Angelica sinensis endogenous hormone content test and analysis method

Abstract

The invention discloses a method for testing and analyzing the content of endogenous hormones in angelica sinensis. The method for testing the content of the endogenous hormones comprises the following steps: in an enzyme-linked immunosorbent assay (ELISA), homogenizing a sample with a 80% methanol solution, extracting at 4 DEG C for 2-10 hours, centrifuging to take supernate, purifying, blow-drying with nitrogen, and dissolving to a constant volume. The liquid chromatography-tandem mass spectrometry (LC-MS) method comprises the following steps: extracting a sample with 80% methanol, filtering, carrying out rotary evaporation, washing with a methanol-water-acetic acid mixed solution, eluting, collecting an eluent, blow-drying with nitrogen, dissolving and feeding the sample. If the result difference value of the two methods is less than or equal to 15%, completing ELISA method result correction; if the difference value is large, adjusting the sample amount and the extraction condition until the difference value is less than or equal to 15%. According to the method, the content of the endogenous hormone is measured by utilizing an ELISA method and an HPLC-MS / MS method, so that the hormone content is rapidly and accurately detected while the operation is simple and convenient.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant physiological and biochemical analysis, and particularly relates to a method for testing and analyzing endogenous hormone content of Angelica sinensis. BACKGROUND

[0002] Angelica sinensis is a perennial herb of the Umbelliferae family, and is praised as "the holy medicine in blood" and has an important position in the field of traditional Chinese medicine. As a traditional and precious Chinese herbal medicine, Angelica sinensis is mainly distributed in Gansu and Sichuan, China. It has the effects of tonifying blood, activating blood, regulating menstruation and relieving pain, moistening the intestines and relieving constipation, and is widely used in the treatment of gynecological diseases and blood deficiency. Modern research shows that Angelica sinensis contains volatile oil, organic acid, polysaccharide and other active ingredients, and has the pharmacological effects of antioxidant, anti-inflammatory and immune regulation. Endogenous hormones, as key factors regulating the growth and development of Angelica sinensis, directly affect the accumulation of medicinal ingredients. Therefore, studying the endogenous hormones of Angelica sinensis is of great significance to improve the cultivation efficiency and ensure the quality of medicinal materials, and provides a scientific basis for the standardized planting and quality improvement of Angelica sinensis.

[0003] Plant endogenous hormones include auxin (IAA), gibberellin (GA), abscisic acid (ABA), etc., which are all endogenous hormones involved in the control of plant flowering. Among them, gibberellin has the greatest influence on the flowering process, and more than 130 types of gibberellins have been found. Plant endogenous hormones are trace amounts in the plant body and are easily variable, and need to be detected by a technology with high selectivity and high sensitivity. At present, the detection of endogenous hormones of Angelica sinensis mainly relies on traditional methods such as enzyme-linked immunosorbent assay (ELISA), but this method has the problems of poor specificity, easy interference by cross reaction, insufficient sensitivity, etc., and it is difficult to accurately determine low content hormones. Although the analysis technology based on chromatography can improve the detection accuracy, the sample pretreatment steps are complicated and involve complex extraction and purification processes. In addition, the existing methods are mainly developed for single plant tissue, and lack of optimized extraction schemes for different parts (top bud, leaf and fleshy root) of Angelica sinensis, which is difficult to meet the demand of synchronous detection of multiple hormones in complex matrix. Therefore, it is urgent to establish a high-efficiency and accurate detection method to improve the reliability and applicability of the analysis of endogenous hormones of Angelica sinensis. SUMMARY

[0004] The purpose of the present application is to solve the problems of low sensitivity and complicated pretreatment steps in the prior art, and to propose a method for testing and analyzing endogenous hormone content of Angelica sinensis.

