Method for detecting blood entering components of Wulan thirteen-ingredient decoction powder for treating black vein disease

Abstract

The invention discloses a method for detecting in-blood components of Wulan thirteen-ingredient decoction powder for treating black vein disease, and relates to the technical field of in-vivo metabolism of drugs, and the method specifically comprises the following steps: S1, collecting a biological sample: respectively collecting blank plasma and drug-containing plasma for later use; s2, preparing a biological sample: treating the blank plasma and the drug-containing plasma to obtain a blank serum sample and a drug-containing serum sample for later use; s3, preparing an in-vitro test sample: treating the Wulan thirteen-ingredient decoction powder to obtain a traditional Chinese medicine sample for later use; and S4, respectively taking the blank serum, the drug-containing serum and 10 microliters of the traditional Chinese medicine sample, carrying out LC-MS / MS analysis, and determining the blood-entering components based on the detection result. The method disclosed by the invention can more comprehensively reveal the characteristics of the drug-derived components after the drug enters the blood, and fills the blank in the fields of qualitative analysis of the components of the Wulan thirteen-ingredient decoction powder entering the blood and quantitative detection of all effective components in the Wulan thirteen-ingredient decoction powder.

Description

Technical Field

[0001] The invention relates to the technical field of drug metabolism in vivo, and in particular to a method for detecting blood-entering components of Ulan Thirteen Flavors Decoction powder used for treating black vein disease. Background Art

[0002] Ulan Thirteen Flavors Soup Powder, referred to as Ulan-13, is a traditional medicine made from a variety of carefully selected herbs. Its main effects and indications include clearing heat and cooling blood, and are suitable for a variety of mature and immature febrile diseases, colds, hypertension, blood heat, and lung heat. In Mongolian medical theory, hypertension is considered a manifestation of "black vein disease". The so-called "black vein" refers to blood vessels, covering the entire blood circulation system. The disease is caused by an abnormal increase in Xie Ri (equivalent to "fire" in traditional Chinese medicine) in the blood, which leads to thickening, swelling, deformation of the arterial wall, and even bleeding as the main clinical manifestations.

[0003] At present, the research on Ulan Thirteen Flavors Soup Powder is still in the preliminary exploratory stage, mainly focusing on the determination of the content of some of its ingredients, such as matrine, oxymatrine and gardenia glycoside. However, the overall component analysis of the soup powder has not yet been carried out, which means that its overall chemical composition and pharmacological effects are not yet fully understood. Although the determination results of some components have been obtained, the research on the pharmacological material basis and the mechanism of action of the active ingredients of Ulan Thirteen Flavors Soup Powder in the treatment of primary hypertension is far from enough, lacking systematicity and comprehensiveness. In addition, the detection methods for the concentration and distribution of these components in the blood are still imperfect. Therefore, future research should focus on in-depth analysis of its pharmacological basis, and the development of quantitative detection technology for blood-entering components is a key technical problem that needs to be solved urgently.

[0004] Liquid chromatography-mass spectrometry (LC-MS) is a very important means and method in metabolomics research. It uses high-performance liquid chromatography to separate metabolites and then uses high-resolution mass spectrometry to detect metabolites. Compared with other detection technologies, LC-MS is more suitable for detecting and analyzing metabolites that are difficult to volatilize or have poor thermal stability in samples. Summary of the invention

[0005] In view of the above problems, the present invention provides a method for detecting blood-entering components of Ulan Thirteen Flavors Decoction Powder for treating black vein disease.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The present invention provides a method for detecting blood components of Ulan Thirteen Flavors Decoction Powder for treating black vein disease, such as Fig.23 , Fig.24 , which specifically includes the following steps: S1: Biological sample collection: blank plasma and drug-containing plasma are collected separately for future use; S2: Biological sample preparation: blank plasma and drug-containing plasma are processed to obtain blank serum and drug-containing serum samples for later use; S3: Preparation of in vitro test samples: Take Wulan Thirteen Flavors Decoction Powder and process it to obtain Chinese medicine samples for later use; S4: Take 10 μL of blank serum, drug-containing serum and Chinese medicine samples for LC-MS / MS analysis, and determine the blood components based on the test results.

