UPLC-MS / MS method for simultaneously determining seven chemical components in cortex fraxini

Abstract

The invention provides a UPLC-MS / MS (Ultra Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) method for simultaneously determining seven chemical components in cortex fraxini. The seven chemical components are aesculin, aesculetin, scopolin, scopoletin, fraxin, fraxetin and fraxetin. The method comprises the following steps: S1, preparation of a sample: S11, preparation of a reference substance solution; s12, preparing a test solution; s2, detecting by adopting a UPLC-MS / MS method, wherein the analysis time of the determination method is short; detection limit and quantitation limit are low, and sensitivity is high; the method can effectively eliminate the interference of complex matrixes of traditional Chinese medicines on the measured components, is high in accuracy, and provides a solid foundation for the research and development of the pharmacological action of coumarin compounds in the cortex fraxini and the comprehensive utilization of the cortex fraxini.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, and in particular to a UPLC-MS / MS method for simultaneously determining seven chemical components in Fraxinus Fraxinus. Background Art

[0002] The Chinese medicinal herb Cortex fraxini is primarily distributed in Liaoning, Jilin, Hebei, Henan, Inner Mongolia, Shaanxi, Shanxi, and Sichuan provinces of my country. It is made from the dried bark or twigs of Fraxinus rhynchophylla Hance, Fraxinus chinensis Roxb., Fraxinus szaboana Lingelsh., or Fraxinus stylosa Lingelsh., all members of the Oleaceae family. As a commonly used Chinese herbal medicine, Cortex fraxini has the effects of clearing heat and dampness, astringing qi, and stopping diarrhea. Clinically, it is widely used to treat a variety of conditions, including damp-heat diarrhea, leucorrhea, red and swollen eyes, and daytime opacities. Fraxinus chinensis contains many active ingredients such as coumarins, lignans and iridoids. Modern pharmacological experiments have shown that Fraxinus chinensis has pharmacological effects such as antibacterial, anti-inflammatory, analgesic, anti-tumor and lowering of blood uric acid, with coumarin compounds as the main active ingredients. Coumarin compounds are a natural antioxidant that can be used to delay human aging and enhance the body's own resistance. There have been many literature reports on the research of coumarin compounds in Fraxinus chinensis, which mostly exist in plant organs in the form of glycoside compounds. Existing technologies have detected or identified the coumarin components contained in Fraxinus chinensis such as fraxinoside, fraxinin, fraxinyl, scopoletin, etc. through various separation / analysis methods. The current "Chinese Pharmacopoeia" 2020 edition uses fraxinyl, fraxinyl and fraxinyl as indicator components for quality control of Fraxinus chinensis medicinal materials.

[0003] At present, the existing technology mostly uses high performance liquid chromatography to determine the coumarin components in the cortex of Fraxinus. For example, Chen Lin et al. (HPLC determination of coumarin components in different varieties of Fraxinus medicinal materials [J]. Chinese Journal of Traditional Chinese Medicine, 2008, 33(23): 2858-2860.) used HPLC to determine the content of five coumarin components in the cortex of Fraxinus medicinal materials; Ye Ying et al. (Study on the determination method of coumarin components in the extract of Fraxinus [J]. Chinese Journal of Traditional Chinese Medicine Information, 2015, 22(08): 83-87) established a method for the determination of total coumarins and four major coumarin components in the extract of Fraxinus by UV spectrophotometry and high performance liquid chromatography. Most of the reported studies used high performance liquid chromatography, and the research objects focused on a few compounds such as aesculin, aesculetin, and aesculin, and rarely involved other components. Since the content of some coumarin compounds in Fraxinus fraxinus is low, it is difficult to meet the sensitivity requirements by high performance liquid chromatography, and it is even more impossible to achieve the simultaneous determination of multiple coumarin compounds with different contents.

[0004] Obtaining a simple and sensitive detection method that can simultaneously detect multiple coumarin components in Fraxinus fraxinus is a technical problem that needs to be solved urgently in this field. Summary of the Invention

[0005] To overcome the defects of the prior art, the present invention aims to provide a UPLC-MS / MS method and application for the simultaneous determination of seven chemical components in Fraxinus fraxinus.