[0005] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: A method for testing and analyzing endogenous hormone content of Angelica sinensis, comprising the following steps: S1, determining the hormone content by enzyme-linked immunosorbent assay: S101. Homogenize the sample with 80% methanol solution and extract at 4°C for 2-10 h. S102, centrifuging to obtain the supernatant, repeatedly extracting the precipitate with methanol solution 1-3 times, and combining the supernatants; S103, Sep-pak C 18 Column purification, nitrogen drying, and dissolution with PBSTG were used for ELISA assay; S2. Liquid chromatography-tandem mass spectrometry: S201, homogenize the sample with 80% methanol for 3 min and extract overnight; S202, using ODS-C 18 The residue was filtered through a small column and extracted with methanol twice more. The filtrates were combined and rotary evaporated at 35°C to 10 ml. S203, washing the column with a mixture of methanol, water, and acetic acid in a volume ratio of 10:89:1, and then eluting with a mixture of methanol, water, and acetic acid in a volume ratio of 80:19:1, collecting the eluate, drying it with nitrogen, and re-dissolving it with methanol; S204, inject 20 μL of the dissolved sample and perform liquid chromatography detection at a wavelength of 222 nm using a mobile phase mixture of 50% methanol and 50% water; S3. ELISA parameter adjustment: Compare the difference between S1 and S2. When the difference is ≤15%, the ELISA calibration is complete. When the difference is >15%, make the following adjustments: When the hormone content tested in S1 is higher than that in S2, the amount of sample in S101 is reduced until the difference reaches the requirement; When the hormone content tested in S1 is lower than that in S2, the sample amount in S101, the extraction time in S101, and the number of extractions in S102 are increased in sequence until the difference is ≤15%. Subsequently, the endogenous hormone content is tested and analyzed using an enzyme-linked immunosorbent assay with improved parameters.

[0006] Preferably, the sample content in S101 is 0.5-1.5 g. Samples within this mass range are homogenized with 80% methanol solution to ensure the accuracy of the test results in subsequent measurements.

[0007] Preferably, the 80% methanol extraction time in step 101 is 2-10 h.

[0008] Preferably, the extraction is repeated 1-3 times in step 102. Since the endogenous hormone content in angelica is extremely low, it is recommended to extend the extraction time to improve extraction efficiency in order to fully release the target components. If the extraction efficiency is insufficient, 1-3 repeated extractions can be performed to ensure sufficient leaching of the hormones.

[0009] Preferably, the liquid chromatography-tandem mass spectrometry in step S201 is used to determine the quality of the angelica sample, which is 5 g. Too much sample can cause serious tailing of the chromatographic peaks of each hormone.

[0010] Preferably, the composition of the methanol mixed solution in the elution in step S203 is methanol, water and acetic acid, and the ratio is 10:89:1. A small amount of acetic acid can improve the chromatographic peak shape of the hormone and improve the reproducibility. The optimization of the elution ratio realizes the removal of impurities and the retention of target substances.

[0011] Preferably, the composition of the methanol mixed solution in the elution in step S203 is methanol, water and acetic acid, and the ratio is 80:19:1. In this weight ratio range, the endogenous hormones in angelica can be fully eluted. In the subsequent liquid phase test, the retention time of the hormone is reduced while having a suitable peak width.

[0012] Preferably, the sample injection amount in step S204 is 20 uL, and the injection flow rate is 0.8 mL·min -1 According to the properties of the hormone, in this volume and flow rate range, the accuracy of the test results can be ensured.

[0013] Preferably, the difference between the results of the two methods in step S3 is within 15%. In this range, the test results of the enzyme-linked immunosorbent assay can be considered accurate.

[0014] Compared with the prior art, the beneficial effects of the present application are: 1. The present application adopts a research system combining qualitative analysis and quantitative analysis, indoor controllable test and field actual cultivation test, and constructs a set of high-precision angelica endogenous hormone detection and correction method. Specifically, first, the enzyme-linked immunosorbent assay (ELISA) is used to realize the rapid and dynamic monitoring of the endogenous hormones of angelica at different growth stages, and real-time hormone content data is obtained. On this basis, the high-performance liquid chromatography-tandem mass spectrometry (HPLC-IT-MS / MS) technology is innovatively introduced as a correction means to realize the rapid and accurate large-scale operation of the endogenous hormone content of angelica samples.

