A method for detecting chemical components of water gentian

Abstract

The present invention provides a detection method and a separation method for the chemical components of water zephyranthes. The present invention uses ultra-high performance liquid chromatography combined with a specific mobile phase and an elution procedure to separate 68 compounds from water zephyranthes, and further uses an ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometer analysis method to quickly identify 50 compounds, including 21 flavonoid compounds, 12 triterpenoid compounds, 10 proanthocyanidin components, 3 cyclopentadiene ether terpenes, 2 phenolic acids and 2 other types of structures. The results show that water zephyranthes contains a variety of biologically active ingredients, has strong ecological significance, and has a high development and utilization value. The present invention enriches the chemical components of water zephyranthes, and lays a foundation for its pharmacological activity and development and utilization research, and at the same time provides a new preparation method for 50 compounds, enriching the source of the compounds.

Description

Technical Field

[0001] The invention relates to the technical field of chemical component analysis, and in particular to a method for detecting chemical components of water gentian. Background Art

[0002] Pemphis acidula JRet Forst. is a multi-branched shrub of the Lythraceae family and the genus Pemphis, which is sometimes in the shape of a small tree. It has a unique dimorphic flower pattern among Lythraceae plants, which is of great significance for studying the formation and evolution of heterotypic plants. Therefore, Pemphis acidula has important conservation value and in-depth research significance. Pemphis acidula extract is rich in polysaccharides, flavonoids and other chemical components, and has multiple biological activities such as in vitro antioxidant, antibacterial, anti-inflammatory and anti-tumor activities. Its medicinal and health care value has gradually attracted people's attention. In this study, UPLC-Q-TOF-MS technology was used to analyze the chemical components of Pemphis acidula extract. Electrospray ionization source (ESI) was used to scan and collect data in positive and negative ion modes. UNIFI scientific information system was used to attribute the main chromatographic peaks and quickly identify the main chemical composition in combination with relevant literature data, which provided a reference for further research on the physiologically active substances of Pemphis acidula and provided a basic basis for scientific and reasonable protection of Pemphis acidula. Summary of the invention

[0003] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for detecting the chemical components of water zephyranthes, which can effectively separate and identify the chemical components in water zephyranthes.

[0004] The first aspect of the present invention is to provide a method for detecting the chemical components of water zephyranthes, taking water zephyranthes as the detection object and adopting ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry for determination.

[0005] The conditions for ultra-high performance liquid chromatography were:

[0006] The chromatographic column is a 100 mm × 2.1 mm, 1.8 μm Waters ACQUITY UPLC HSS T3 column; column temperature: 38-42°C; volume flow rate: 0.2-0.4 mL / min; mobile phase: mobile phase A is a 0.05-0.15% volume concentration of formic acid in water; mobile phase B is a 0.005-0.015% volume concentration of formic acid in acetonitrile; gradient elution, the elution gradient is as follows:

[0007] 0-2 min, 99% → 92% mobile phase A, 1% → 8% mobile phase B;

[0008] 2-7 min, 92% → 84% mobile phase A, 8% → 16% mobile phase B;

[0009] 7-10 min, 84% → 80% mobile phase A, 16% → 20% mobile phase B;

[0010] 10-11 min, 80% → 64% mobile phase A, 20% → 36% mobile phase B;

[0011] 11-14 min, 64% → 24% mobile phase A, 36% → 76% mobile phase B;

[0012] 14-15 min, 24% → 1% mobile phase A, 76% → 99% mobile phase B;

[0013] 15-19 min, 1% mobile phase A, 99% mobile phase B;

[0014] 19-20 min, 1% → 99% mobile phase A, 99% → 1% mobile phase B;

[0015] 20-23min, 99% mobile phase A, 1% mobile phase B.

[0016] Wherein, before the ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry is used for determination, the detection object is extracted with alcohol, and the alcohol is preferably methanol and / or ethanol.

[0017] Preferably, the column temperature is 40°C.

[0018] Preferably, the volume flow rate is 0.25-0.35 mL / min, more preferably 0.3 mL / min.

[0019] Preferably, the mobile phase A is a formic acid aqueous solution with a volume concentration of 0.08-0.12%, more preferably a formic acid aqueous solution with a volume concentration of 0.1%.