[0006] The present invention is achieved through the following technical solutions:

[0007] The first aspect of the present invention provides a UPLC-MS / MS method for simultaneously determining seven chemical components in Fraxinus Fraxinus, wherein the seven chemical components are: aesculin, aesculetin, scopoletin, scopoletin, scopolamine, aesculin, aesculetin, and aesculetinidine;

[0008] Furthermore, the UPLC-MS / MS method for simultaneously determining seven chemical components in Fraxinus fraxinus comprises the following steps:

[0009] S1: Sample preparation, including:

[0010] Preparation of S11 reference solution: Accurately weigh each component of the reference substance, add methanol to prepare a reference solution of appropriate concentration, accurately measure an appropriate amount of the reference solution of each component, place it in the same volumetric flask, and add methanol to the volume to obtain a mixed reference solution;

[0011] Preparation of S12 test solution: Take the slices of Fraxinus chinensis, crush and sieve, accurately weigh, add appropriate amount of 70v / v% methanol aqueous solution, extract by ultrasonic, cool and then make up to volume, filter and obtain;

[0012] S2: UPLC-MS / MS method was used for detection. The test solution and the reference solution were injected separately and the chromatograms were compared.

[0013] S21 UPLC chromatographic conditions are as follows:

[0014] Column: WATERS ACQUITY BEH C 18 column;

[0015] Mobile phase: 0.1 v / v% formic acid aqueous solution as phase A, methanol as phase B;

[0016] Elution program: Gradient elution program was used for elution;

[0017] S22 mass spectrometry conditions are as follows:

[0018] Ion source: electrospray ionization source (ESI + ); desolvation temperature: 500℃; capillary voltage: 1.5kV; cone voltage: 50V; cone gas flow rate: 150L·Hr -1 ; Atomizing gas flow rate: 1000L·Hr -1 ; Scan mode: multiple reaction monitoring (i.e., MRM);

[0019] Furthermore, in S11, the preparation of the reference solution includes: accurately weighing an appropriate amount of each component reference solution, adding methanol, and preparing the solution to a concentration of 0.5 to 2 mg·mL -1 The reference solution of each component was accurately measured and placed in the same volumetric flask, and methanol was added to make up the volume to obtain a mixed reference solution. The concentrations of each component in the mixed reference solution were 4-6, 4-6, 4-6, 4-6, 1-3, 1-3, and 0.5-2 μg·mL -1 ; Further, the concentration of each component reference substance solution is 1 mg·mL -1 ; Further, the concentrations of each component in the mixed control solution were 5, 5, 5, 5, 2, 2, and 1 μg·mL -1 ;

[0020] Furthermore, in S12, the preparation of the test solution includes: crushing the Fraxinus chinensis slices, sieving them through 40-60 mesh, accurately weighing, adding 60%-80% v / v methanol, ultrasonically extracting, cooling and replenishing the lost weight, shaking, and preparing a Fraxinus chinensis medicinal material test solution with a concentration of 4-6 mg / mL, and filtering through a 0.2-0.4 μm filter membrane to obtain the obtained solution; further, the concentration of the test solution is 5 mg·mL -1 ;

[0021] Furthermore, in S21, the gradient elution program is as follows: 0-1.5 min, 20 v / v% B; 1.5-5.0 min, 20 v / v% B-55 v / v% B; 5.0-5.2 min, 55 v / v% B-90 v / v% B; 5.2-9.7 min, 90 v / v% B; 9.7-10.0 min, 90 v / v% B-20 v / v% B; 10.0-13.0 min, 20 v / v% B;

[0022] Furthermore, in S22, the retention times, detection ion pairs, and collision energies of the seven chemical components are as follows:

[0023] name Retention time (min) Precursor ion (m / z) Product ion (m / z) Collision energy (v) Aesculin 1.58 341 179.1* / 123 21 / 31 Esculetin 2.6 179 123.1* / 133.1 31 / 26 Scopoletin 2.01 193 133* / 178 27 / 19 Scopoletin 3.92 193 133.1 / 178.1 27 / 20 Fraxinoside 2.63 209 194.1* / 149.1 21 / 20 Fraxinus 3.41 209 149.1* / 194 20 / 20 Fraxinidine 4.44 223.1 190.1* / 162.1 33 / 30

[0024] Beneficial effects: This study used a highly selective and highly sensitive ultra-high performance liquid chromatography-mass spectrometry method to simultaneously determine seven coumarin components in Fraxinus fraxinus, including fraxinic acid, fraxinic acid ethyl, fraxinic acid, fraxinoside, fraxinoside pyridine, scopoletin and scopolamine. In particular, there is no report on the content determination method of scopoletin and fraxinoside pyridine in Fraxinus fraxinus. This experiment established their determination method in Fraxinus fraxinus for the first time and studied the content of the two in Fraxinus fraxinus medicinal materials; secondly, the present invention used the liquid chromatography-mass spectrometry method for the first time to simultaneously determine the content of seven components. The analysis time of this determination method is short, which is 13 minutes. The instrumental analysis time of the liquid chromatography method reported in the existing technology is all over 40 minutes. This method can achieve rapid determination of samples; thirdly, the content of fraxinoside pyridine in Fraxinus fraxinus is about one part per million, and the concentration in the test solution of the method of the present invention is about 5ng / mL. The liquid chromatography-ultraviolet method is not sensitive enough to determine it, and the liquid chromatography-mass spectrometry method can accurately quantify it.