[0015] 2. Under the enzyme-linked immunosorbent assay and liquid chromatography-tandem mass spectrometry described above, the present application realizes simple, rapid and accurate testing of the endogenous hormones in angelica samples, optimizes the pretreatment conditions (sample content, extraction time and frequency, etc.) and chromatographic separation conditions (chromatographic column type, sample injection amount, sample injection flow rate, etc.), and detects the hormone (gibberellin GA3, gibberellin GA4, auxin IAA, abscisic acid ABA and zeatin riboside ZR (Z+ZR)) content at different stages of angelica roots, sprouts and stems. Provide accurate data basis for the correlation between the flower bud differentiation rate, early bolting rate and stem elongation rate of angelica and the endogenous hormone content. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a flow chart of a method for testing and analyzing the content of angelica intrinsic hormones. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments.

[0018] Embodiment 1: S1, enzyme-linked immunosorbent assay for determining the content of hormones: S101, homogenate 0.5 g sample with 80% methanol solution, extract at 4℃ for 4 h; S102, centrifuge, obtain supernatant, and then repeat the extraction of the precipitate with methanol solution once, and combine the supernatants; S103, purify through Sep-pak C 18 column, dry with nitrogen, dissolve in PBSTG for constant volume for ELISA determination; S2, liquid chromatography-tandem mass spectrometry determination: S201, homogenate the sample with 80% methanol for 3 min, and extract overnight; S202, filter through ODS-C 18 small column, and then repeat the extraction of the residue with methanol twice, combine the filtrates, and rotary evaporate at 35℃ to 10 ml; S203, wash the small column with a mixture of methanol, water and acetic acid at a volume ratio of 10:89:1, and then elute with a mixture of methanol, water and acetic acid at a volume ratio of 80:19:1, collect the eluate, dry with nitrogen, and redissolve with methanol; S204, inject 20 μL of the dissolved sample, and perform liquid phase detection at 222 nm wavelength with a mixture of 50% methanol and 50% water as the mobile phase; S3, enzyme-linked immunosorbent assay parameter adjustment: Compare the difference between S1 and S2, when the difference is >15%, and the hormone content tested by S1 is lower than that by S2, increase the sample mass in S101 to 0.8 g, repeat the above S1-S3 steps. The difference is >15%, the sample mass in S101 is increased to 1.0 g, and the above S1-S3 steps are repeated. The difference is close to 15%, the extraction time in S101 is increased to 6 h, the difference is ≤15%, and the correction of the enzyme-linked immunosorbent assay is completed.

[0019] Embodiment 2: S1, enzyme-linked immunosorbent assay for determining the content of hormones: S101, homogenate 1.5 g sample with 80% methanol solution, extract at 4℃ for 4 h; S102, centrifugation, after obtaining the supernatant, the precipitate is repeatedly extracted once with methanol solution, and the supernatants are combined; S103, Sep-pak C 18 Column purification, nitrogen blowing, PBSTG dissolution constant volume for ELISA determination; S2, liquid chromatography-tandem mass spectrometry determination: S201, homogenize the sample with 80% methanol for 3 min, and extract overnight; S202, ODS-C 18 Small column filtration, the residue is repeatedly extracted twice with methanol, the filtrate is combined, and rotary evaporation is performed at 35°C to 10 ml; S203, the small column is washed with a mixture of methanol, water and acetic acid in a volume ratio of 10:89:1, and then eluted with a mixture of methanol, water and acetic acid in a volume ratio of 80:19:1, the eluate is collected, nitrogen blowing, using methanol redissolved; S204, 20 μL of the dissolved sample is injected, 50% methanol and 50% water are mixed as the mobile phase, and liquid phase detection is carried out at 222 nm wavelength; S3, enzyme-linked immunosorbent assay parameter adjustment: Compare the difference between S1 and S2, when the difference is greater than 15%, adjust as follows: when the hormone content of S1 test is higher than S2, reduce the sample amount in S101 to 1.2 g, repeat S1-S3 steps. When the difference is close to 15%, change the extraction time of S102 to 8 h, repeat S1-S3 steps. When the difference between S1 and S2 is less than or equal to 15%, the correction of enzyme-linked immunosorbent assay is completed.