[0020] Preferably, the mobile phase B is a formic acid acetonitrile solution with a volume concentration of 0.008-0.012%, preferably a formic acid acetonitrile solution with a volume concentration of 0.01%.

[0021] The mass spectrometry conditions of the mass spectrometer are as follows: using an electrospray ionization source (ESI) to collect MS under positive and negative ion conditions E data.

[0022] Preferably, the mass spectrometry conditions of the mass spectrometer also include: scanning range m / z 50-1200, scanning time 0.2s, detection time 20min; low energy collision voltage (CE) 6V, high energy collision voltage 20-60V; capillary voltages of positive and negative ion modes are 3.8kV and 2kV respectively, cone voltage is 45V, ion source temperature is 120°C, auxiliary spray ionization and desolvation gas is high purity N2, desolvation temperature is 350°C, cone gas flow rate is 50L·h-1 The desolvation gas flow rates in the positive and negative ion modes were 650 L·h -1 and 600L·h -1 .

[0023] The detection method of the present invention can identify 50 compounds from water caltrop, including 21 flavonoids, 12 triterpenoids, 10 proanthocyanidins, 3 iridoids, 2 phenolic acids and 2 other types of structures. The identification results are shown in Table 1 and the compound results are shown in Table Figure 3 and Figure 4 .

[0024] The second aspect of the present invention is to provide a method for separating the chemical components of water zephyranthes, taking water zephyranthes as the separation object and adopting ultra-high performance liquid chromatography for separation.

[0025] The conditions for ultra-high performance liquid chromatography were:

[0026] The chromatographic column is a 100 mm × 2.1 mm, 1.8 μm Waters ACQUITY UPLC HSS T3 column; column temperature: 38-42°C; volume flow rate: 0.2-0.4 mL / min; mobile phase: mobile phase A is a 0.05-0.15% volume concentration of formic acid in water; mobile phase B is a 0.005-0.015% volume concentration of formic acid in acetonitrile; gradient elution, the elution gradient is as follows:

[0027] 0-2 min, 99% → 92% mobile phase A, 1% → 8% mobile phase B;

[0028] 2-7 min, 92% → 84% mobile phase A, 8% → 16% mobile phase B;

[0029] 7-10 min, 84% → 80% mobile phase A, 16% → 20% mobile phase B;

[0030] 10-11 min, 80% → 64% mobile phase A, 20% → 36% mobile phase B;

[0031] 11-14 min, 64% → 24% mobile phase A, 36% → 76% mobile phase B;

[0032] 14-15 min, 24% → 1% mobile phase A, 76% → 99% mobile phase B;

[0033] 15-19 min, 1% mobile phase A, 99% mobile phase B;

[0034] 19-20 min, 1% → 99% mobile phase A, 99% → 1% mobile phase B;

[0035] 20-23min, 99% mobile phase A, 1% mobile phase B.

[0036] Wherein, before separation by ultra-high performance liquid chromatography, the separation object is extracted by alcohol, and the alcohol is preferably methanol and / or ethanol.

[0037] Preferably, the column temperature is 40°C.

[0038] Preferably, the volume flow rate is 0.25-0.35 mL / min, more preferably 0.3 mL / min.

[0039] Preferably, the mobile phase A is a formic acid aqueous solution with a volume concentration of 0.08-0.12%, more preferably a formic acid aqueous solution with a volume concentration of 0.1%.

[0040] Preferably, the mobile phase B is a formic acid acetonitrile solution with a volume concentration of 0.008-0.012%, preferably a formic acid acetonitrile solution with a volume concentration of 0.01%.

[0041] By adopting the separation method of the present invention, 68 compounds can be separated from water gentian.

[0042] The third aspect of the present invention is to provide a method for preparing a compound, wherein the compound is one or more of the compounds in the following table:

[0043]

[0044]

[0045]

[0046] It is separated from water genkwa by ultra-high performance liquid chromatography, and the conditions of ultra-high performance liquid chromatography are:

[0047] The chromatographic column is a 100 mm × 2.1 mm, 1.8 μm Waters ACQUITY UPLC HSS T3 column; column temperature: 38-42°C; volume flow rate: 0.2-0.4 mL / min; mobile phase: mobile phase A is a 0.05-0.15% volume concentration of formic acid in water; mobile phase B is a 0.005-0.015% volume concentration of formic acid in acetonitrile; gradient elution, the elution gradient is as follows:

[0048] 0-2 min, 99% → 92% mobile phase A, 1% → 8% mobile phase B;

[0049] 2-7 min, 92% → 84% mobile phase A, 8% → 16% mobile phase B;

[0050] 7-10 min, 84% → 80% mobile phase A, 16% → 20% mobile phase B;

[0051] 10-11 min, 80% → 64% mobile phase A, 20% → 36% mobile phase B;

[0052] 11-14 min, 64% → 24% mobile phase A, 36% → 76% mobile phase B;

[0053] 14-15 min, 24% → 1% mobile phase A, 76% → 99% mobile phase B;

[0054] 15-19 min, 1% mobile phase A, 99% mobile phase B;

[0055] 19-20 min, 1% → 99% mobile phase A, 99% → 1% mobile phase B;

[0056] 20-23min, 99% mobile phase A, 1% mobile phase B.

[0057] Wherein, before separation by ultra-high performance liquid chromatography, the separation object is extracted by alcohol, and the alcohol is preferably methanol and / or ethanol.

[0058] Preferably, the column temperature is 40°C.

[0059] Preferably, the volume flow rate is 0.25-0.35 mL / min, more preferably 0.3 mL / min.

[0060] Preferably, the mobile phase A is a formic acid aqueous solution with a volume concentration of 0.08-0.12%, more preferably a formic acid aqueous solution with a volume concentration of 0.1%.

[0061] Preferably, the mobile phase B is a formic acid acetonitrile solution with a volume concentration of 0.008-0.012%, preferably a formic acid acetonitrile solution with a volume concentration of 0.01%.

[0062] The present invention uses ultra-high performance liquid chromatography combined with a specific mobile phase and elution program to separate 68 compounds from water zephyranthes, and further uses an ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometer analysis method to quickly identify 50 compounds, including 21 flavonoid compounds, 12 triterpenoid compounds, 10 proanthocyanidins, 3 cyclopentadiene ether terpenes, 2 phenolic acids and 2 other types of structures. The results show that water zephyranthes contains a variety of biologically active ingredients, has strong ecological significance, and has a high development and utilization value. The present invention enriches the chemical components of water zephyranthes, and lays a foundation for its pharmacological activity and development and utilization research, and at the same time provides a new preparation method for 50 compounds, enriching the source of the compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1The TIC flow chart of water gentian in positive and negative ion modes.

[0064] Figure 2 TIC diagram of Blank in positive and negative ion modes.

[0065] Figure 3 and Figure 4 This is the structure diagram of the compound identified from Water gentian. DETAILED DESCRIPTION

[0066] The present invention will be further described below with reference to the accompanying drawings in conjunction with specific embodiments to better understand the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. Where the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be obtained commercially.

[0067] 1Instruments and consumables

[0068] Instrument: LC-MS instrument mainly includes I-Class system, Xevo G2-XS Q-Tof mass spectrometer system, autosampler, binary solvent manager (Waters, USA); ACQUITY UPLC HSS T3 (100×2.1 mm1.8 μm) chromatographic column (Waters, USA), Secura 513-1CN precision balance (Sartorius, Germany), KQ3200DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), Milli-Q ultrapure water instrument (Millipore, USA), 5810R desktop high-speed refrigerated centrifuge (Eppendorf Centrifuge, Germany).

[0069] Consumables: 0.2μm, 13mm Pall Syringe filter membrane (Pall Corporation, USA), 1 mL disposable sterile syringe with needle (Fenglin Medical Instrument Co., Ltd.), Labmed pipette tips 1000 μL, 200 μL, 20 μL (Labmed Biotech Corporation, USA), KG2211W 1.5 mL centrifuge tube (KiRGEN Corporation, USA), 2 mL transparent screw cap sample bottle (Waters Corporation, USA), Oasis HLB 3cc (60 mg) Extraction Cartridges (Waters Corporation, USA).

[0070] Software: Masslynx V4.1 mass spectrometry software (Waters, USA), Scientific Information System (Waters Corporation, USA).