[0025] In addition, the present invention also conducted a methodological investigation, which proved that the analytical method of the present invention has good precision, accuracy, and detection limit. The present invention also optimized the mass spectrometry conditions, chromatographic conditions, and sample pretreatment, which proved that the detection method of the present invention achieved good technical results. The detection limit and quantification limit are low, and the sensitivity is high. It can effectively eliminate the interference of the complex matrix of traditional Chinese medicine on the measured components, and the accuracy is high. It provides a solid foundation for the research and development of the pharmacological effects of coumarin compounds in Fraxinus cortex and the comprehensive utilization of Fraxinus cortex. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Typical MRM chromatograms showing seven coumarins

[0027] Figure 2 Indicates the total amount of coumarin compounds extracted at different times using extraction solvents with different material-liquid ratios DETAILED DESCRIPTION

[0028] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0029] Example

[0030] 1. Materials

[0031] 1.1 Instrument

[0032] IClass XEVO TQ-S liquid spectrometer-mass spectrometer (WATERS, USA); BT25S electronic analytical balance (precision 0.01 mg, Sartorius, Germany); BSA124-CW electronic analytical balance (precision 0.1 mg, Sartorius, Germany); KQ-700VDB dual-frequency CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); 1000Y multifunctional pulverizer (Wuyi Haina Electric Co., Ltd.); No. 3 standard sieve (Zhangxing Yarn Screen Factory, Shangyu, Shaoxing).

[0033] 1.2 Drug testing

[0034] Samples: Qinpi decoction pieces (Sichuan 180301, Sichuan 191001, Shaanxi 180401, Fujian 8190512101, Hubei 210803, Shaanxi 190901, Henan 210921, Anhui 201011) were purchased from different pharmacies and identified by Lei Chengkang, chief pharmacist of Xi'an Food and Drug Inspection Institute, as dried branch bark or dry bark of Fraxinus chinensis Roxb., a plant of the Oleaceae family.

[0035] Reference substances: aesculin (batch number 19F-XLD-40-5, purity 99.8%; Panphy), aesculetin (batch number D0012157, purity 99.5%; Bepure), scopoletin (batch number 27M-XSG-16-0, purity 99.5%, Panphy), scopoletin (batch number 2353102, purity 99.9%, Anpeltrace), aesculin (batch number 27M-UAL-01-8, purity 99.2%; Panphy), aesculin (batch number B0003473, purity 99.8%; Bepure), aesculetinidine (batch number A14HB191436, purity 99.8%, Shanghai Yuanye Biotechnology Co., Ltd.).

[0036] Reagents: ethanol, methanol, acetonitrile (chromatographic grade, Merck, Germany); formic acid (chromatographic grade, Shanghai Aladdin Biochemical Technology Co., Ltd.); ultrapure water was used in the experiments.

[0037] 2. Methods and Results

[0038] 2.1 Solution Preparation

[0039] 2.1.1 Preparation of reference solution

[0040] Reference substance stock solution: Accurately weigh appropriate amounts of aesculin, aesculetin, scopoletin, scopoletin, scopolamine, esculin, aesculetin, and aesculetin pyrimidine reference substances, place them in 10 mL volumetric flasks, dissolve them in methanol, make up to volume, and shake well to make a concentration of approximately 1 mg mL -1 Store the reference solution at 2-8°C away from light.

[0041] 2.1.2 Preparation of sample solution

[0042] The Qinpi slices were crushed with a multifunctional grinder and passed through a No. 3 pharmacopoeia sieve (50 mesh, 335 μm ± 13 μm). About 0.2 g of the powder was taken and accurately weighed. The powder was placed in a stoppered conical flask. 40 mL of 70% methanol was accurately added, the flask was sealed, the weight was determined, and the flask was ultrasonicated for 30 min. The flask was cooled and weighed again. The lost weight was supplemented with 70% methanol. The filtrate was shaken and filtered through a 0.22 μm organic microporous membrane. The filtrate was diluted to an appropriate concentration and injected into a liquid chromatography-mass spectrometry (LC-MS / MS) instrument.

