Method for establishing andrographis paniculata quality control by using quantitative analysis of multi-components by single marker

Abstract

The invention discloses a method for establishing andrographis paniculata quality control by using a QAMS (quantitative analysis of multi-components by single marker) method. Comprising the following steps: preparing a test sample solution, preparing a reference solution, determining chromatographic conditions and mass spectrum conditions, determining main components of a test sample, establishing a standard curve, performing methodological investigation by using an external standard method, determining an internal reference and a relative correction factor of the quantitative analysis of multi-components by single marker, and investigating the durability of the relative correction factor and positioning of a chromatographic peak. And comparing and verifying the content of the measured sample. The invention further discloses an andrographis paniculata quality control method. According to the method, relative correction factors of an internal reference substance and other to-be-detected components are established on the basis of an ultra-high performance liquid chromatography-tandem mass spectrometry-based quantitative analysis of multi-components by single marker, and the contents of four components including desoxidized andrographolide, andrographolide, dehydroandrographolide and andrographolide in andrographis paniculata are determined as a quality control method of the andrographis paniculata medicinal material, so that the quality of the andrographis paniculata medicinal material is improved. The calculated value and the measured value meet the requirements of quantitative methodology, the requirements of reference substances are reduced, and the quality control level of andrographis paniculata is improved.

Description

Technical Field

[0001] The invention relates to a method for determining the content of main components of Andrographis paniculata by a one-test-multiple-evaluation method and establishing quality control, and belongs to the technical field of traditional Chinese medicine and inspection. Background Art

[0002] Andrographis paniculata is the dried aerial part of the plant Andrographis paniculata (Burm.f.) Nees of the Acanthaceae family. It has the effects of clearing away heat and detoxifying, antibacterial and anti-inflammatory, protecting the liver and promoting bile secretion. Andrographis paniculata is rich in various ingredients, and the content determination of such ingredients can better control its quality. The identification and content determination items in the Andrographis paniculata medicinal material standard in the 2020 edition of the Pharmacopoeia of the People's Republic of China only include the thin layer chromatography identification of Andrographis paniculata medicinal materials and the HPLC determination of andrographolide, which cannot fully reflect the intrinsic quality of Andrographis paniculata. In order to improve the quality control level of Andrographis paniculata meat, a study has developed an HPLC-MS method to determine the content of several components of Andrographis paniculata (andrographolide, andrographolide, dehydroandrographolide nucleoside, and deoxyandrographolide) and established a fingerprint spectrum. At the same time, it is pointed out that andrographolide, andrographolide, dehydroandrographolide nucleoside, and deoxyandrographolide are relatively stable and high-content components. Literature reports that the detection of Andrographis paniculata mainly adopts liquid chromatography and liquid chromatography-mass spectrometry. However, the quantitative methods reported in the above literature all adopt the external standard method, and the types and quantities of reference substances required are relatively large. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a method for establishing quality control of Andrographis paniculata using a one-test-multiple-evaluation method, thereby solving the problem that there is currently no quality control method for Andrographis paniculata; a method for quality control of Andrographis paniculata is also provided, thereby solving the problem that a variety of reference substances need to be used when determining the content of the main components of Andrographis paniculata, and a plurality of method establishments need to be performed for methodological investigations.

[0004] The technical solution of the present invention is as follows: a method for establishing quality control of Andrographis paniculata using a one-test-multiple-evaluation method, the method comprising the following steps:

[0005] (1) Preparation of test solution: Accurately weigh the homogenized sample of crushed Andrographis paniculata, place it in a volumetric flask, add 80% methanol to dilute to the mark, shake well, vortex mix, ultrasonically extract, centrifuge, take the supernatant and place it in another volumetric flask, add 80% methanol to dilute to the mark, shake well, vortex mix, take the supernatant and filter it through a 0.22 μm filter membrane into a sample injection vial to obtain the test solution;