[0007] Furthermore, in step S1, biological sample collection is specifically as follows: after one week of adaptive feeding of SHR rats, 1 mL of blood is collected from the orbits of each rat before administration, and the blood is centrifuged at 3500 r / min for 10 minutes to separate the plasma to obtain blank plasma, which is frozen at -80°C for later use; after continuous administration of Ulan Shisanwei Tangsan for 7 days, 1 mL of blood is collected from the orbits of each rat 1 hour after administration on the last day, and the blood is centrifuged at 3500 r / min for 10 minutes to separate the plasma to obtain drug-containing plasma, which is frozen at -80°C for later use.

[0008] Furthermore, the dosage of the Ulan Thirteen Flavors Decoction Powder is 1.35 g / (kg·d).

[0009] Furthermore, in step S2, the biological sample preparation is specifically as follows: 100 μL of blank plasma and drug-containing plasma are taken respectively, 400 μL of pre-cooled pure methanol is added, oscillated for 1 min, ultrasonicated in an ice bath for 20 min, allowed to stand for 1 h, centrifuged at 16000g4°C for 20 min, the supernatant is taken, dried with nitrogen, 100 μL of pre-cooled 50% methanol is added for re-dissolution, centrifuged at 20000g4°C for 10 min, the supernatant is taken, and blank serum and drug-containing serum samples are obtained for later use.

[0010] Furthermore, in step S3, the in vitro test sample preparation is specifically as follows: weigh 100 mg of Ulan Thirteen Flavors Decoction Powder, add 1 mL of pure water to dissolve, add 3 mL of ethanol, vortex oscillate for 3 minutes, ultrasonicate in an ice bath for 10 minutes, stand at room temperature for 12 hours, centrifuge at 7000g and 4°C for 20 minutes, take the supernatant, blow dry the ethanol with nitrogen to obtain a refined solution, add ultrapure water to make up the volume to 1 mL, filter through a 0.22 μm filter membrane, and obtain a Chinese medicine sample for later use.

[0011] Furthermore, in step S4, the chromatographic conditions are as follows: the chromatographic column is an ACQUITY UPLC® HSS T3 chromatographic column, 2.1×100 mm, 1.8 µm; the column temperature is 40°C, and the flow rate is 0.3 mL / min; the chromatographic mobile phase A: 0.1% formic acid aqueous solution, B: 0.1% formic acid acetonitrile; the chromatographic gradient elution program is as follows: 0~2 min, 0% B; 2~4 min, 25% B; 4~22 min, B changes linearly from 25% to 100%; 22~25 min, B is maintained at 100%; 25~25.1 min, B changes linearly from 100% to 0%; 25.1~30 min, B is maintained at 0%.

