are collected. Each of the collected eluates is analyzed by HPLC, and the hyuganin D fractions with the same retention time and a purity between 90% and 95% and those with a purity above 95% are combined, and concentrated under reduced pressure to obtain hyuganin D fractions with a purity between 90% and 95% and above 95%.

[0013] As a preferred embodiment, the method for preparing high-purity hyuganin D of the present invention comprises the following steps: step (1) using methanol in an amount 8 to 15 times the weight of the medicinal material, and ultrasonically extracting for 1 to 3 times, each time for 30 to 60 minutes.

[0014] As a preferred embodiment, the method for preparing high-purity hyuganin D of the present invention comprises the following steps: (1) performing silica gel column chromatography using a gradient elution of petroleum ether-ethyl acetate in a volume ratio of 1:0-0:1; and then collecting the elution of petroleum ether-ethyl acetate in a volume ratio of 10:1.

[0015] As a preferred embodiment, the method for preparing high-purity Hyuganin D described in the present invention, the preparative high performance liquid chromatography column is a C-18 column, methanol-water with a volume ratio of 70:25 is used as the mobile phase for elution, the detection wavelength is 321nm, the flow rate is 1mL / min, and the column temperature is 35°C.

[0016] As a preferred embodiment, the present invention provides a method for preparing high-purity Hyuganin D, wherein the HPLC analysis conditions are: the chromatographic column is a C-18 column; the mobile phase is methanol-water = 70:25; the detection wavelength is 321nm; and the flow rate is 1ml / min.

[0017] The present invention also provides a method for detecting Hyuganin D, which adopts a thin layer chromatography analysis method: taking an appropriate amount of Hyuganin D, using methanol to prepare a Hyuganin D solution of a certain concentration, spotting on the same silica gel plate according to different spotting amounts, placing the plate in a developing cylinder for development, taking the plate out, drying it, and inspecting it under an ultraviolet lamp with a wavelength of 365 nm. As a result, a single blue fluorescent spot can be seen in a GoodLook-1000 thin layer chromatography imaging system.

[0018] The developing solvent system of thin layer chromatography includes: system (1) petroleum ether (60-90°C)-ethyl acetate in a ratio of 2:1; system (2) ethyl acetate-methanol-water in a ratio of 8:1:1; system (3) chloroform-methanol in a ratio of 4:1.

[0019] In addition, the present invention also provides a method for detecting Hyuganin D, which uses a high performance liquid chromatography analysis method to determine the content: accurately weigh an appropriate amount of Hyuganin D reference substance, add a methanol aqueous solution to prepare a Hyuganin D solution of a certain concentration, inject it into a liquid chromatograph, calculate the content by an area normalization method, and the result system determines the content of the reference substance;

[0020] Peak purity test: Take an appropriate amount of Hyuganin D reference substance and perform peak purity check on a high performance liquid chromatograph using a diode array DAD detector according to the mobile phase system.

[0021] As a preferred embodiment, the chromatographic conditions of the detection method are: chromatographic column Amethyst C18-H, specification 4.6×250mm, 5μm, mobile phase: acetonitrile-water or methanol-water, gradient elution, flow rate: 1ml / min, injection volume: 10ul.

[0022] As a preferred solution, when the content is detected, when the mobile phase is acetonitrile (A)-water (B), the gradient elution mode is: 0-20min, 20%-30% A; 20-30min, 30%-60% A; 30-50min, 60% A; 50-60min, 60%-30% A; 60-90min, 30%-20% A; the detection wavelength is 334nm;

[0023] Or when the mobile phase is methanol (A)-water (B), the gradient elution mode is: 0-20 min, 50% A; 20-30 min, 50%-75% A; 30-50 min, 75% A; 50-60 min, 75%-50% A; 60-90 min, 50% A; detection wavelength: 334 nm;

[0024] During peak purity detection, when the mobile phase is methanol (A)-water (B), the gradient elution method is: 0-20 min, 50% A; 20-30 min, 50%-75% A; 30-50 min, 75% A; 50-60 min, 75%-50% A; 60-90 min, 50% A; detection wavelength: 321 nm.

[0025] The chemical reference substance of Hyuganin D separated and purified by the present invention has its chemical structure confirmed by infrared spectrum, ultraviolet spectrum, nuclear magnetic resonance, mass spectrum and physical and chemical detection. It is detected by three development systems, five TLCs of different concentrations, two mobile phase systems and two HPLCs of different wavelengths, and the purity of the chromatographic peak is checked by DAD, which shows that it meets the requirements of chemical reference substances for the determination of the content of traditional Chinese medicine, and the content is greater than 95%.