[0006] (2) Preparation of reference substance solution: Accurately weigh reference substances forsythiaside, dehydroandrographolide succinate half ester, deoxyandrographolide, oleanolic acid, caffeic acid, andrographolide, dehydroandrographolide, andrographolide sodium bisulfite into a volumetric flask, dissolve and dilute to scale with 80% methanol, shake well to obtain reference substance stock solution; Accurately weigh 1 mL of reference substance stock solution into a 50 mL volumetric flask, dissolve and dilute to scale with 80% methanol, shake well to obtain reference substance solution;

[0007] (3) Determine the chromatographic and mass spectrometric conditions;

[0008] (4) Determine the main components of the test sample: Place the test sample solution and the reference solution in an Agilent ultra-high performance liquid chromatography-triple quadrupole mass spectrometer for determination under the same analytical conditions. If the test sample solution shows chromatographic peaks of quantitative ion pairs and qualitative ion pairs, the retention time of the characteristic ion peak of the measured component is consistent with the retention time of the reference solution, and the relative abundance ratio of the selected qualitative ion does not exceed the maximum deviation of the relative abundance ratio of the qualitative ion of the reference solution of equivalent concentration, then the corresponding component can be determined to be present in the sample; andrographolide, deoxyandrographolide, dehydroandrographolide and andrographolide were found to be present in the test sample solution;

[0009] (5) Establish a standard curve: Take 5 portions of reference solution with different volumes and increasing in volume, place them in volumetric flasks of the same volume, add 80% methanol water to dilute to the mark, shake well, and obtain 5 portions of reference solution with the same volume and different concentrations. Perform injection analysis according to the chromatographic conditions of step (3), and draw standard curves for the four components of andrographolide, deoxyandrographolide, dehydroandrographolide, and andrographolide, with the peak area Y as the ordinate and the mass concentration X, μg / mL as the abscissa;

[0010] (6) Methodological investigation using external standard method:

[0011] a. Instrument precision test: Take the reference solution and place it in a volumetric flask, add 80% methanol water to dilute to the scale, shake well, and obtain the corresponding reference solution to be tested; accurately pipette 5 μL of each reference solution to be tested, repeat the injection 6 times according to the conditions of step (3), and calculate the relative standard deviation (RSD) of the peak area of ​​andrographolide, deoxyandrographolide, dehydroandrographolide, and andrographolide reference solution;

[0012] b. Repeatability study: Take the same batch of Andrographis paniculata medicinal materials and prepare 6 test samples in parallel. Prepare the test sample solution according to step (1), and perform the determination according to the conditions of step (3). Calculate the RSD value of andrographolide, deoxyandrographolide, dehydroandrographolide and andrographolide in each test sample solution.

[0013] c. Recovery rate investigation: accurately weigh 9 portions of the test sample with the measured content, 0.05 g each, prepare the test sample solution according to step (1), divide it into 3 groups, 3 portions in each group, accurately add the same volume of reference stock solution to the 3 portions in each group, and increase the volume of reference stock solution added to each group. Perform the test according to the conditions of step (3), and calculate the average recovery rate and RSD;

[0014] (7) Determine the internal reference and relative correction factor of the one-test-multiple-evaluation method: Select a component with a moderate peak area as the internal reference. In this method, andrographolide is selected as the internal reference. The relative correction factor calculation formula is: fs / i=fs / fi=(Wi×As) / (Ws×Ai), where: fs / i is the relative correction factor between the internal reference and other components; As is the peak area of ​​the internal reference; Ws is the concentration of andrographolide internal reference; Ai is the peak area of ​​other components; Wi is the concentration of other components; The content of each component in the test sample is calculated as follows: Wi= fs / i×Ws×Ai / As, where Ai is the peak area of ​​the component to be tested, Wi is the concentration of the component to be tested, As is the peak area of ​​the internal reference, Ws is the concentration of the internal reference, and fs / i is the relative correction factor of the internal reference to the component to be tested;

[0015] (8) Investigating the durability of the relative correction factor and the positioning of the chromatographic peak: In order to investigate the durability of the relative correction factor for instruments and chromatographic columns from different manufacturers, three chromatographic columns from different manufacturers and two instruments of different models were selected to investigate the durability of the correction factor;