[0012] Furthermore, the mass spectrometry conditions were as follows: electrospray ionization (ESI) was used for positive (+) and negative (-) ionization mode detection, and HESI source was used for ionization, and the ionization conditions were as follows: spray voltage: 3.8 kV (+) and 3.2 kV (-); capillary temperature: 320 °C (±); sheath gas: 30 arb (±); auxiliary gas: 5 arb (±); probe heater temperature: 350 °C (±); S-LensRF Level: 50; mass spectrometry acquisition settings were as follows: mass spectrometry acquisition time: 30 min; parent ion scan range: 90-1350 m / z, primary mass spectrometry resolution: 70,000@m / z 200, AGC target: 3e6, primary Maximum IT: 100 ms; secondary mass spectrometry analysis was acquired according to the following method: after each full scan, the secondary mass spectra (MS2 scan) of the 10 highest intensity parent ions were triggered for acquisition, and the secondary mass spectrometry resolution: 17,500@m / z 200, AGC target:1e5, Secondary Maximum IT:50ms, MS2 Activation Type:HCD, Isolation window:2m / z, Normalized collision energy (Setpped):20,30,40.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The present invention constructs a method for detecting the blood-entering components of Ulan Shisanwei Tangsan based on liquid chromatography-mass spectrometry (LC-MS). Through detection and comparative analysis by this method, a total of 810 substances were identified in the positive and negative ion modes, including 686 traditional Chinese medicine components and 40 blood-entering components. This method can more comprehensively reveal the characteristics of the drug-derived components after the drug enters the blood, filling the gap in the field of qualitative analysis of the blood-entering components of Ulan Shisanwei Tangsan and quantitative detection of all effective components in Ulan Shisanwei Tangsan, laying a solid foundation for in-depth exploration of the metabolic cycle and metabolic mechanism of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is the Base Peak spectrum of the positive (upper) and negative (lower) ion modes of the Chinese medicine group ZY-1 of the present invention; Figure 2 It is the Base Peak spectrum of the positive (upper) and negative (lower) ion modes of the Chinese medicine group ZY-2 of the present invention; Figure 3 It is the Base Peak spectrum of the positive (upper) and negative (lower) ion modes of the Chinese medicine group ZY-3 of the present invention; Figure 4 It is the Base Peak spectrum of the positive (upper) and negative (lower) ion modes of the blank serum group K-1 of the present invention; Figure 5 It is the Base Peak spectrum of the positive (upper) and negative (lower) ion modes of the blank serum group K-2 of the present invention; Figure 6 It is the Base Peak spectrum of the blank serum group K-3 in positive (upper) and negative (lower) ion modes of the present invention; Figure 7 It is the Base Peak spectrum of the blank serum group K-4 in positive (upper) and negative (lower) ion modes of the present invention; Figure 8 It is the Base Peak spectrum of the blank serum group K-5 in positive (upper) and negative (lower) ion modes of the present invention; Fig. 9 It is the Base Peak spectrum of the blank serum group K-6 in positive (upper) and negative (lower) ion modes of the present invention; Fig.10 It is the Base Peak spectrum of the positive (upper) and negative (lower) ion modes of the administration serum group R-1 of the present invention; Fig.11 It is the Base Peak spectrum of positive (upper) and negative (lower) ion mode of the administration serum group R-2 of the present invention; Fig.12 It is the Base Peak spectrum of positive (upper) and negative (lower) ion mode of the administration serum group R-3 of the present invention; Fig.13 It is the Base Peak spectrum of positive (upper) and negative (lower) ion mode of the administration serum group R-4 of the present invention; Fig.14 It is the Base Peak spectrum of positive (upper) and negative (lower) ion mode of the administration serum group R-5 of the present invention; Fig.15 It is the Base Peak spectrum of positive (upper) and negative (lower) ion mode of the administration serum group R-6 of the present invention; Fig.16 It is a Venn diagram of the medicinal substances and the substances entering the blood of the present invention; Fig.17 It is the Upset diagram of the Chinese medicine ZY of the present invention and related medicinal materials; Fig.18 is a compound classification ring diagram of the Chinese medicine ZY of the present invention; Fig.19 It is a classification ring diagram of blood-entering components of the present invention; Fig. 20 The KEGG enrichment of the blood-entering components of the present invention in the model plant Arabidopsis thaliana; Fig.21 It is the KEGG enrichment of the blood components of the present invention in rats; Fig. 22 This is the KEGG pathway diagram of the blood-entering components of the present invention in rats; Fig.23 It is a technical roadmap for identifying the components of Chinese medicines of the present invention; Fig.24 It is a flow chart for analyzing the components of Chinese medicine entering blood of the present invention. DETAILED DESCRIPTION

[0015] In order to make the purpose and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0016] The instruments, reagents, materials, etc. involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.

[0017] Example 1 1. Experimental instruments and reagents Table 1 Experimental instruments and reagents

[0018] 2. Experimental methods (1) Experimental animal grouping and drug administration Eight SHR rats, half male and half female, weighing 180±20g, were used. After one week of adaptive feeding, blood was collected from the eye sockets before administration to obtain blank plasma. The rats were then administered with 1.35 g / (kg·d) of Ulan Shisanwei Tangsan for 7 consecutive days, and blood was collected from the eye sockets to obtain drug-containing plasma.