[0026] Compared with the prior art, the present invention has the following outstanding substantive features and significant improvements:

[0027] 1. The present invention prepares Hyuganin D from Peucedanum sphenantherae for the first time, the purity of which meets the requirements of chemical reference substances (content above 95%), which can solve the supply problem of Hyuganin D chemical reference substances and provide a scientific basis and guarantee for the quality detection of Peucedanum sphenantherae.

[0028] 2. The extraction process of the present invention is reasonably designed and simple. It uses alcohol-water solvent extraction, and then recrystallizes after silica gel column chromatography to obtain Hyuganin D with high purity. Finally, a Hyuganin D chemical reference substance with a purity of more than 95% is prepared by preparative high performance liquid chromatography. The method is simple and easy.

[0029] 3. The present invention has fast separation speed, short production cycle, low cost, is suitable for industrial production, and has good application prospects and social value.

[0030] 4. The present invention adopts thin layer chromatography and high performance liquid chromatography to carry out purity inspection, content determination and quality inspection to ensure the quality of the product.

[0031] By studying Hyuganin D chemical reference substances, the batch extraction process, purity, content and impurity inspection analysis and determination methods of Hyuganin D chemical reference substances are established, thereby establishing the technical standards of Hyuganin D chemical reference substances, providing a scientific basis and guarantee for its use as a Chinese medicine chemical reference substance, as well as the quality standard research of medicinal materials and preparations. The present invention can provide a more complete basic chemical basis for Hyuganin D chemical reference substances, master its chemical information and analytical testing technology, and facilitate the further development and utilization of related products.

[0032] The research on the chemical composition of Chinese herbal medicines and the standardization of quality standards are the only way for the modernization of Chinese medicine. They are of great significance for clarifying the material basis of the effects of Chinese herbal medicines, formulating the production and processing technology of Chinese herbal medicine preparations, and identifying counterfeit and inferior products. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a process flow chart for the preparation of high-purity Hyuganin D;

[0034] Figure 2 This is the UV absorption spectrum of the chromatographic peak of Hyuganin D;

[0035] Figure 3 This is a thin layer chromatogram of Hyuganin D;

[0036] Figure 4 This is the HPLC chromatogram of the purity check of the Hyuganin D DAD peak;

[0037] Figure 5 This is a HPLC chromatogram of the quality test of Hyuganin D. DETAILED DESCRIPTION

[0038] Example 1

[0039] A method for preparing high-purity Hyuganin D comprises the following steps:

[0040] according to Figure 1The process flow comprises the following steps: taking 2 kg of the dried root of Peucedanum sphenantherum of the genus Peucedanum of the family Apiaceae, crushing the root, passing through a No. 3 sieve, using 8 times the amount of methanol for each kg of the dried root of Peucedanum sphenantherum, ultrasonically extracting the root for 30 min, repeating the process twice, filtering, combining the filtrate, concentrating under reduced pressure, and drying to obtain an extract; mixing the extract with silica gel, and then applying the extract to a silica gel column for chromatography by dry method, first eluting with a gradient of petroleum ether-ethyl acetate (volume ratio of 1:10-10:1), collecting the eluted portion of petroleum ether-ethyl acetate (volume ratio of 10:1), combining and concentrating the fraction containing Hyuganin D, and obtaining crude Hyuganin D crystals with a purity of 80-90% by weight; separating and purifying the crude crystals by preparative high performance liquid chromatography, wherein the chromatographic conditions are as follows: a C-18 column, isocratic elution using methanol-water (volume ratio of 70:25) as the mobile phase, a detection wavelength of 321 nm, and a flow rate of 1 mL / min, and collecting Hyuganin D; D component, and each collected eluate was detected by HPLC (chromatographic column: C-18 column, mobile phase methanol-water volume ratio 70:25, detection wavelength 321nm, flow rate according to 1mL / min), and Hyuganin D with the same retention time and a purity between 90% and 95% and a purity above 95% were combined, and concentrated under reduced pressure to obtain 4.64g of Hyuganin D with a purity between 90% and 95% and 18.56g of Hyuganin D with a purity above 95%.