[0043] 2.2 Instrument conditions

[0044] 2.2.1 Chromatographic conditions

[0045] WATERS ACQUITY BEH C18 (100 mm × 2.1 mm, 1.7 μm) mobile phase: 0.1% formic acid in water (A)-methanol (B), flow rate: 0.3 mL min -1 , injection volume: 2 μL, column temperature: 40°C. LC gradient elution program: 0–1.5 min, 20% B; 1.5–5.0 min, 20% B–55% B; 5.0–5.2 min, 55% B–90% B; 5.2–9.7 min, 90% B; 9.7–10.0 min, 90% B–20% B; 10.0–13.0 min, 20% B.

[0046] 2.2.2 Mass spectrometry conditions

[0047] Ion source: electrospray ionization (ESI+); desolvation temperature: 500°C; capillary voltage: 1.5 kV; cone voltage: 50 V; cone gas flow rate: 150 L·Hr -1 ; Atomizing gas flow rate: 1000L·Hr -1Scanning mode: multiple reaction monitoring (MRM). The chemical structures of the seven components are shown in Formula I. The retention time, monitoring ion pairs, collision energy and other information are shown in Table 1. The typical MRM chromatogram is shown in Figure 1 , among which, 1: esculin; 2: esculetin; 3: scopolin; 4: scopoletin; 5: fraxin; 6: fraxetin; 7: fraxidin;

[0048]

[0049] A: esculin; B: esculetin; C: scopolin; D: scopoletin; E: fraxin; F: fraxetin; G: fraxidin.

[0050] Table 1 Mass spectrometric parameters of seven coumarins

[0051]

[0052] 2.3 Results

[0053] 2.3.1 Linearity and range

[0054] Accurately measure 0.5, 0.5, 0.5, 0.5, 0.2, 0.2, and 0.1 mL of aesculin, aesculetin, scopoletin, scopolamine, scopolamine lactone, esculin, aesculin, aesculinidine, and reference stock solutions, respectively, into a 100 mL volumetric flask and dilute to volume with methanol to obtain a mixed control solution. These solutions were serially diluted 10, 20, 50, 100, 200, 500, and 1000 times with 70% methanol to obtain a mixed series of working solutions. A 2 μL injection of each mixed series of working solutions was performed and the samples were analyzed according to the chromatographic and mass spectrometric conditions described in "2.2." Linear regression standard curves were plotted with the mass concentration (X) of each component as the abscissa and the peak area (Y) as the ordinate, and the regression equation was calculated. The linear regression equation, linear range, and correlation coefficient (r) for each component are shown in Table 2.

[0055] Table 2 Linear range, linear equation and correlation coefficient of 7 coumarins

[0056]

[0057] 2.3.2 Limit of detection and limit of quantification

[0058] The mixed control solution in Item 2.3.1 was serially diluted with 70% methanol and assayed. The concentrations of each compound at chromatographic peak signal-to-noise ratios (S / N) of 3 and 10 were used as the limits of detection and quantification. The results are shown in Table 2.

[0059] 2.3.3 Precision

[0060] The concentration is about 20 ng·mL -1 The mixed control solution was measured six times in succession according to the chromatographic and mass spectrometric conditions in Section 2.2, and the relative standard deviations (RSDs) of the chromatographic peak retention times and peak areas of the seven components were calculated. The results showed that the RSDs of the retention times of aesculin, aesculetin, scopoletin, scopoletin, scopolamine, fraxin, aesculetin, and aesculetinidine were all less than 0.4%, and the RSDs of their peak areas were 4.4%, 2.1%, 2.3%, 3.0%, 2.8%, 2.7%, and 1.5%, respectively, indicating good instrument precision.

[0061] 2.3.4 Repeatability

[0062] Six 0.2g samples of Fraxinus chinensis (Batch No. 180301) were weighed and prepared according to the method in "2.1.2". The contents of the seven components in the samples were determined according to the chromatographic and mass spectrometric conditions in "2.2". The repeatability of the method was evaluated using the relative standard deviations (RSDs) of the contents. The results are shown in Table 3. The RSDs for each component were ≤6.5%, demonstrating good repeatability of the method.