[0019] (2) Collection of biological samples: After one week of adaptive feeding, 1 mL of blood was collected from the eye sockets of SHR rats before administration, and the blood was centrifuged at 3500 r / min for 10 min to separate the plasma. The blank plasma was obtained and frozen at -80°C for future use. After continuous administration of Wulan Shisanwei Tangsan for 7 days, 1 mL of blood was collected from the eye sockets of SHR rats 1 hour after administration on the last day, and the blood was centrifuged at 3500 r / min for 10 min to separate the plasma. The drug-containing plasma was obtained and frozen at -80°C for future use. (3) Biological sample preparation: 100 μL of blank plasma and drug-containing plasma were taken respectively, and 400 μL of pre-cooled pure methanol was added. The mixture was shaken for 1 min, ultrasonicated in an ice bath for 20 min, allowed to stand for 1 h, centrifuged at 16000 g and 4°C for 20 min, the supernatant was taken, dried with nitrogen, and reconstituted with 100 μL of pre-cooled 50% methanol. The mixture was centrifuged at 20000 g and 4°C for 10 min. 60 μL of the supernatant was taken for later use to obtain blank serum and drug-containing serum samples. When mass spectrometry was performed, 10 μL of each sample was injected, and each sample was injected once. The blank serum group was designated as group K, including K-1, K-2, K-3, K-4, K-5, and K-6, and the drug-containing serum group was designated as group R, including R-1, R-2, R-3, R-4, R-5, and R-6. (4) Preparation of in vitro test samples: Weigh 100 mg of Ulan Shisanwei Tangsan powder, add 1 mL of pure water to dissolve, add 3 mL of ethanol, vortex for 3 min, ultrasonicate in an ice bath for 10 min, stand at room temperature for 12 h, centrifuge at 7000 g for 20 min, take the supernatant, blow dry the ethanol with nitrogen to obtain the refined solution, add ultrapure water to make up the volume to 1 mL, filter through a 0.22 μm filter membrane, obtain the traditional Chinese medicine sample, set aside, inject 10 μL of the sample into the mass spectrometer, repeat 3 times, the traditional Chinese medicine group is recorded as ZY group, including ZY-1, ZY-2, and ZY-3; (5) LC-MS / MS analysis a. Chromatographic separation During the entire analysis, the sample was placed in an autosampler at 4°C. The sample was analyzed using a SHIMADZU-LC30 ultra-high performance liquid chromatography system (UHPLC) using an ACQUITY UPLC® HSS T3 (2.1×100 mm, 1.8 µm) (Waters, Milford, MA, USA) column. The column temperature was 40°C, the flow rate was 0.3 mL / min; the chromatographic mobile phase A was 0.1% formic acid in water, and B was 0.1% formic acid in acetonitrile. The chromatographic gradient elution program was as follows: 0-2 min, 0% B; 2-4 min, 25% B; 4-22 min, B changed linearly from 25% to 100%; 22-25 min, B was maintained at 100%; 25-25.1 min, B changed linearly from 100% to 0%; 25.1-30 min, B was maintained at 0%.

[0020] b. Mass Spectrum Acquisition Each sample was detected by electrospray ionization (ESI) in positive (+) and negative (-) modes. After UPLC separation, the samples were analyzed by mass spectrometry using a QE Plus mass spectrometer (Thermo Scientific). The HESI source was used for ionization with the following ionization conditions: spray voltage: 3.8 kV (+) and 3.2 kV (-); capillary temperature: 320 °C (±); sheath gas: 30 arb (±); auxiliary gas: 5 arb (±); probe heater temperature: 350 °C (±); S-Lens RF Level: 50; mass spectrometry acquisition settings were as follows: mass spectrometry acquisition time: 30 min; parent ion scan range: 90-1350 m / z, primary mass spectrometry resolution: 70,000@m / z 200, AGC target: 3e6, primary maximum IT: 100 ms; secondary mass spectrometry analysis was acquired according to the following method: after each full scan, the secondary mass spectra (MS2 scan) of the 10 highest intensity parent ions were triggered for acquisition, the secondary mass spectra resolution: 17,500@m / z 200, AGC target:1e5, secondary Maximum IT:50ms, MS2 Activation Type:HCD, Isolation window:2m / z, normalized collision energy (Setpped):20,30,40.

[0021] 3. Data preprocessing Libraries were established for Inula officinalis, Sophora flavescens, Rubus chinensis, Acorn, Gardenia jasminoides, Eriobotrya japonica, Rubia cordifolia, Toosendan fructus, Terminalia chebula, Trollius, Kaempferia galanga, and Lithospermum officinale (because Lithospermum officinale is an insect secretion, its components have not been studied in the prior art, and it is impossible to determine its high molecular compound, and it has not been detected by mass spectrometry, so its library was not established). Related medicinal materials with special marks (origin, subspecies) were uniformly classified as a certain medicinal material category in the results.

[0022] The collected mass spectrometry raw data were used for peak alignment, retention time correction and peak area extraction using MS DIAL software. Metabolite structure identification parameters: The mass deviation was set to primary spectrum matching mass tolerance <0.01 Da, secondary spectrum matching mass tolerance <0.02 Da, and secondary mass spectrum matching score greater than 70%.

[0023] 4. Data visualization The identified metabolites were plotted into super class and class material classification fan and ring diagrams using R language. Pathway enrichment was performed using the KEGG ID of the identified target component, and the significantly enriched functions and pathways were screened with Fisher's exact test p < 0.05, and the ggplot2 package was used to draw the graph.