[0041] Example 2

[0042] A method for preparing high-purity Hyuganin D comprises the following steps:

[0043] 2 kg of dried root of Peucedanum sphenantherum of the genus Peucedanum of the family Apiaceae is taken, crushed, passed through a No. 3 sieve, 8 times the amount of methanol is used for each kg of dried root of Peucedanum sphenantherum, ultrasonically extracted for 30 minutes, repeated twice, filtered, the filtrate is combined, concentrated under reduced pressure, and dried to obtain an extract, the extract is mixed with silica gel and then subjected to dry silica gel column chromatography, first eluted with petroleum ether-ethyl acetate (volume ratio 1:10-10:1) gradient, the petroleum ether-ethyl acetate (volume ratio 10:1) elution portion is collected, and the fraction containing Hyuganin D is combined and concentrated to obtain Hyuganin with a purity of 80-90% by weight. D crude crystals; the crude crystals were separated and purified by preparative high performance liquid chromatography, the chromatographic conditions were as follows: a C-18 column, a gradient elution using methanol (A)-water (B) as the mobile phase, an elution procedure (0-20 min, 50% A; 20-30 min, 50%-75% A; 30-50 min, 75% A; 50-60 min, 75%-50% A; 60-90 min, 50% A), a detection wavelength of 334 nm, a flow rate of 2 mL / min, and Hyuganin was collected. D component, and each collected eluate was detected by HPLC (chromatographic column: C-18 column, detection wavelength 334nm, flow rate according to 2mL / min), methanol (A)-water (B) gradient elution, elution procedure (0-20min, 70% A; 20-30min, 70%-50% A; 30-50min, 50% A; 50-60min, 50%-70% A; 60-90min, 70% A), and Hyuganin D with the same retention time and a purity between 90% and 95% and a purity above 95% were combined, and concentrated under reduced pressure to obtain 4.54g of Hyuganin D with a purity between 90% and 95% and 18.24g of Hyuganin D with a purity above 95%.

[0044] Example 3

[0045] A method for preparing high-purity Hyuganin D comprises the following steps:

[0046] 2 kg of dried root of Peucedanum sphenantherum of the genus Peucedanum of the family Apiaceae is taken, crushed, passed through a No. 3 sieve, 8 times the amount of methanol is used for each kg of dried root of Peucedanum sphenantherum, ultrasonically extracted for 30 minutes, repeated twice, filtered, the filtrate is combined, concentrated under reduced pressure, and dried to obtain an extract, the extract is mixed with silica gel and then subjected to dry silica gel column chromatography, first eluted with petroleum ether-ethyl acetate (volume ratio 1:10-10:1) gradient, the petroleum ether-ethyl acetate (volume ratio 10:1) elution portion is collected, and the fraction containing Hyuganin D is combined and concentrated to obtain Hyuganin with a purity of 80-90% by weight. D crude crystals; the crude crystals were separated and purified by preparative high performance liquid chromatography, the chromatographic column was a C-18 column, methanol (A)-water (B) was used as the mobile phase for gradient elution, the elution procedure was (0-20 min, 60% A; 20-30 min, 60%-75% A; 30-50 min, 75% A; 50-60 min, 75%-60% A; 60-90 min, 60% A), the detection wavelength was 321 nm, the flow rate was 2 mL / min, and Hyuganin was collected D component, and each collected eluate was detected by HPLC (chromatographic column: C18 column, detection wavelength 321nm, flow rate according to 2mL / min), methanol (A)-water (B) gradient elution, elution procedure (0-20min, 80%-70% A; 20-30min, 70%-50% A; 30-50min, 50% A; 50-60min, 50%-70% A; 60-90min, 70% A), and Hyuganin D with the same retention time and a purity between 90% and 95% and a purity above 95% were combined, and concentrated under reduced pressure to obtain 4.46g of Hyuganin D with a purity between 90% and 95% and 17.84g of Hyuganin D with a purity above 95%.

[0047] Example 4

[0048] The quality detection methods of Hyuganin D include thin layer chromatography and high performance liquid chromatography.

