Marker for rapidly identifying various sulfur-fumigated traditional Chinese medicinal materials and method thereof

Abstract

The application discloses a kind of marker for quickly identifying multiple sulfur fumigation traditional Chinese medicinal materials and method thereof.2-sulfo-tryptophan can be used as a sulfur fumigation marker to identify sulfur fumigation traditional Chinese medicinal materials, and the traditional Chinese medicinal materials are Ophiopogon japonicus, Fritillaria, Houttuynia cordata, Anemarrhena asphodeloides or Lily.The method for identifying sulfur fumigation traditional Chinese medicinal materials provided by the application comprises the following steps:S1, the sample to be measured is crushed and dissolved in a methanol solution, centrifuged after mixing, and filtered to obtain a filtrate;S2, the filtrate is analyzed by UPLC-Q-TOF-MS to obtain a total ion current chromatogram, and if the total ion current chromatogram includes the molecular ion peak of 2-sulfo-tryptophan, the sample to be measured is a sulfur fumigation traditional Chinese medicinal material.The application uses UPLC-Q-TOF-MS combined with metabolomics statistical methods to find a universal sulfur fumigation marker in sulfur fumigation Houttuynia cordata, Fritillaria, Ophiopogon japonicus, Anemarrhena asphodeloides and Lily, which can be used for the rapid identification of multiple sulfur fumigation traditional Chinese medicinal materials.

Description

Technical Field

[0001] This invention relates to a marker and method for rapidly identifying various sulfur-fumigated Chinese medicinal materials, belonging to the field of analytical technology. Background Technology

[0002] Sulfur fumigation (hereinafter referred to as "sulfur fumigation") is a commonly used primary processing method for Chinese medicinal materials. It is often used to dry Chinese medicinal materials with high starch and polysaccharide content. However, due to its simple operation and low cost, it has been overused in recent years. The phenomenon of sulfur fumigation of Chinese medicinal materials still exists in the market (Mao Chunqin, Lu Tulin, Shan Xin, et al. Research progress on the historical evolution and current status of sulfur fumigation of Chinese medicinal materials [A]. China Association of Traditional Chinese Medicine, 2014:2.; Xu Weiyi, Yu Jiangyong, Jin Hongyu, et al. Survey on sulfur fumigation of Chinese medicinal materials and decoction pieces in my country from 2013 to 2017 and recommendations on the limit standard for sulfur dioxide residue [J]. China Pharmacy, 2019, 30(24):3330-3336.; Sun X, Cui XB, Wen HM, ex al. Influence of sulfur fumigation on the chemical profiles of Atractylodes macrocephala Koidz. evaluated by UFLC-QTOF-MS combined with multivariate statistical analysis [J]. Journal of pharmaceutical and biomedical analysis, 2017, 141:19-31.). Tryptophan is an important precursor for auxin biosynthesis in plants. Its structure is similar to that of IAA and it is widely present in higher plants. Among them, Ophiopogon japonicus (Wang Yancui. Study on chemical and nutritional components and in vitro activity of Ophiopogon japonicus [D]. Northwest A&F University, 2016.), Fritillaria cirrhosa (Lu Qi, Xue Shujing, Yang De, et al. Comparative analysis of components and physicochemical properties of Fritillaria cirrhosa from Hubei and Fritillaria thunbergii [J]. Food Industry Technology, 2023, 44(03):49-55.), and Houttuynia cordata (Chen Li, Wu Wei, Zheng Youliang. Amino acid composition and content of Houttuynia cordata germplasm resources) Quantitative analysis [J]. Guangxi Plants, 2006(06):697-701.), Anemarrhena asphodeloides (Xiao Xichen, Jia Penghui, Huang Qi, et al. Preliminary experiment on chemical components of Anemarrhena asphodeloides fibrous roots [J]. Guangzhou Chemical Industry, 2016, 44(22):42-43+61.), and Lilium brownii (Zhu Jianping, Chen Yuzhen, Gao Mengmeng, et al. Comparative study on chemical components of fresh and dried Lilium brownii root decoction based on UPLC-Q-TOF / MS method [J]. Journal of Hunan University of Traditional Chinese Medicine, 2021, 41(11):1698-1704.) are all contained in these medicinal materials.