[0024] 5. Analysis of experimental results (1) Base Peak graph of the sample After UPLC-Q-Exactive Plus (LC-MS / MS) analysis, two mass spectrometry raw files (positive ion mode and negative ion mode) were obtained for each sample. Figure 1-15 The mass spectrometry Base Peak graphs of the traditional Chinese medicine group ZY-1~ZY-3, the blank serum group K-1~K-6, and the drug-treated serum group R-1~R-6 in the positive and negative ion detection modes are shown respectively.

[0025] (2) Identification results of Chinese medicine components entering blood The results of the components of Chinese medicine entering blood were obtained by comparing the Chinese medicine ZY group, the blank serum K group, and the drug serum R group. The analysis principle is as follows: After comparative analysis, a total of 810 substances were detected in the positive and negative modes, including 686 Chinese medicine components and 40 blood components, as shown in Table 2 and Table 3.

[0026] Table 2 List of the top 30 substances in terms of substance identification strength ,

[0027] Table 3 Three-line table of blood components ,

[0028] (3) Venn diagram analysis of blood components Fig.16 The Venn plot of the blank serum group, the drug-treated serum group and the Chinese medicine group was displayed, and a preliminary statistical analysis of the entry of medicinal ingredients into the blood was performed.

[0029] (4) Ownership of Chinese medicine ingredients and medicinal materials Fig.17 The Upset image of the traditional Chinese medicine ZY and the medicinal materials Inula, Sophora flavescens, Gardenia jasminoides, loquat leaves, Rubia cordifolia, Toosendan fructus, Terminalia chebula, Nasturtium, Kaempferia galanga, and Lithospermum officinale is displayed.

[0030] The left bar graph on the figure shows the number of Chinese medicine components detected in each sample, the upper bar graph shows the number of common components in each sample, and the dotted line below shows the sample where the common components are located. This figure does not include the subclass identification results, and the number of common components between Chinese medicine and medicinal materials is related to the number of known compounds in the specific medicinal materials. From bottom to top: ZY is a compound, Kushen is Sophora flavescens, Jinlianhua is Golden Lotus, Chuanlianzi is Chuanlianzi, Zhizi is Gardenia jasminoides, Qiancao is Rubia cordifolia, Pipaye is Loquat Leaf, Shannai is Kaempferia galanga, Tumuxiang is Inula officinalis, Zicao is Lithospermum officinale, and Hezi is Terminalia chebula.

[0031] (5) Classification of Chinese medicine substances Fig.18 , Fig.19 The classification ring diagrams of traditional Chinese medicine and blood-entering components are shown respectively.

[0032] (6) KEGG analysis of substances entering blood The 40 blood-entering components were analyzed using Arabidopsis and rats as references to analyze the synthesis of compounds in plants and the metabolic pathways in animals. Fig. 20 , Fig.21 The KEGG enrichment results of blood-entering components in the model plant Arabidopsis and rats are shown. The pathway with the most significant P value is selected and the pathway diagram is drawn as shown below. Fig. 22 .

[0033] This embodiment uses liquid chromatography-mass spectrometry (LC-MS) and ultra-high performance liquid chromatography (UPLC) combined with quadrupole-orbital trap (Q-Exactive) mass spectrometry to accurately identify and quantitatively analyze the main components of Wulan Thirteen Flavors Soup Powder and its blood-entering components. By this method, the present invention can accurately identify various compounds in Wulan Thirteen Flavors Soup Powder and quantitatively determine their contents. These compounds cover a variety of bioactive molecules, which play a vital role in the pharmacological mechanism of Wulan Thirteen Flavors Soup Powder, and lay a solid foundation for subsequent drug metabolism research.

[0034] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting blood-entering components of Ulan Thirteen Flavors Decoction Powder for treating black vein disease, characterized in that: The specific steps include: S1: Biological sample collection: blank plasma and drug-containing plasma are collected separately for future use; S2: Biological sample preparation: blank plasma and drug-containing plasma are processed to obtain blank serum and drug-containing serum samples for later use; S3: Preparation of in vitro test samples: Take Wulan Thirteen Flavors Decoction Powder and process it to obtain Chinese medicine samples for later use; S4: Take 10 μL of blank serum, drug-containing serum and Chinese medicine samples for LC-MS / MS analysis, and determine the blood components based on the test results.