[0071] Reagents: chromatographic grade methanol and isopropanol (Merck, Germany), mass spectrometry grade acetonitrile and formic acid (Merck, Germany); ACS grade sodium hydroxide (Sigma, USA); leucine enkephalin (Standards Kit for TofG2-S, Waters, USA); distilled water (Guangzhou Watsons Food and Beverage Co., Ltd.); ultrapure water prepared by Milli-Q ultrapure water instrument (Millipore, USA); other reagents were of analytical grade.

[0072] 2 Methods

[0073] 2.1 Test sample preparation

[0074] All liquid samples must be prepared and used immediately, and stored in a refrigerator at 4°C for no more than 24 hours.

[0075] Preparation of test solution: Take the raw material of Daphne daphne, dry it naturally, crush it, and pass it through an 80-mesh sieve. Accurately weigh 1g of the sample, add 20ml of chromatographic methanol, ultrasonically extract for 30min, let it stand, take 1mL of the extract, refrigerate centrifuge (12000rpm, 15min, 10℃), take the supernatant and pass it through a 0.2μm microporous filter membrane, and store it in a 2mL transparent sample bottle, marked as SYH.

[0076] 2.2 UPLC-Q-TOF-MS detection conditions

[0077] (1) Chromatographic conditions

[0078] Waters ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm 1.8 μm), volume flow rate 0.3 mL min -1 , injection volume 1 μL, column temperature 40°C; mobile phase 0.1% formic acid aqueous solution (A)-0.01% formic acid acetonitrile solution (B), gradient elution, the elution gradient is as follows:

[0079] 0-2 min, 99% → 92% mobile phase A, 1% → 8% mobile phase B;

[0080] 2-7 min, 92% → 84% mobile phase A, 8% → 16% mobile phase B;

[0081] 7-10 min, 84% → 80% mobile phase A, 16% → 20% mobile phase B;

[0082] 10-11 min, 80% → 64% mobile phase A, 20% → 36% mobile phase B;

[0083] 11-14 min, 64% → 24% mobile phase A, 36% → 76% mobile phase B;

[0084] 14-15 min, 24% → 1% mobile phase A, 76% → 99% mobile phase B;

[0085] 15-19 min, 1% mobile phase A, 99% mobile phase B;

[0086] 19-20 min, 1% → 99% mobile phase A, 99% → 1% mobile phase B;

[0087] 20-23min, 99% mobile phase A, 1% mobile phase B.

[0088] (2) Mass spectrometry conditions

[0089] Electrospray ionization (ESI) was used to collect MS under positive and negative ion conditions. E data.

[0090] The scanning range was m / z 50-1200, the scanning time was 0.2 s, and the detection time was 20 min.

[0091] The low energy collision voltage (CE) was 6 V, and the high energy collision voltage was 20-60 V. The capillary voltages for the positive and negative ion modes were 3.8 kV and 2 kV, respectively. The cone voltage was 45 V, the ion source temperature was 120 °C, the auxiliary spray ionization and desolvation gas was high-purity N2, the desolvation temperature was 350 °C, and the cone gas flow rate was 50 L·h -1 The desolvation gas flow rates in the positive and negative ion modes were 650 L·h -1 and 600L·h -1 .

[0092] 2.3 Data processing

[0093] Masslynx V4.1 software intelligent control system is used to collect UPLC / Q-TOF-MS E Data; With the help of UNIFI scientific information system, comprehensive literature report data, combined with TCM Chinese [UNIFI1.7], ChemSpider online database, compound component identification was carried out.

[0094] 3 Results and analysis

[0095] Based on UPLC / Q-TOF-MS technology, under the premise of optimizing chromatographic conditions, the mass spectrometry peaks and ion responses in positive and negative ion modes were investigated to conduct qualitative analysis of the test samples. By observing the total ion current (TIC), it was found that in the negative ion mode, the mass spectrometry ion current had better separation and higher response ( Figure 1 ).