[0020] Example 3: S1, enzyme-linked immunosorbent assay for determination of hormone content: S101, homogenize 1.0 g sample with 80% methanol solution, extract at 4°C for 4 h; S102, centrifugation, after obtaining the supernatant, the precipitate is repeatedly extracted once with methanol solution, and the supernatants are combined; S103, Sep-pak C 18 Column purification, nitrogen blowing, PBSTG dissolution constant volume for ELISA determination; S2, liquid chromatography-tandem mass spectrometry determination: S201, homogenize the sample with 80% methanol for 3 min, and extract overnight; S202, ODS-C 18 Small column filtration, the residue is repeatedly extracted twice with methanol, the filtrate is combined, and rotary evaporation is performed at 35°C to 10 ml; S203, washing the column with a mixture of methanol, water, and acetic acid in a volume ratio of 10:89:1, and then eluting with a mixture of methanol, water, and acetic acid in a volume ratio of 80:19:1, collecting the eluate, drying it with nitrogen, and re-dissolving it with methanol; S204, inject 20 μL of the dissolved sample and perform liquid chromatography detection at a wavelength of 222 nm using a mobile phase mixture of 50% methanol and 50% water; S3. ELISA parameter adjustment: Comparing the differences between the S1 and S2 methods, the hormone content tested by S1 was lower than that of S2, with a difference greater than 15%, and the difference for some hormones was less than 15%. When the extraction time in S102 was changed to 8 h, the difference was ≤15%. Subsequently, the endogenous hormone content was tested and analyzed using the enzyme-linked immunosorbent assay with improved parameters.

[0021] Example 4: S1. Determination of hormone content using enzyme-linked immunosorbent assay: S101, homogenize 1.0 g of sample with 80% methanol solution and extract at 4°C for 8 h; S102, centrifuging to obtain the supernatant, extracting the precipitate with methanol solution again once, and combining the supernatants; S103 was purified by Sep-pak C18 column, dried by nitrogen, and dissolved in PBSTG to a fixed volume for ELISA assay; S2. Liquid chromatography-tandem mass spectrometry: S201, homogenize the sample with 80% methanol for 3 min and extract overnight; S202, filter using ODS-C18 cartridge, extract the residue twice with methanol, combine the filtrates, and evaporate to 10 ml at 35°C; S203, washing the column with a mixture of methanol, water, and acetic acid in a volume ratio of 10:89:1, and then eluting with a mixture of methanol, water, and acetic acid in a volume ratio of 80:19:1, collecting the eluate, drying it with nitrogen, and re-dissolving it with methanol; S204, inject 20 μL of the dissolved sample and perform liquid chromatography detection at a wavelength of 222 nm using a mobile phase mixture of 50% methanol and 50% water; S3. ELISA parameter adjustment: Compare the difference between S1 and S2 methods. When the hormone content tested by S1 is lower than that of S2, and the difference of some hormone determinations is greater than 15%, change the number of S102 extractions to 2, repeat S1-S3 operations, and the difference is ≤15%, it is considered that the enzyme-linked immunosorbent assay calibration is completed.

[0022] Example 5: S1. Determination of hormone content using enzyme-linked immunosorbent assay: S101, homogenate 1.0 g sample with 80% methanol solution, extract at 4°C for 8 h; S102, centrifuge, take supernatant, and then repeat extraction of the precipitate with methanol solution for 2 times, and combine the supernatants; S103, purify through Sep-pak C18 column, dry with nitrogen, dissolve in PBSTG for ELISA determination; S2, liquid chromatography-tandem mass spectrometry determination: S201, homogenate sample with 80% methanol for 3 min, and extract overnight; S202, filter through ODS-C18 column, and then repeat extraction of the residue with methanol for 2 times, combine the filtrates, and rotary evaporate at 35°C to 10 ml; S203, wash the column with a mixture of methanol, water and acetic acid at a volume ratio of 10:89:1, and then elute with a mixture of methanol, water and acetic acid at a volume ratio of 80:19:1, collect the eluate, dry with nitrogen, and redissolve with methanol; S204, inject 20 μL of the redissolved sample, and perform liquid phase detection at 222 nm wavelength with a mixture of 50% methanol and 50% water as mobile phase; S3, enzyme-linked immunosorbent assay parameter adjustment: Compare the difference between S1 and S2, and if the difference is ≤15%, then use the improved parameters of enzyme-linked immunosorbent assay for subsequent testing and analysis of endogenous hormone content.