[0049] 1. Thin layer identification method:

[0050] Take an appropriate amount of Hyuganin D prepared in Example 1, prepare a 0.383 mg / mL solution with methanol, and spot the sample on the same silica gel plate (Qingdao Ocean Silica Gel Plate) according to different spotting amounts, and the spotting amounts are 1 μg, 1.5 μg, 2 μg, 2.5 μg, and 3 μg respectively; place the sample in a developing cylinder for development, and the developing solvent system of the thin layer chromatography includes: system (1) petroleum ether (60-90°C)-ethyl acetate in a ratio of 2:1; system (2) ethyl acetate-methanol-water in a ratio of 8:1:1; system (3) chloroform-methanol in a ratio of 4:1. Spread distance: 10 cm, positioning: take out, dry, and examine under ultraviolet light, and select a wavelength of 365 nm. The results are shown in the GoodLook-1000 thin layer chromatography imaging system. Figure 3 The development was carried out under the conditions of system (1), and a single blue fluorescent spot was observed. The other two developing agent systems were gradient spotted at 5 different concentrations, and the results were all single spots, with no impurity spots observed.

[0051] 2. Content determination and purity inspection

[0052] High performance liquid chromatography was used, and the chromatographic conditions of the high performance liquid chromatography were: chromatographic column AmethystC18-H (4.6×250 mm, 5 μm), flow rate: 1 ml / min, injection volume: 10 ul, and quantitative analysis by area normalization method:

[0053] System conditions (1) Mobile phase: acetonitrile (A)-water (B), gradient elution mode: 0-20 min, 20%-30% A; 20-30 min, 30%-60% A; 30-50 min, 60% A; 50-60 min, 60%-30% A; 60-90 min, 30%-20% A; detection wavelength 334 nm;

[0054] System conditions (2) Mobile phase: methanol (A)-water (B), gradient elution mode: 0-20 min, 50% A; 20-30 min, 50%-75% A; 30-50 min, 75% A; 50-60 min, 75%-50% A; 60-90 min, 50% A; Detection wavelength: 334 nm;

[0055] System conditions (3) Mobile phase: methanol (A)-water (B), gradient elution mode: 0-20 min, 50% A; 20-30 min, 50%-75% A; 30-50 min, 75% A; 50-60 min, 75%-50% A; 60-90 min, 50% A; detection wavelength: 321 nm.

[0056] Content and purity determination: Accurately weigh an appropriate amount of Hyuganin D, add methanol and water solution to make a 0.383 mg / ml solution. Under the above determination conditions, inject 10ul into the liquid chromatograph, and use the above three mobile phase solvent systems to record the chromatogram until the peak retention time of the main component is more than 2.5 times. Calculate the content by area normalization method. According to the system conditions (3), the test results are as follows: Figure 5 As shown, the content of the reference substance was above 95%; for impurity inspection, in the chromatograms recorded in different systems, except for the solvent peak, the sum of the impurity peak areas was less than 5.0%.

[0057] Peak purity test: Take an appropriate amount of Hyuganin D reference substance and perform peak purity test on a high performance liquid chromatograph using a DAD detector according to the mobile phase system conditions (3). The results are as follows: Figure 4 As shown, the HPLC chromatographic peak of Hyuganin D is >95%, and its chromatographic peak UV absorption spectrum completely overlaps, indicating that it is a single pure substance peak.

[0058] from Figure 4 It can be seen that the DAD peak purity check HPLC chromatogram of Hyuganin D is a single peak, indicating that it is a pure substance peak. At the same time, it can be seen from Table 1 below that the purity angles are all less than the purity threshold, indicating that it is a single substance.

[0059] Table 1

[0060]

[0061]

[0062] from Figure 5 It can be seen that the main component Hyuganin D has a retention time of about 32 minutes, the content of the main component is greater than 96%, and the sum of the peak areas of each impurity does not exceed 4.0%. The peak result data information of the HPLC detection chromatogram of its quality detection is as follows in Table 2:

[0063] Table 2

[0064] Serial number Retention time (minutes) Peak area result(%) 1 7.642 14375 0.18 2 9.319 25452 0.31 3 10.446 14222 0.17 4 23.444 22924 0.28 5 29.313 165273 2.03 6 30.103 28140 0.35 7 32.269 2086 0.03 8 32.680 7838517 96.18 9 35.393 38632 0.47 .