[0096] Based on the peak situation, the data was imported into UNIFI, and the relevant method parameters were set. According to the accurate relative molecular mass given by the mass spectrum, the mass spectrum fragment information was extracted to conduct a qualitative analysis of the chemical composition of the sample. Combined with the relevant literature and database inference, the identification results are shown in Table 1. 68 compounds were obtained from the analysis of water lily, and the structures of 50 of them were identified ( Figure 3 and Figure 4 , the number below the compound structure in the figure corresponds to No. in Table 1). It includes 21 flavonoids, 12 triterpenoids, 10 proanthocyanidins, 3 cyclopentadiene, 2 phenolic acids and 2 other types of structures. Flavonoids are the main components of water scutellaria, including flavonoids, flavonoid glycosides and dihydroflavonoid aglycones and their glycosides. Flavonoids have obvious antioxidant, antibacterial and anti-inflammatory functions. Characteristic proanthocyanidins were also identified in this material, mainly B-type catechins formed by catechin and epicatechin connected by a CC bond. Proanthocyanidins are natural antioxidants, indicating that water scutellaria has strong antioxidant activity and has good development and application prospects. Cyclopentadiene has a good protective effect on the nervous system and also has an immune-enhancing effect. It is also one of the main components of water scutellaria. Another major component in water calendula is triterpenes. This study identified 12 triterpenoid components, mainly oleanane-type triterpenes. Triterpenes are a major class of plant secondary metabolites with multiple functions such as anti-inflammatory, whitening and liver protection.

[0097] Table 1 UPLC / Q-TOF-MS / MS mass spectrometry data and identification of SYH

[0098]

[0099]

[0100]

[0101]

[0102]

[0103] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modification and substitution of the practical is also within the scope of the present invention. Therefore, the equalization transformation and modification made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A method for detecting the chemical components of water genkwa, characterized in that: Taking Water gentian as the detection object, ultra-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry was used for determination; The conditions for ultra-high performance liquid chromatography were: The chromatographic column was a 100 mm×2.1 mm, 1.8 μm Waters ACQUITY UPLC HSS T3 column; column temperature: 38-42°C; volume flow rate: 0.2-0.4 mL / min; mobile phase: mobile phase A was a 0.05-0.15% volume concentration of formic acid in water; mobile phase B was a 0.005-0.015% volume concentration of formic acid in acetonitrile; gradient elution, the elution gradient was as follows: 0-2 min, 99%→92% mobile phase A, 1%→8% mobile phase B; 2-7 min, 92%→84% mobile phase A, 8%→16% mobile phase B; 7-10 min, 84%→80% mobile phase A, 16%→20% mobile phase B; 10-11 min, 80%→64% mobile phase A, 20%→36% mobile phase B; 11-14 min, 64%→24% mobile phase A, 36%→76% mobile phase B; 14-15 min, 24% → 1% mobile phase A, 76% → 99% mobile phase B; 15-19 min, 1% mobile phase A, 99% mobile phase B; 19-20 min, 1%→99% mobile phase A, 99%→1% mobile phase B; 20-23 min, 99% mobile phase A, 1% mobile phase B.

2. The detection method according to claim 1, characterized in that: The test objects were extracted with alcohol before being measured by ultra-performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry.

3. The detection method according to claim 2, characterized in that: The alcohol is methanol and / or ethanol.

4. The detection method according to claim 1 or 2, characterized in that: In the conditions of ultra-high performance liquid chromatography, the column temperature is 40°C; the volume flow rate is 0.25-0.35 mL / min; the mobile phase A is a formic acid aqueous solution with a volume concentration of 0.08-0.12%; and the mobile phase B is a formic acid acetonitrile solution with a volume concentration of 0.008-0.012%.

5. The detection method according to claim 4, characterized in that: The volume flow rate is 0.3 mL / min, the mobile phase A is a 0.1% by volume formic acid aqueous solution, and the mobile phase B is a 0.01% by volume formic acid acetonitrile solution.

6. The detection method according to claim 1, characterized in that: The mass spectrometry conditions of the mass spectrometer are as follows: using an electrospray ion source to collect MS under positive and negative ion conditions E data.

7. The detection method according to claim 6, characterized in that: Scan range m / z 50~1200, scanning time 0.2s, detection time 20min; low energy collision voltage (CE) 6V, high energy collision voltage 20~60V; capillary voltage of positive and negative ion mode was 3.8kV and 2kV, cone voltage was 45V, ion source temperature was 120℃, auxiliary spray ionization and desolvation gas was high purity N2, desolvation temperature was 350℃, cone gas flow rate was 50L·h -1 The desolvation gas flow rates in the positive and negative ion modes were 650 L·h -1 and 600 L·h -1 .