[0023] Comparative Example 1: S1, determine hormone content by enzyme-linked immunosorbent assay: S101, homogenate 1.0 sample with 80% methanol solution, extract at 4°C for 8 h; S102, centrifuge, and combine the supernatants; S103, purify through Sep-pak C18 column, dry with nitrogen, dissolve in PBSTG for ELISA determination; S2, liquid chromatography-tandem mass spectrometry determination: S201, homogenate sample with 80% methanol for 3 min, and extract overnight; S202, filter through ODS-C18 column, and then repeat extraction of the residue with methanol for 2 times, combine the filtrates, and rotary evaporate at 35°C to 10 ml; S203, wash the column with a mixture of methanol, water and acetic acid at a volume ratio of 10:89:1, and then elute with a mixture of methanol, water and acetic acid at a volume ratio of 80:19:1, collect the eluate, dry with nitrogen, and redissolve with methanol; S204, 20 μL of the dissolved sample was injected, and liquid phase detection was performed at 222 nm using 50% methanol and 50% water as the mobile phase mixture; S3, enzyme-linked immunosorbent assay parameter adjustment: When the difference between S1 and S2 was greater than 15%, and the hormone content of S1 was lower than that of S2, the sample amount in S101 was increased to 1.2 g, and the difference between S1 and S2 was reduced but still greater than 15%. The sample amount in S1 was further increased to 1.5 g, but the difference did not change significantly, and correction could not be completed.

[0024] Comparative Example 2: Comparative Example 3 differed from Example 1 in that the Sep-pak C18 column purification step was removed, and the parameter adjustment step was as in Comparative Example 1.

[0025] Correlation calculation: difference = (a is the liquid chromatography-tandem mass spectrometry test result, and b is the enzyme-linked immunosorbent assay test result.) The test results (represented by the difference between the two test methods) are shown in Table 1: Table 1: Difference between the two test methods

[0026] Data analysis: As can be seen from Table 1, for the determination of the endogenous hormone content of Angelica sinensis, there is a certain gap between the enzyme-linked immunosorbent assay and the liquid chromatography-tandem mass spectrometry method, which is caused by the determination conditions, the nature and state of the hormone, and is unavoidable. The enzyme-linked immunosorbent assay has poor repeatability and many interference factors, and the result accuracy is lower than that of the liquid chromatography-tandem mass spectrometry method. Therefore, the liquid chromatography-tandem mass spectrometry method is used as the standard detection method, and the enzyme-linked immunosorbent assay determination result is corrected. When the difference between the two results is less than 15%, the enzyme-linked immunosorbent assay determination result is considered accurate. Otherwise, the sample amount, extraction time, and extraction times of the enzyme-linked immunosorbent assay are changed in turn until the difference is less than 15%.

[0027] From Table 1, the absolute value of the difference between the two methods of Example 1 is more than 25%, the error is large, and the sample quality is improved to 0.8 g, the difference is reduced, and the sample quality is continuously increased, the experiment is repeated, the difference between S1 and S2 is close to 15%, the extraction time is prolonged, and the difference between each hormone is less than 15%, and the correction of enzyme-linked immunosorbent assay is completed. Compared with Example 1, the angelica sample quality is increased in Example 2, the total content of hormones is increased, the signal is high, the liquid chromatography-mass spectrometry condition is unchanged, the enzyme-linked immunosorbent assay test result is high, the S1 sample quality is reduced to 1.2 g, the extraction time is prolonged to 8 h, and the difference between S1 and S2 is less than 15%. Compared with Example 2, the homogenate sample quality is reduced in Example 3, the number of enzyme-antibody captured hormone antigens is reduced, the signal is low, and the overall difference is reduced. Compared with Example 1, the extraction time is prolonged and the extraction times are increased in Examples 4 and 5, which leads to an increase in the total content of hormones, an increase in the enzyme-linked immunosorbent assay determination result, a decrease in the difference between the liquid chromatography-tandem mass spectrometry method result, and an increase in the difference between the two methods. In Example 5, the difference between GA3, GA4, IAA, ABA and ZR (Z+ZR) is less than 15%, which indicates that the enzyme-linked immunosorbent assay determination result is accurate at this time, and the difference between the above-mentioned five hormones can also be found. The higher the content of endogenous hormones in angelica, the smaller the difference between the test results of the two methods. In the same experiment, the difference between the trace hormones GA4 and ZR (Z+ZR) is greater than that of the other three hormones. This is because the detection limit of enzyme-linked immunosorbent assay is 0.1 ng / mL, the detection limit of liquid chromatography-mass spectrometry is also close to it, and the types of endogenous hormones in angelica are complex, which leads to a large deviation between the test results of the two methods.