[0016] (9) Comparative verification of the content of the determined samples: Accurately weigh different batches of Andrographis paniculata samples, prepare the test sample solution according to step (1), and prepare three replicates for each batch. Perform sample injection analysis according to the conditions of step (3). The external standard method uses a linear regression equation to calculate the content, and the one-measurement multiple evaluation method uses the average value to calculate the content. After determination and analysis, there is no significant difference between the results of the external standard method and the one-measurement multiple evaluation method for the content calculation of the four components of andrographolide, deoxyandrographolide, dehydroandrographolide, and andrographolide, and the detection sensitivity of the two detection methods is consistent, indicating that there is no significant difference in the results of the four components in Andrographis paniculata detected by the external standard method and the one-measurement multiple evaluation method. This method is established to establish the quality control method of Andrographis paniculata using the one-measurement multiple evaluation method. The quality control of Andrographis paniculata can be carried out by determining the components of andrographolide, deoxyandrographolide, dehydroandrographolide, and andrographolide in Andrographis paniculata by the one-measurement multiple evaluation method.

[0017] The maximum deviation between the relative abundance ratio of the selected qualifier ion and the relative abundance ratio of the qualifier ion of the reference solution of equivalent concentration is: when the relative ion abundance is greater than 50, the allowable relative deviation is ±20%; when the relative ion abundance is greater than 20-50, the allowable relative deviation is ±25%; when the relative ion abundance is greater than 10-20, the allowable relative deviation is ±30%; when the relative ion abundance is ≤10, the allowable relative deviation is ±50%.

[0018] This application explores and investigates a variety of influencing factors, including different pre-treatment solvents, chromatographic conditions, mass spectrometry conditions, external standard method methodology, determination of internal reference materials, durability investigation, and comparison between the external standard method and the one-test-multiple-evaluation method. A method for quality control of Andrographis paniculata using the one-test-multiple-evaluation method based on ultra-performance liquid chromatography-tandem mass spectrometry is established. This application determines the contents of four components in Andrographis paniculata pulp, including deoxyandrographolide, andrographolide, dehydroandrographolide, and andrographolide, as a quality evaluation method for Andrographis paniculata medicinal materials.

[0019] This application selects andrographolide as the reference substance for the internal reference, establishes the relative correction factor (RCF) between the internal reference substance and other components to be tested, and through methodology and cross-validation, makes its calculated value and measured value meet the requirements of quantitative methodology. The newly established "one test, multiple evaluations" method for andrographolide components can reduce the demand for reference substances, make it possible to apply multi-index quality control and evaluation models to actual production, and improve the quality control level of andrographolide slices. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the MRM chromatogram of the 9 components in this application. DETAILED DESCRIPTION

[0021] A method for establishing quality control of Andrographis paniculata using a one-test-multiple-evaluation method, the method comprising the following steps:

[0022] (1) Preparation of test solution:

[0023] Pretreatment conditions were determined: pure water, methanol, 10% methanol-water, 25% methanol-water, 35% methanol-water, 50% methanol-water, and 80% methanol-water were selected as extraction solvents, respectively. The results showed that methanol had the highest dissolution rate of the nine components, but it contained many impurities and had large matrix interference in mass spectrometry. The 80% methanol solution was clear in color and had good separation of the nine components. Therefore, this study used 80% methanol to extract the sample and then dilute it to reduce matrix interference for detection.

[0024] Accurately weigh 0.1 g of the crushed Andrographis paniculata medicinal material homogenized sample, place it in a 50 mL volumetric flask, add 80% methanol to dilute to the scale, shake well, vortex mix for 5 min, ultrasonically extract for 60 min, centrifuge at 5000 r / min, take 0.2 mL of the supernatant and place it in a 10 mL volumetric flask, add 80% methanol to dilute to the scale, shake well, vortex mix for 5 min, take the supernatant and pass it through a 0.22 μm filter membrane into a sampling vial to obtain the test solution.