[0003] Current research on sulfur fumigation markers mostly focuses on single or similar Chinese medicinal materials (Kang Chuanzhi. Effects of sulfur fumigation on the quality of Gastrodia elata and Achyranthes bidentata [D]. China Academy of Chinese Medical Sciences, 2018.; Deng Aiping, Kang Chuanzhi, Zhang Yue, et al. Effects of sulfur fumigation on the chemical components and pharmacological effects of Chinese medicinal materials [J]. Journal of Pharmaceutical Analysis, 2019, 39(09):1542-1559.; Deng Aiping. Effects of sulfur fumigation on the quality of Chinese medicinal materials containing α,β-unsaturated carbonyl compounds [D]. China Academy of Chinese Medical Sciences, 2022.; Zhang Rong, Yang Yaling, Deng Aiping, et al. Effects of sulfur fumigation on the quality and safety of Lilium brownii [J]. Chinese Materia Medica, 2023, 48(03):660-671.). There are no simple and universal markers for the detection of multiple varieties of sulfur-fumigated Chinese medicinal materials. Therefore, there is a need to provide a method that can simultaneously identify multiple sulfur-fumigated Chinese medicinal materials. Summary of the Invention

[0004] The purpose of this invention is to provide a marker and method for rapid identification of various sulfur-fumigated Chinese medicinal materials. This invention utilizes UPLC-Q-TOF-MS combined with metabolomics statistical methods to isolate common sulfur-fumigated characteristic components in sulfur-fumigated Chinese medicinal materials (Houttuynia cordata), which are used for rapid identification of sulfur-fumigated Chinese medicinal materials. It has the characteristics of being fast, simple, and highly specific.

[0005] The biomarker provided by this invention for identifying various sulfur-fumigated Chinese medicinal materials is 2-sulfotryptophan, with the structural formula shown in Formula I:

[0006]

[0007] As shown in Formula I, 2-sulfotryptophan is used as a sulfur fumigation marker. Only sulfur-fumigated Chinese medicinal materials contain this component, which can be used to distinguish between sulfur-fumigated and sulfur-free Chinese medicinal materials.

[0008] Formula I shows that 2-sulfotryptophan can be used to identify a variety of different Chinese medicinal herbs, such as houttuynia cordata, fritillaria cirrhosa, ophiopogon japonicus, anemarrhena asphodeloides, and lily bulb. It is fast, simple, and universally applicable, and has great significance for promotion. Among them, the fritillaria cirrhosa involved in this invention can be fritillaria thunbergii, fritillaria sylvestris, fritillaria hupehensis, fritillaria yishen, and fritillaria chuanxiong.

[0009] The experiments of this invention have confirmed that when 2-sulfotryptophan shown in Formula I is used as a sulfur fumigation marker for identification, the compound (peak at 284.04) can also be detected in the Fritillaria sample with the lowest sulfur dioxide residue of 35.1 mg / kg, demonstrating the high sensitivity of the method of this invention.

[0010] Using 2-sulfotryptophan as a sulfur fumigation marker, as shown in Formula I, the identification of sulfur-fumigated Chinese medicinal materials includes the following steps:

[0011] S1. After crushing the sample to be tested, dissolve it in methanol solution, mix well, centrifuge, and filter to obtain the filtrate;

[0012] S2. Perform UPLC-Q-TOF-MS analysis on the filtrate to obtain a total ion chromatogram. If the total ion chromatogram includes a molecular ion peak of 2-sulfotryptophan, then the sample to be tested is a sulfur-fumigated Chinese medicinal material.

[0013] In the above method, in step S1, the particle size of the sample to be tested after pulverization is 250±9.9μm;

[0014] The ratio of the sample to be tested to the methanol solution is 0.1 g / 1.5 to 2 mL;

[0015] The volume content of the methanol solution is 70% to 85%;

[0016] It is then subjected to ultrasonic treatment before centrifugation.

[0017] In the above method, in step S1, the molecular ion peak of 2-sulfotryptophan is 283.03±0.01.

[0018] In the above method, the liquid phase detection conditions for UPLC-Q-TOF-MS analysis in step S2 are as follows:

[0019] Mobile phase: 0.01% formic acid aqueous solution as phase A, 0.01% formic acid acetonitrile solution as phase B;

[0020] Chromatographic column: Waters ACQUITY UPLC-HSS-T3 (2.1mm×100mm, 1.8μm);

[0021] The gradient elution program is as follows: 0–6.0 min, 5%–16% B; 6.0–13.0 min, 16%–47% B; 13.0–18.0 min, 47%–55% B; 18.0–20.0 min, 55%–95% B; 20.0–23.0 min, 95% B; 23.0–24.0 min, 95%–5% B; 24.0–27.0 min, 5% B.