2. The method for detecting blood components of Ulan Thirteen Flavors Decoction Powder for treating black vein disease according to claim 1, characterized in that: In the step S1, biological sample collection is specifically as follows: after one week of adaptive feeding of SHR rats, 1 mL of blood is collected from the orbits of each rat before administration, and the blood is centrifuged at 3500 r / min for 10 minutes to separate the plasma to obtain blank plasma, which is frozen at -80°C for later use; after continuous administration of Ulan Shisanwei Tangsan for 7 days, 1 mL of blood is collected from the orbits of each rat 1 hour after administration on the last day, and the blood is centrifuged at 3500 r / min for 10 minutes to separate the plasma to obtain drug-containing plasma, which is frozen at -80°C for later use.

3. The method for detecting blood components of Ulan Thirteen Flavors Decoction Powder for treating black vein disease according to claim 2, characterized in that: The dosage of the Ulan Thirteen Flavors Decoction Powder is 1.35 g / (kg·d).

4. The method for detecting blood components of Ulan Thirteen Flavors Decoction Powder for treating black vein disease according to claim 1, characterized in that: In step S2, the biological sample preparation is specifically as follows: 100 μL of blank plasma and drug-containing plasma are taken respectively, 400 μL of pre-cooled pure methanol is added, oscillated for 1 min, ultrasonicated in an ice bath for 20 min, allowed to stand for 1 h, centrifuged at 16000 g and 4° C. for 20 min, the supernatant is taken, dried with nitrogen, 100 μL of pre-cooled 50% methanol is added for re-dissolution, and centrifuged at 20000 g and 4° C. for 10 min, the supernatant is taken, and blank serum and drug-containing serum samples are obtained for later use.

5. The method for detecting blood components of Ulan Thirteen Flavors Decoction Powder for treating black vein disease according to claim 1, characterized in that: In the step S3, the in vitro test sample preparation is specifically as follows: weigh 100 mg of Ulan Thirteen Flavors Decoction Powder, add 1 mL of pure water to dissolve, add 3 mL of ethanol, vortex oscillate for 3 min, ultrasonicate in an ice bath for 10 min, stand at room temperature for 12 h, centrifuge at 7000 g and 4 ° C for 20 min, take the supernatant, blow dry the ethanol with nitrogen to obtain a refined solution, add ultrapure water to make up the volume to 1 mL, filter through a 0.22 μm filter membrane to obtain a Chinese medicine sample for later use.

6. The method for detecting blood components of Ulan Thirteen Flavors Decoction Powder for treating black vein disease according to claim 1, characterized in that: In step S4, the chromatographic conditions are as follows: the chromatographic column is an ACQUITY UPLC® HSS T3 chromatographic column, 2.1×100 mm, 1.8 µm; the column temperature is 40°C, and the flow rate is 0.3 mL / min; the chromatographic mobile phase A is 0.1% formic acid in water, and B is 0.1% formic acid in acetonitrile; the chromatographic gradient elution program is as follows: 0-2 min, 0% B; 2-4 min, 25% B; 4-22 min, B changes linearly from 25% to 100%; 22-25 min, B is maintained at 100%; 25-25.1 min, B changes linearly from 100% to 0%; 25.1~30min, B is maintained at 0%.

7. The method for detecting blood components of Ulan Thirteen Flavors Decoction Powder for treating black vein disease according to claim 1, characterized in that: In step S4, the mass spectrometry conditions are: electrospray ionization is used for positive ion (+) and negative ion (-) mode detection, and HESI source is used for ionization, and the ionization conditions are as follows: spray voltage: 3.8 kV (+) and 3.2 kV (-); capillary temperature: 320 ° C (±); sheath gas: 30 arb (±); auxiliary gas: 5 arb (±); probe heater temperature: 350 ° C (±); S-Lens RF Level: 50; mass spectrometry acquisition settings are as follows: mass spectrometry acquisition time: 30min; parent ion scanning range: 90-1350m / z, primary mass spectrometry resolution: 70,000@m / z200, AGCtarget: 3e6, primary MaximumIT: 100ms; secondary mass spectrometry analysis is acquired according to the following method: after each full scan, trigger the acquisition of the secondary mass spectra of the 10 highest intensity parent ions, secondary mass spectrometry resolution: 17,500@m / z200, AGCtarget:1e5, secondary MaximumIT: 50ms, MS2ActivationType: HCD, Isolationwindow: 2m / z, normalized collision energy: 20, 30, 40.

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