[0025] (2) Preparation of reference substance solution: accurately weigh reference substance forsythiaside 0.02625 g, dehydroandrographolide succinate ester 0.03750 g, deoxyandrographolide 0.02128 g, oleanolic acid 0.02375 g, caffeic acid 0.02748 g, andrographolide 0.02751 g, dehydroandrographolide 0.01750 g, andrographolide 0.0401 g, and sodium bisulfite andrographolide 0.03125 g, and place them in a 100 mL volumetric flask. Dissolve and dilute to the mark with 80% methanol to obtain the reference substance stock solution. Shake well. Accurately measure 1 mL and place in a 50 mL volumetric flask. Dissolve and dilute to the mark with 80% methanol. Shake well to obtain the reference substance solution.

[0026] In the prepared reference solution, the content of forsythin was 5.25 μg / mL, the content of deoxyandrographolide was 4.17 μg / mL, the content of oleanolic acid was 4.75 μg / mL, the content of caffeic acid was 5.48 μg / mL, the content of andrographolide was 5.48 μg / mL, the content of dehydroandrographolide was 3.43 μg / mL, the content of andrographolide was 7.89 μg / mL, the content of sodium bisulfite andrographolide was 6.25 μg / mL, and the content of dehydroandrographolide succinate half ester was 7.35 μg / mL.

[0027] (3) Determine the chromatographic and mass spectrometric conditions;

[0028] This method compares the mobile phase systems consisting of water, 0.1% formic acid water, 5mmol ammonium acetate aqueous solution, 10mmol ammonium acetate aqueous solution with methanol and acetonitrile, and comprehensively considers and takes into account the retention time, resolution, peak shape and response of the 9 components separated by SB-C18. The results show that using the gradient method of water-methanol+acetonitrile = 70:30, the 9 components have more suitable peak time, better peak shape and higher response in low concentration spiked samples. Figure 1 Therefore, a gradient system of water-methanol+acetonitrile = 70:30 was selected as the mobile phase.

[0029] a. Chromatographic conditions: Chromatographic column: ACQUITY UPLC BEH C18 (50 mm×2.1mm, 1.8mm), column temperature of 35℃, mobile phase system: A: aqueous solution, B: methanol+acetonitrile (7+3); flow rate of 0.3mL / min; gradient elution conditions: 0~0.5min, 45%A; 0.5~1min, 45%~100% A; 1.0~3.0min, 100%A; 3.0~5.0min, 45%A, injection volume of 5μL.

[0030] b. Mass spectrometry conditions: Ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry was performed in electrospray negative ionization mode (ESI-), multiple reaction monitoring (MRM) mode. Agilent 6460 high performance liquid chromatography-tandem mass spectrometer (Agilent Technologies, USA), nebulizer pressure: 30PSI; dryer temperature: 300℃; dryer flow rate: 8 L / min; capillary total voltage: 4000 V, collision gas is high purity nitrogen, Shimadzu LCMS-8050 high performance liquid chromatography-tandem mass spectrometer nebulizer flow rate 2 L / min, heater flow rate 10 L / min, interface temperature 250℃, desolvation temperature 444℃, DL temperature 200℃, heating module temperature 200℃, dryer flow rate 10 L / min, collision gas is high purity argon.

[0031] (4) Determine the main components of the test sample: The test sample solution and the reference solution were placed in an Agilent ultra-high performance liquid chromatography-triple quadrupole mass spectrometer for determination under the same analytical conditions. If the test sample solution showed chromatographic peaks of quantitative ion pairs and qualitative ion pairs, the retention time of the characteristic ion peak of the measured component was consistent with the retention time of the reference solution, and the relative abundance ratio of the selected qualitative ion did not exceed the maximum deviation of the relative abundance ratio of the qualitative ion of the reference solution of equivalent concentration, then the corresponding component could be determined to be present in the sample. It was determined that the test sample solution contained four components: andrographolide, deoxyandrographolide, dehydroandrographolide, and andrographolide.