Claims

1. A method for preparing high-purity hyuganin D, characterized in that: The following steps are involved: (1) The dried root of Peucedanum sphenantherum of the Umbelliferae family was crushed, extracted with methanol, filtered, the filtrate was combined, concentrated under reduced pressure, and dried to obtain an extract, which was subjected to silica gel column chromatography and gradient elution with petroleum ether-ethyl acetate to collect the fraction containing hyuganin D, and concentrated to obtain crude hyuganin D crystals; (2) The crude hyuganin D crystals obtained in step (1) are separated and purified by preparative high performance liquid chromatography, and the hyuganin D fractions are collected. Each of the collected eluates is analyzed by HPLC, and the hyuganin D fractions with the same retention time and a purity between 90% and 95% and those with a purity above 95% are combined, and concentrated under reduced pressure to obtain hyuganin D fractions with a purity between 90% and 95% and above 95%.

2. The method for preparing high-purity hyuganin D according to claim 1, characterized in that: In step (1), 8 to 15 times the volume of the medicinal material is used as methanol, and ultrasonic extraction is performed for 1 to 3 times, each time for 30 to 60 minutes.

3. The method for preparing high-purity hyuganin D according to claim 1, characterized in that: Step (1) silica gel column chromatography is performed using a gradient elution of petroleum ether-ethyl acetate in a volume ratio of 1:0-0:1; and then the petroleum ether-ethyl acetate elution fraction in a volume ratio of 10:1 is collected.

4. The method for preparing high-purity Hyuganin D according to claim 1, characterized in that: The chromatographic column of the preparative high performance liquid chromatography method is a C-18 column, using methanol-water with a volume ratio of 75:25 as the mobile phase for elution, a detection wavelength of 321nm, a flow rate of 1mL / min, and a column temperature of 35°C.

5. The method for preparing high-purity Hyuganin D according to claim 1, characterized in that: The HPLC analysis conditions are as follows: the chromatographic column is a C-18 column; the mobile phase is methanol-water with a volume ratio of 75:25; the detection wavelength is 321 nm; and the flow rate is 1 ml / min.

6. A method for detecting high-purity Hyuganin D prepared by the preparation method according to claim 1, characterized in that: The thin layer chromatography analysis method was adopted: an appropriate amount of Hyuganin D was taken, and a Hyuganin D solution of a certain concentration was prepared with methanol. On the same silica gel plate, the samples were spotted in different gradients according to different spotting amounts, and the samples were developed in a developing cylinder. The samples were taken out, dried, and examined under ultraviolet light with a wavelength of 365 nm. As a result, a single blue fluorescent spot was visible in the GoodLook-1000 thin layer chromatography imaging system.

7. The detection method according to claim 6, characterized in that: The developing agent system includes: system (1) petroleum ether (60-90°C)-ethyl acetate in a ratio of 2:1; system (2) ethyl acetate-methanol-water in a ratio of 8:1:1; system (3) chloroform-methanol in a ratio of 4:

1.

8. A detection control method for high-purity Hyuganin D prepared by the preparation method according to claim 1, characterized in that: The content was determined by high performance liquid chromatography analysis: an appropriate amount of Hyuganin D product was accurately weighed, and a methanol-water solution was added to prepare a Hyuganin D solution of a certain concentration, which was injected into a liquid chromatograph, and the content was calculated by the area normalization method. The result system was used to determine the content of the reference product; Peak purity test: Take an appropriate amount of Hyuganin D reference substance and perform peak purity check on a high performance liquid chromatograph using a diode array DAD detector according to the mobile phase system.

9. The detection method according to claim 8, characterized in that: The chromatographic conditions were as follows: chromatographic column Amethyst C18-H, specification 4.6×250 mm, 5 μm, mobile phase: acetonitrile-water or methanol-water, flow rate: 1 ml / min, injection volume: 10 ul.

10. The detection method according to claim 9, characterized in that: When the content is detected, when the mobile phase is acetonitrile (A)-water (B), the gradient elution mode is: 0-20min, 20%-30% A; 20-30min, 30%-60% A; 30-50min, 60% A; 50-60min, 60%-30% A; 60-90min, 30%-20% A; Detection wavelength 334nm; Or when the mobile phase is methanol (A)-water (B), the gradient elution mode is: 0-20 min, 50% A; 20-30 min, 50%-75% A; 30-50 min, 75% A; 50-60 min, 75%-50% A; 60-90 min, 50% A; detection wavelength: 334 nm; During peak purity detection, when the mobile phase is methanol (A)-water (B), the gradient elution method is: 0-20 min, 50% A; 20-30 min, 50%-75% A; 30-50 min, 75% A; 50-60 min, 75%-50% A; 60-90 min, 50% A; detection wavelength: 321 nm.

Images