[0063] Table 3 Repeatability of 7 coumarin components

[0064]

[0065] 2.3.5 Accuracy

[0066] Six 0.2 g samples of Fraxinus fraxinus (Batch No. 180301) were weighed and added to each sample. An appropriate amount of the mixed reference solution was added. Sample solutions were prepared according to the method in "2.1.2." The components were analyzed using the chromatographic and mass spectrometric conditions in "2.2." The recoveries of each component were calculated and the results are shown in Table 4. The recoveries of each component ranged from 85.3% to 99.7%, with RSDs of 6.1% or less, indicating good accuracy of the method.

[0067] Table 4 Recovery rates of 7 coumarin components

[0068]

[0069]

[0070] 2.3.6 Stability

[0071] A test solution of Fraxinus officinalis (Batch No. 180301) was prepared and placed at room temperature. The solution was analyzed at 0, 2, 4, 8, 12, and 24 hours using the chromatographic and mass spectrometric conditions described in "2.2." The RSDs of the peak areas of the seven components were calculated. The RSDs of the peak areas of aesculin, aesculetin, scopoletin, scopoletin, fraxinoside, aesculetin, and aesculetin pyridine were 4.2%, 4.5%, 4.5%, 4.8%, 3.7%, 4.6%, and 4.0%, respectively, indicating that the Fraxinus officinalis test solution was stable within 24 hours of placement at room temperature.

[0072] 2.3.7 Sample determination

[0073] Eight batches of test solutions of Fraxinus fraxinus were prepared according to the method in "2.1.2" and assayed according to the chromatographic and mass spectrometric conditions in "2.2." The contents are shown in Table 5. The results showed that the contents of Fraxinus fraxinus varied significantly from different origins, with aesculin and quercetin being relatively high in Fraxinus fraxinus, indicating that coumarins exist primarily in the form of glycosides in Fraxinus fraxinus.

[0074] Table 5 Contents of seven coumarins in each batch of Qinpi decoction pieces

[0075]

[0076] 3 Discussions

[0077] 3.1 Optimization of mass spectrometry conditions

[0078] In this experiment, the responses of the seven coumarins in positive ion mode were better than those in negative ion mode. During the optimization of the monitoring ion of aesculin, it was found that its quasi-molecular ion ([M+H] + ) peak response is low, while the ion peak response with a mass-to-charge ratio (m / z) of 179 is larger, which is exactly the quasi-molecular ion of aesculetin. In terms of chemical structure, aesculetin is a glycoside form formed by the combination of aesculetin and glucose. It is known from the literature that in-source cleavage occurs in the electrospray ion source, resulting in the breakage of chemical bonds such as glycosidic bonds and disulfide bonds. Therefore, it is speculated that under the high temperature and ionization of the ion source, the quasi-molecular ion of aesculetin undergoes in-source cleavage, as shown in Formula II, and its glycosidic bond breaks to form the quasi-molecular ion of aesculetin. Therefore, when entering the triple quadrupole mass analyzer, the two compounds have the same parent ion and daughter ion. At the same time, due to the strong polarity of glycoside compounds, they are weakly retained in the reversed-phase chromatography system and have a short retention time. Therefore, aesculetin and aesculetin can be distinguished by retention time. The other two groups of compounds, scopoletin and scopoletin, and aesculetin and aesculetin also have the above-mentioned rules.

[0079]

[0080] Using batch number 180401 of Fraxinus chinensis as the research object, 10 0.2g samples were accurately weighed and placed in stoppered conical flasks. Two samples were added to each flask, and 40mL of ethanol, methanol, water, 70v / v% ethanol, and 70v / v% methanol were accurately added. The flasks were sealed and weighed. Ultrasonication was performed for 30 minutes. The flasks were cooled and weighed again. The weight loss was made up with the corresponding solvent, shaken, and filtered through a 0.22μm organic microporous membrane. The filtrate was diluted to the appropriate concentration and analyzed by chromatography and mass spectrometry under the conditions described in "2.2". The total amount of seven coumarins was calculated to be 2.686%, 3.801%, 3.485%, 4.115%, and 4.288%, respectively. The results showed that 70% methanol had the best extraction efficiency compared to 70% ethanol, followed by methanol and water. Ethanol had the lowest extraction efficiency, so 70% methanol was ultimately selected as the extraction solvent.

[0081] 3.3 Optimization of test solution preparation method

[0082] After the extraction solvent was determined, the extraction effects of different extraction times (ultrasonication 20, 30, 45, 60 min) and different solid-liquid ratios (1:50, 1:100, 1:200) were investigated. Figure 2 The results showed that a solid-liquid ratio of 1:200 resulted in the highest extraction rate of coumarins. Furthermore, an optimal ultrasonication time of 30 minutes was recommended; prolonged ultrasonication would result in an overall decrease in the content of coumarins. The final extraction conditions were determined to be a solid-liquid ratio of 1:200 and an ultrasonication time of 30 minutes.