[0032] The maximum deviation between the relative abundance ratio of the selected qualifier ion and the relative abundance ratio of the qualifier ion of the reference solution of equivalent concentration is: when the relative ion abundance is greater than 50, the allowable relative deviation is ±20%; when the relative ion abundance is greater than 20-50, the allowable relative deviation is ±25%; when the relative ion abundance is greater than 10-20, the allowable relative deviation is ±30%; when the relative ion abundance is ≤10, the allowable relative deviation is ±50%.

[0033] (5) Establish a standard curve: Take 5 portions of reference solution, 0.1, 0.2, 0.4, 1.0, 2.0, 4.0 mL, respectively, and place them in 10 mL volumetric flasks. Add 80% methanol water to dilute to the mark, shake well, and obtain 5 portions of reference solution with the same volume and different concentrations. Perform sample injection analysis according to the chromatographic conditions of step (3).

[0034] The contents of deoxyandrographolide in the reference test solution were 0.0417, 0.0833, 0.1666, 0.4165, 0.8333, and 1.666 μg / mL; the contents of andrographolide were 0.0548, 0.1096, 0.2191, 0.5478, 1.0956, and 2.1912 μg / mL; the contents of dehydroandrographolide were 0.0343, 0.6860, 0.1372, 0.3430, and 1.3720 μg / mL; and the contents of andrographolide were 0.0789, 0.1578, 0.3155, 0.7888, 1.5776, and 3.1552 μg / mL.

[0035] Standard curves of the four components of andrographolide, deoxyandrographolide, dehydroandrographolide and andrographolide were drawn with the peak area Y as the ordinate and the mass concentration X, μg / mL as the abscissa.

[0036] The linear regression equations, correlation coefficients and linear ranges of the reference substances for the external standard method of each standard substance are shown in Table 1. From the data in Table 1, it can be seen that there is a good linear relationship between the injection amount and peak area of ​​the four components of deoxyandrographolide, andrographolide, dehydroandrographolide and andrographolide, and their correlation coefficients r are all greater than 0.995.

[0037] Table 1 Linear relationships and ranges of the four components of Andrographis paniculata

[0038] Element Standard curve regression equation Correlation coefficient r Linear range / (μg / mL) Andrographolide Y=973.190212X+149.825094 0.998 0.0548~2.1912 Dehydroandrographolide Y=4423.131410X+184.121408 0.998 0.0343~1.3720 Andrographolide Y=2417.751242X+80.684019 0.999 0.0789~3.15524 Deoxyandrographolide Y=6876.385636X+508.249098 0.998 0.0417~1.6660

[0039] (6) Methodological investigation using external standard method:

[0040] a. Instrument precision test: Take 2.0 mL of the reference solution and place it in a volumetric flask. Add 80% methanol water to dilute to the mark and shake well to obtain the corresponding reference solution. Accurately pipette 5 μL of each reference solution and repeat the injection 6 times according to the conditions of step (3). The relative standard deviations (RSDs) of the peak areas of andrographolide, deoxyandrographolide, dehydroandrographolide and andrographolide were calculated to be 1.92%, 1.27%, 1.87% and 1.99%, respectively, indicating that the instrument has good precision.

[0041] b. Repeatability study: Take the same batch of Andrographis paniculata medicinal materials and prepare 6 test samples in parallel. Prepare the test sample solution according to step (1) and perform the determination according to the conditions of step (3). The RSD values ​​of andrographolide, deoxyandrographolide, dehydroandrographolide and andrographolide in each test sample solution are calculated to be 2.53%, 2.87%, 1.95% and 1.76%, respectively, indicating that the repeatability of this method is good.

[0042] c. Recovery rate investigation: 9 portions of the test sample with the measured content were accurately weighed, each portion was 0.05 g, and the test sample solution was prepared according to step (1), and divided into 3 groups, each with 3 portions. The 3 portions in each group were accurately added with the same volume of the reference stock solution, respectively, 3 portions of 0.2 mL in the first group, 3 portions of 0.4 mL in the second group, and 3 portions of 1.0 mL in the third group. The determination was carried out according to the conditions of step (3). After calculation, the average recovery rate was 99.2%, and the RSD was 4.51%. The results are shown in Table 2. The results show that this method is reliable and has good accuracy.