[0028] Compared with Example 5, the repeated extraction step is removed in Comparative Example 1, the total content of hormones flows out, the enzyme-linked immunosorbent assay result is low, and the difference is increased. In subsequent parameter adjustment, the S1 sample quality is also increased, which cannot make the difference between S1 and S2 decrease greatly, which indicates that the step of repeated extraction with methanol cannot be omitted. Compared with Example 5, the purification step is removed in Comparative Example 2, the impurity content is high when the enzyme-linked immunosorbent assay is determined, the hormone content is low, the result is low, the difference is increased, and the same, in subsequent parameter adjustment, the S1 sample quality is also increased, which cannot make the difference between S1 and S2 decrease greatly, which indicates that the step of purification cannot be omitted.

[0029] The above is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can make equivalent replacement or change according to the technical scheme and the inventive concept of the present application within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A method for testing and analyzing the endogenous hormone content of Angelica sinensis, characterized in that: The following steps are involved: S1. Determination of hormone content using enzyme-linked immunosorbent assay: S101. Homogenize the sample with 80% methanol solution and extract at 4°C for 2-10 h. S102, centrifuging to obtain the supernatant, repeatedly extracting the precipitate with methanol solution 1-3 times, and combining the supernatants; S103, Sep-pak C 18 Column purification, nitrogen drying, and dissolution with PBSTG were used for ELISA assay; S2. Liquid chromatography-tandem mass spectrometry: S201, homogenize the sample with 80% methanol for 3 min and extract overnight; S202, using ODS-C 18 Filter the small column, and extract the residue with methanol twice again. Combine the filtrates and evaporate to 10 ml at 35℃. S203, washing the column with a mixture of methanol, water, and acetic acid in a volume ratio of 10:89:1, and then eluting with a mixture of methanol, water, and acetic acid in a volume ratio of 80:19:1, collecting the eluate, drying it with nitrogen, and re-dissolving it with methanol; S204, inject 20 μL of the dissolved sample and perform liquid chromatography detection at a wavelength of 222 nm using a mixture of 50% methanol and 50% water as the mobile phase; S3. ELISA parameter adjustment: Compare the difference between S1 and S2. When the difference is ≤15%, the ELISA calibration is complete. When the difference is >15%, make the following adjustments: When the hormone content tested in S1 is higher than that in S2, the amount of sample in S101 is reduced until the difference reaches the requirement; When the hormone content tested in S1 is lower than that in S2, the sample amount in S101, the extraction time in S101, and the number of extractions in S102 are increased in sequence until the difference is ≤15%. Subsequently, the endogenous hormone content is tested and analyzed using an enzyme-linked immunosorbent assay with improved parameters.

2. The method for testing and analyzing the endogenous hormone content of Angelica sinensis according to claim 1, characterized in that: The mass of the sample required for S101 is 0.5-1.5 g.

3. The method for testing and analyzing the endogenous hormone content of Angelica sinensis according to claim 1, characterized in that: The centrifugal speed required for S102 is 4000 r·min -1 , the centrifugation time was 10 min.

4. The method for testing and analyzing the endogenous hormone content of Angelica sinensis according to claim 1, wherein: The S103 was dissolved in PBSTG to a volume of 2 mL for ELISA determination.

5. The method for testing and analyzing the endogenous hormone content of Angelica sinensis according to claim 1, characterized in that: The sample mass required for S201 is 5 g.

6. The method for testing and analyzing the endogenous hormone content of Angelica sinensis according to claim 1, characterized in that: The volume of methanol used in the S202 is 10 mL.

7. The method for testing and analyzing the endogenous hormone content of Angelica sinensis according to claim 1, characterized in that: The volume of methanol used to dissolve the eluent sample in S203 is 1 mL.

8. The method for testing and analyzing the endogenous hormone content of Angelica sinensis according to claim 1, characterized in that: The injection volume of the dissolved sample of S204 was 20 μL, and the injection flow rate was 0.8 mL·min -1 .

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