[0083] The above is only a preferred embodiment of the present invention, but the present invention should not be limited to the contents disclosed in this embodiment. Therefore, any equivalent or modified implementations that do not depart from the spirit disclosed in the present invention fall within the scope of protection of the present invention.

Claims

1. A UPLC-MS / MS method for the simultaneous determination of seven chemical components in Fraxinus fraxinus, characterized in that: The seven chemical components are: aesculin, aesculetin, scopoletin, scopoletin, aesculin, aesculetin and aesculetinidine.

2. The UPLC-MS / MS method for simultaneously determining seven chemical components in Fraxinus Fraxinus according to claim 1, characterized in that: The UPLC-MS / MS method for simultaneously determining seven chemical components in Fraxinus Fraxinus includes the following steps: S1: Sample preparation, including: Preparation of S11 reference solution: Accurately weigh each component of the reference substance, add methanol to prepare a reference solution of appropriate concentration, accurately measure an appropriate amount of the reference solution of each component, place it in the same volumetric flask, and add methanol to the volume to obtain a mixed reference solution; Preparation of S12 test solution: Take the slices of Fraxinus chinensis, crush and sieve, accurately weigh, add appropriate amount of 70v / v% methanol aqueous solution, extract by ultrasonic, cool and then make up to volume, filter and obtain; S2: UPLC-MS / MS method was used for detection, and the test solution and reference solution were injected separately, wherein, S21 UPLC chromatographic conditions are as follows: Column: WATERS ACQUITY BEH C 18 column; Mobile phase: 0.1 v / v% formic acid aqueous solution as phase A, methanol as phase B; Elution program: Gradient elution program was used for elution; S22 mass spectrometry conditions are as follows: Ion source: electrospray ionization source (ESI + ); desolvation temperature: 500℃; capillary voltage: 1.5kV; cone voltage: 50V; cone gas flow rate: 150L·Hr -1 ; Atomizing gas flow rate: 1000L·Hr -1 ; Scanning mode: multiple reaction monitoring (i.e. MRM).

3. The UPLC-MS / MS method for simultaneously determining seven chemical components in Fraxinus Fraxinus according to claim 1, characterized in that: In S11, the preparation of the reference solution includes: accurately weighing the appropriate amount of each component reference substance, adding methanol, and preparing the solution to a concentration of 0.5 to 2 mg·mL -1 The reference solution of each component was accurately measured and placed in the same volumetric flask, and methanol was added to make up the volume to obtain a mixed reference solution. The concentrations of each component in the mixed reference solution were 4-6, 4-6, 4-6, 4-6, 1-3, 1-3, and 0.5-2 μg·mL -1 ; Further, the concentration of each component reference substance solution is 1 mg·mL -1 ; Further, the concentrations of each component in the mixed control solution were 5, 5, 5, 5, 2, 2, and 1 μg·mL -1 .

4. The UPLC-MS / MS method for simultaneously determining seven chemical components in Fraxinus Fraxinus according to claim 1, characterized in that: In S12, the preparation of the test solution includes the following steps: crushing the Fraxinus chinensis slices and sieving them through 40-60 mesh, accurately weighing, adding 60%-80% v / v methanol, ultrasonically extracting, cooling and replenishing the lost weight, shaking, and preparing a Fraxinus chinensis medicinal material test solution with a concentration of 4-6 mg / mL, and filtering through a 0.2-0.4 μm filter membrane to obtain the obtained solution; further, the concentration of the test solution is 5 mg·mL -1 .

5. The UPLC-MS / MS method for simultaneously determining seven chemical components in Fraxinus Fraxinus according to claim 1, characterized in that: In S21, the gradient elution procedure is as follows: 0-1.5 min, 20 v / v% B; 1.5-5.0 min, 20 v / v% B-55 v / v% B; 5.0-5.2 min, 55 v / v% B-90 v / v% B; 5.2-9.7 min, 90 v / v% B; 9.7-10.0 min, 90 v / v% B-20 v / v% B; 10.0-13.0 min, 20 v / v% B.

6. The UPLC-MS / MS method for simultaneous determination of seven chemical components in Fraxinus Fraxinus according to claim 1, characterized in that: In S22, the retention times, detection ion pairs, and collision energies of the seven chemical components are as follows:

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