[0022] Injection volume: 2 μL, flow rate: 0.3 mL / min, column temperature: 40℃.

[0023] In the above method, the mass spectrometry detection conditions for the UPLC-Q-TOF-MS analysis in step S2 are as follows:

[0024] Data was collected using an ESI ion source in negative ion mode.

[0025] Ion source parameters: Detection data format: continuum; Ion source temperature: 100℃; Desolventizing gas: nitrogen; Cone voltage: 40V; Collision energy: 30~45eV; Capillary voltage: 2kV; Cone gas flow rate: 50L / Hr; Desolventizing gas flow rate: 900L / Hr; Data acquisition range: 50~1500Da; The calibration solution (lock mass) is leucine enkephalin, and the accurate molecular weight in negative ion mode is set to 554.2620.

[0026] This invention uses UPLC-Q-TOF-MS combined with metabolomics statistical methods to discover a common sulfur fumigation marker in sulfur-fumigated houttuynia cordata, fritillaria cirrhosa, ophiopogon japonicus, anemarrhena asphodeloides and lily bulb, which can be used for rapid identification of various sulfur-fumigated Chinese medicinal materials. Attached Figure Description

[0027] Figure 1 This is the mass spectrometric cleavage pathway for 2-sulfotryptophan.

[0028] Figure 2 The 1H NMR spectrum (500MHz, CD3OD-D2O) of 2-sulfotryptophan is shown.

[0029] Figure 3 For 2-sulfotryptophan 13 C NMR (100MHz, CD3OD-D2O) spectrum.

[0030] Figure 4 The DEPT spectrum is for 2-sulfotryptophan.

[0031] Figure 5 The HMBC spectrum of 2-sulfotryptophan.

[0032] Figure 6 The HSQC spectrum of 2-sulfotryptophan.

[0033] Figure 7 The key HMBC for 2-sulfotryptophan and 1 H, 1 H-COSY correlation.

[0034] Figure 8 The total ion chromatogram of Houttuynia cordata is shown.

[0035] Figure 9 The peak containing 283.03 was extracted from different varieties of sulfur-fumigated Chinese medicinal materials. Detailed Implementation

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0037] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0038] The instruments and reagents used in the following examples are as follows: ACQUITY I-Class ultra-high performance liquid chromatograph-Waters Xevo-G2-S QTof MS mass spectrometer (Waters Corporation, USA), ACQUITY UPLC™ I-Class system with electrospray ionization source, Waters ACQUITY UPLC-CORTECS T3 (2.1mm × 100mm, 1.6μm, Waters Corporation, USA), MRI400 nuclear magnetic resonance spectrometer (Agilent Technologies, USA), Mettler Toledo New classic MF analytical balance (Mettler Toledo, Switzerland), BSA224S analytical balance (Sartorius, Germany), Centrifuge 5415D centrifuge (Eppendorf, Germany), SB-800-DTD ultrasonic cleaner with 500W ultrasonic power (Ningbo Xinzhi Biotechnology Co., Ltd.), Pacific The system used was a T-II type ultrapure water system (Thermo Fisher Scientific, USA), with hydrophobic PTFE microporous filter heads (0.22μm and 0.45μm, Millipor, USA). Water was ultrapure, methanol was used, acetonitrile was chromatographic grade (Merck, Germany), and all other reagents were analytical grade.

[0039] Experimental materials used in the following examples:

[0040] The Houttuynia cordata sample was collected in April 2023 from the Houttuynia cordata base of Guangxi Zhongzhi Pharmaceutical Co., Ltd. The sample was identified by Researcher Guo Lanping of the Center for Medicinal Plant Resources, China Academy of Chinese Medical Sciences, as the fresh whole herb of Houttuynia cordata Thunb., a plant of the Saururaceae family.

[0041] The Zhejiang Fritillaria thunbergii sample was collected on April 30, 2021, in Pan'an County, Jinhua City, Zhejiang Province; the Ping Fritillaria thunbergii sample was collected on June 5, 2021, in Xinbin Manchu Autonomous County, Fushun City, Liaoning Province; the Hubei Fritillaria thunbergii, Yi Fritillaria thunbergii, Anemarrhena asphodeloides and Lilium brownii samples were purchased from Bozhou Medicinal Materials Market on August 21, 2021; the Sichuan Fritillaria thunbergii sample was purchased from Beijing Tongrentang (Group) Co., Ltd. on August 26, 2021; and the Ophiopogon japonicus sample was collected on March 31, 2021, from Mianyang Academy of Agricultural Sciences, Mianyang City, Sichuan Province.