8. A method for separating the chemical components of water genkwa, characterized in that: Taking Daphne daphne as the separation object, ultra-high performance liquid chromatography was used for separation; The conditions for ultra-high performance liquid chromatography were: The chromatographic column was a 100 mm×2.1 mm, 1.8 μm Waters ACQUITY UPLC HSS T3 column; column temperature: 38-42°C; volume flow rate: 0.2-0.4 mL / min; mobile phase: mobile phase A was a 0.05-0.15% volume concentration of formic acid in water; mobile phase B was a 0.005-0.015% volume concentration of formic acid in acetonitrile; gradient elution, the elution gradient was as follows: 0-2 min, 99%→92% mobile phase A, 1%→8% mobile phase B; 2-7 min, 92%→84% mobile phase A, 8%→16% mobile phase B; 7-10 min, 84%→80% mobile phase A, 16%→20% mobile phase B; 10-11 min, 80%→64% mobile phase A, 20%→36% mobile phase B; 11-14 min, 64%→24% mobile phase A, 36%→76% mobile phase B; 14-15 min, 24% → 1% mobile phase A, 76% → 99% mobile phase B; 15-19 min, 1% mobile phase A, 99% mobile phase B; 19-20 min, 1%→99% mobile phase A, 99%→1% mobile phase B; 20-23 min, 99% mobile phase A, 1% mobile phase B.

9. The separation method according to claim 8, characterized in that Before separation by ultra-high performance liquid chromatography, the separation object is extracted with alcohol.

10. The separation method according to claim 9, characterized in that The alcohol is methanol and / or ethanol.

11. The separation method according to claim 8 or 9, characterized in that: In the conditions of ultra-high performance liquid chromatography, the column temperature is 40°C; the volume flow rate is 0.25-0.35 mL / min; the mobile phase A is a formic acid aqueous solution with a volume concentration of 0.08-0.12%; and the mobile phase B is a formic acid acetonitrile solution with a volume concentration of 0.008-0.012%.

12. The separation method according to claim 11, characterized in that The volume flow rate is 0.3 mL / min; mobile phase A is a 0.1% by volume formic acid aqueous solution; and mobile phase B is a 0.01% by volume formic acid acetonitrile solution.

13. A method for preparing a compound, characterized in that: The compound is one or more of the compounds in the following table: It is separated from water genkwa by ultra-high performance liquid chromatography, and the conditions of ultra-high performance liquid chromatography are: The chromatographic column was a 100 mm×2.1 mm, 1.8 μm Waters ACQUITY UPLC HSS T3 column; column temperature: 38-42°C; volume flow rate: 0.2-0.4 mL / min; mobile phase: mobile phase A was a 0.05-0.15% volume concentration of formic acid in water; mobile phase B was a 0.005-0.015% volume concentration of formic acid in acetonitrile; gradient elution, the elution gradient was as follows: 0-2 min, 99%→92% mobile phase A, 1%→8% mobile phase B; 2-7 min, 92%→84% mobile phase A, 8%→16% mobile phase B; 7-10 min, 84%→80% mobile phase A, 16%→20% mobile phase B; 10-11 min, 80%→64% mobile phase A, 20%→36% mobile phase B; 11-14 min, 64%→24% mobile phase A, 36%→76% mobile phase B; 14-15 min, 24% → 1% mobile phase A, 76% → 99% mobile phase B; 15-19 min, 1% mobile phase A, 99% mobile phase B; 19-20 min, 1%→99% mobile phase A, 99%→1% mobile phase B; 20-23 min, 99% mobile phase A, 1% mobile phase B.

14. The preparation method according to claim 13, characterized in that: Before separation by ultra-high performance liquid chromatography, the separation object is extracted with alcohol.

15. The preparation method according to claim 14, characterized in that: The alcohol is methanol and / or ethanol.

16. The preparation method according to claim 13 or 14, characterized in that: In the conditions of ultra-high performance liquid chromatography, the column temperature is 40°C; the volume flow rate is 0.25-0.35 mL / min; the mobile phase A is a formic acid aqueous solution with a volume concentration of 0.08-0.12%; and the mobile phase B is a formic acid acetonitrile solution with a volume concentration of 0.008-0.012%.

17. The preparation method according to claim 16, characterized in that The volume flow rate was 0.3 mL / min; mobile phase A was a 0.1% by volume formic acid aqueous solution; and mobile phase B was a 0.01% by volume formic acid acetonitrile solution.

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