[0042] Example 1: Preparation and structural identification of 2-sulfotryptophan shown in Formula I

[0043] I. Preparation of 2-sulfotryptophan as shown in Formula I

[0044] Approximately 400 ml of aqueous solution was taken, and 80 g of sodium metabisulfite solid powder was added. The solution was heated to dissolve, and 6 g of L-tryptophan was added. The mixture was stirred and refluxed at 80 °C for 48 h. After cooling to room temperature, the solution was concentrated under reduced pressure to a small volume. 300 ml of methanol was added, and the solution was sonicated for 30 min and filtered. The solid was discarded, and the filtrate was collected and concentrated under reduced pressure to 50 ml. 50 ml of water was added, and the solution was filtered through a 0.45 μm organic filter membrane. HPLC preparative separation was performed using a 250 × 150 × 9 μm column with a flow rate of 150 mL / min and a UV detection wavelength of 220 nm, with methanol:water = 15:85 as the mobile phase. The separation was performed at 150 mL / min using a 250 × 150 × 9 μm column and a UV detection wavelength of 220 nm. First, each product fraction was collected and analyzed by UPLC-QTOF-MS to identify the target product fraction and its purity. The fraction was identified as the target product based on retention time, molecular weight, and secondary fragmentation. The purity was judged to be above 95% based on the peak area. Second, the target fraction was collected on a large scale and concentrated to dryness under reduced pressure to obtain the compound shown in Formula I.

[0045] UPLC-QTOF-MS detection conditions:

[0046] The liquid chromatography detection conditions are as follows:

[0047] Using 0.01% formic acid aqueous solution (A) and 0.01% formic acid acetonitrile (B) as elution solvents, the gradient elution program was as follows: 0–6.0 min, 5%–16% B; 6.0–13.0 min, 16%–47% B; 13.0–18.0 min, 47%–55% B; 18.0–20.0 min, 55%–95% B; 20.0–23.0 min, 95% B; 23.0–24.0 min, 95%–5% B; 24.0–27.0 min, 5% B. Injection volume: 2 μL, flow rate: 0.3 mL / min, column temperature: 40 °C.

[0048] The mass spectrometry detection conditions are as follows:

[0049] The prepared samples and standard solutions were injected into the UPLC-Q-TOF-MS system. Data was acquired using an ESI ion source in negative ion mode and a MassLynx 4.1 signal acquisition system. Ion source parameters were as follows: SurveyData Format: continuous; Ion Source Temperature: 100℃; Desolventizing Gas: Nitrogen; Samplecone Voltage: 40V; Ramp High Energy: 30–45eV; Capillary Voltage: 2kV; Cone Gas Flow: 50L / Hr; Desolventizing Gas Flow: 900L / h; Acquisition Mass Range: 50–1500 Da; Lock Mass: Leucine enkephalin; Accurate Molecular Mass was set to 554.2620 in negative ion mode.

[0050] II. Structural identification of 2-sulfotryptophan shown in Formula I

[0051] Nuclear magnetic resonance analysis was performed using CD3OD+D2O (5:1) dissolved in an NMR tube.

[0052] The compound obtained in step one was a white crystal. UV (acetonitrile-water) spectral density: λmax (logε) 216.9 nm; ESI-MS: m / z 283.0305 ([MH]). - C 11 H 11 N2O5S - Compared to tryptophan, compound 1 has one more SO3 molecule in its molecular formula; therefore, it is speculated that compound 1 is formed by a substitution reaction between tryptophan and H2SO3. Its mass spectrometric fragmentation pathway is as follows: Figure 1 As shown.

[0053] The nuclear magnetic resonance (NMR) spectral data of this compound are shown in Tables 1 and 2. (Combined with...) 1 H-NMR ( Figure 2 ), 13 C-NMR ( Figure 3 DEPT Figure 4 ), HMBC ( Figure 5 ) and HSQC ( Figure 6 The spectrum shows that this compound has 8 hydrogen signals, including 4 aromatic hydrogens (δ-hydrogens) from the benzene ring. H 7.13,t,J=7.5Hz,H-5; 7.26,t,J=7.6Hz,H-6; 7.46,d,J=8.3Hz,H-7; 7.69,d,J=8.1Hz,H-4), 2 methylene hydrogens (δ H3.62,dd,J=7.95,15.07Hz,H-8a; 3.75,dd,J=15.1,4.9Hz,H-8b), 1 methine hydrogen (δ H 4.23,dd,J=7.9,4.9Hz,H-9) and 1 secondary amine hydrogen (δ H 10.91,s,NH-1). 13 CNMR and DEPT spectra indicate that the compound contains one methylene group, five methine groups (four aromatic ring carbons), and five quaternary carbons (one carbonyl group). Comparison of these signals with literature data (Yan, XJ, Suzuki M, Ohnishi-Kameyama M, et al. Extraction and identification of antioxidants in the roots of yacon (Smallanthussonchifolius)[J]. Journal of Agricultural and Food Chemistry, 1999, 47(11):4711-4713.) suggests that the basic skeleton of this compound is tryptophan. The planar structure of this compound was further determined by 2D-NMR. Figure 7 ), 1 H, 1 The H-COSY spectrum shows correlations between H-4 / H-5 / H-6 / H-7 and H-8 / H-9, thus confirming the presence of a benzene ring and alanine side chain. Combined with key correlation signals from the HMBC spectrum: NH-1 correlates with C-2, C-3, C-3a, and C-7a; H-4 correlates with C-3, C-6, and C-7a; H-6 correlates with C-4 and C-7a; H-8 correlates with C-2, C-3a, and C-10 (δc 172.8); and H-9 correlates with C-3 and C-10, the planar structure of compound 1 is determined to be tryptophan substituted with a sulfonic acid group at the C-2 position, i.e., 2-sulfotryptophan.

[0054] The compounds shown in Formula I in Table 1 1 H-NMR (500MHz) data

[0055]

[0056] The compounds shown in Formula I in Table 2 13 C-NMR (100MHz) data

[0057]

[0058]

[0059] Example 2: Identification of Sulfur-Fumigated Chinese Medicinal Materials

[0060] I. Preparation of Sulfur Fumigation Samples

[0061] Fresh houttuynia cordata was washed and divided into a sulfur-free group and a sulfur-fumigated group. The sulfur-fumigated group used a sulfur-to-sample ratio of 1:10 (3 samples per group, approximately 200g per sample). The samples were placed in two separate plastic boxes, and each box was ignited with 12ml of alcohol. After 12 hours, the samples were removed, freeze-dried, ground into powder, and stored at 4℃.

[0062] II. Preparation of the test solution

[0063] Take about 0.1g of each of the above sample powders (particle size 250±9.9μm), add 2.0ml of methanol solution (80%), mix well, sonicate for 1h, make up the weight difference, centrifuge at 12rpm for 10min, filter through a 0.22μm filter membrane, and obtain the sample solutions respectively.

[0064] 3. UPLC-Q-TOF-MS analysis

[0065] 1. Liquid phase conditions

[0066] Using 0.01% formic acid aqueous solution (A) and 0.01% formic acid acetonitrile (B) as elution solvents, the gradient elution program was as follows: 0–6.0 min, 5%–16% B; 6.0–13.0 min, 16%–47% B; 13.0–18.0 min, 47%–55% B; 18.0–20.0 min, 55%–95% B; 20.0–23.0 min, 95% B; 23.0–24.0 min, 95%–5% B; 24.0–27.0 min, 5% B. Injection volume: 2 μL, flow rate: 0.3 mL / min, column temperature: 40 °C.

[0067] 2. Mass spectrometry conditions

[0068] Data was acquired using an ESI ion source in negative ion mode, employing a MassLynx 4.1 signal acquisition system. Ion source parameters were as follows: Survey Data Format: continuous; Ion source temperature: 100℃; Desolventizing gas: nitrogen; Sample cone voltage: 40V; Ramp High Energy: 30–45eV; Capillary voltage: 2kV; Cone gas flow rate: 50L / Hr; Desolventizing gas flow rate: 900L / h; Acquisition mass range: 50–1500 Da; Lock mass: leucine enkephalin; Accurate molecular weight set to 554.2620 in negative ion mode.

[0069] IV. Identification of Sulfur-Fumigated Samples

[0070] Using 2-sulfotryptophan as a specific component, as shown in Formula I, sulfur-fumigated samples and sulfur-free samples can be distinguished.

[0071] like Figure 1 As shown, the characteristic ion of this compound is 283.03, and the total ion chromatogram of Houttuynia cordata is as follows. Figure 8 As shown, the compound is only present in sulfur-fumigated Houttuynia cordata samples and cannot be detected in unfumigated Houttuynia cordata samples, indicating that the compound has good specificity in identifying sulfur-fumigated Chinese medicinal materials.

[0072] V. Identification of Different Varieties of Sulfur-Fumigated Chinese Medicinal Herbs

[0073] Sulfur-fumigated samples and test solutions of Fritillaria cirrhosa, Ophiopogon japonicus, Anemarrhena asphodeloides, and Lilium brownii were prepared according to procedures one and two, respectively. Analysis was performed according to conditions three, yielding the results. Figure 9 The total ion current chromatogram shown is shown.

[0074] like Figure 9 As shown, 283.03 was extracted from the total ion chromatograms of different sulfur-fumigated Chinese medicinal materials. The results showed that the peak of 283.03 in sulfur-fumigated Fritillaria cirrhosa, Ophiopogon japonicus, Anemarrhena asphodeloides, and Lilium brownii all had high responses, indicating that this method can rapidly and universally identify a variety of sulfur-fumigated Chinese medicinal materials. Among all sulfur-fumigated medicinal materials, the peak of 283.03 could also be detected in Fritillaria cirrhosa sample with the lowest sulfur dioxide residue of 35.1 mg / kg, indicating that the method has high sensitivity.

[0075] The method of extracting the peak at 283.03 from the total ion chromatogram of sulfur-fumigated medicinal materials using high-resolution mass spectrometry has the advantages of being simple, rapid, universal, and highly sensitive in distinguishing between sulfur-fumigated and sulfur-free Chinese medicinal materials containing tryptophan.

Claims

Application of 1,2-sulfotryptophan in the identification of sulfur-fumigated Chinese medicinal materials; The sulfur dioxide residue in the sulfur-fumigated Chinese medicinal materials is as low as 35 mg / kg; The Chinese medicinal materials mentioned are Ophiopogon japonicus, Fritillaria cirrhosa, Houttuynia cordata, Anemarrhena asphodeloides, or Lilium brownii.

2. The application according to claim 1, characterized in that: The 2-sulfotryptophan is used as a sulfur fumigation marker.

3. A method for identifying sulfur-fumigated Chinese medicinal materials, comprising the following steps: S1. After crushing the sample to be tested, dissolve it in methanol solution, mix well, centrifuge, and filter to obtain the filtrate; S2. Perform UPLC-Q-TOF-MS analysis on the filtrate to obtain a total ion chromatogram. If the total ion chromatogram includes a molecular ion peak of 2-sulfotryptophan, then the sample to be tested is a sulfur-fumigated Chinese medicinal material. The residual sulfur dioxide content in the test sample was as low as 35 mg / kg; The medicinal herbs mentioned are houttuynia cordata, fritillaria cirrhosa, ophiopogon japonicus, anemarrhena asphodeloides, and lily bulb.

4. The method according to claim 3, characterized in that: In step S1, the particle size of the sample to be tested after pulverization is 250±9.9 µm; The ratio of the sample to be tested to the methanol solution is 0.1 g / 1.5~2 mL; The volume content of the methanol solution is 70% to 85%; After ultrasonic treatment, it is then centrifuged.

5. The method according to claim 3 or 4, characterized in that: In step S1, the molecular ion peak of 2-sulfotryptophan is 283.03±0.

01.

6. The method according to claim 3 or 4, characterized in that: In step S2, the liquid phase detection conditions for the UPLC-Q-TOF-MS analysis are as follows: Mobile phase: 0.01% formic acid aqueous solution as phase A, 0.01% formic acid acetonitrile solution as phase B; The gradient elution program is as follows: 0–6.0 min, 5%–16% phase B; 6.0–13.0 min, 16%–47% phase B; 13.0–18.0 min, 47%–55% phase B; 18.0–20.0 min, 55%–95% phase B; 20.0–23.0 min, 95% phase B; 23.0–24.0 min, 95%–5% phase B; 24.0–27.0 min, 5% phase B.

7. The method according to claim 3 or 4, characterized in that: In step S2, the mass spectrometry detection conditions for the UPLC-Q-TOF-MS analysis are as follows: Data was collected using an ESI ion source in negative ion mode. Ion source parameters: Detection data format: continuum; Ion source temperature: 100 ℃; Desolvation gas: nitrogen; Cone voltage: 40 V; Collision energy: 30~45 eV; Capillary voltage: 2 kV; Cone gas flow rate: 50 L / Hr; Desolvation gas flow rate: 900 L / Hr; Data acquisition range: 50~1500 Da.

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