[0043] Table 2 Recovery results (n=9)

[0044] Element Deoxyandrographolide Andrographolide Dehydroandrographolide Andrographolide Average recovery rate / % 100.41 101.64 101.58 108.93 RSDs / % 1.21 1.03 1.12 2.44

[0045] (7) Determine the internal reference and relative correction factor of the one-test-multiple-evaluation method: Select a component with a moderate peak area as the internal reference. In this method, andrographolide is selected as the internal reference. The relative correction factor calculation formula is: fs / i=fs / fi=(Wi×As) / (Ws×Ai), where: fs / i is the relative correction factor between the internal reference and other components; As is the peak area of ​​the internal reference; Ws is the concentration of the andrographolide internal reference; Ai is the peak area of ​​other components; Wi is the concentration of other components; The calculation formula for the content of each component in the test sample is as follows: Wi=fs / i×Ws×Ai / As, where Ai is the peak area of ​​the component to be tested, Wi is the concentration of the component to be tested, As is the peak area of ​​the internal reference, Ws is the concentration of the internal reference, and fs / i is the relative correction factor of the internal reference to the component to be tested.

[0046] With andrographolide (1.0956 μg / mL) as the internal reference, the relative correction factors of deoxyandrographolide (0.8333 μg / mL), dehydroandrographolide (0.6860 μg / mL) and andrographolide (1.5776 μg / mL) were calculated respectively. The results showed that the correction factors met the detection requirements, and the RSD values ​​were all less than 5% (see Table 3).

[0047] Table 3 Relative correction factors using andrographolide as internal reference

[0048] Relative correction factor <![CDATA[f 穿心莲新酯 / 穿心莲内酯 ]]> <![CDATA[f 穿心莲内酯 / 穿心莲内酯 ]]> <![CDATA[f 脱水穿心莲内酯 / 穿心莲内酯 ]]> <![CDATA[f 去氧穿心莲内酯 / 穿心莲内酯 ]]> average value 0.3374 1 0.2604 0.1123 RSDs / % 4.80 0.00 4.91 4.59

[0049] (8) Examining the durability of the relative correction factor and the positioning of the chromatographic peaks:

[0050] The durability of the relative correction factor refers to the degree to which it can withstand small changes in the content determination conditions. In order to make the relative correction factors of each component to be measured applicable to routine testing, the durability of the RCF of the component to be measured should be comprehensively investigated. In order to investigate the durability of RCF for instruments and chromatographic columns from different manufacturers, three different manufacturers' chromatographic columns and two different models of instruments were selected to investigate the durability of the correction factor; 5 μL of the reference solution was accurately aspirated for determination, and three chromatographic columns were investigated, namely: American Waters AT3 chromatographic column 50 mm×2.1 mm, 1.8 μm, China Amp CNW-Eclipse-plus-C18 chromatographic column 50 mm×2.1 mm, 1.8 μm, and American Agilent ZORBAX SB-C18 chromatographic column 50mm×2.1 mm, 1.8 μm. Two types of liquid chromatography-mass spectrometry instruments were used: Agilent 1260 liquid chromatograph, equipped with Agilent G6460 triple tandem mass spectrometer; LC-40B XR ultra-high performance liquid chromatograph, equipped with LCMS-8050 triple quadrupole mass spectrometer. The results showed that under different chromatographic column and instrument conditions, the relative correction factor (f) was relatively stable, and the RSD value was less than 5%, as shown in Table 4, with good adaptability.

[0051] Table 4 Comparison of relative correction factors of 9 index components for andrographolide under different chromatographic columns and instrument conditions

[0052]

[0053] (9) Comparative verification of the content of the determined samples: Weigh 20 batches of Andrographis paniculata samples accurately, prepare the test sample solution according to step (1), and perform three replicates for each batch. Perform sample injection analysis according to the conditions of step (3). The external standard method uses a linear regression equation to calculate the content, and the one-measurement multiple evaluation method uses the average value to calculate the content, as shown in Table 5. After determination and analysis, there was no significant difference between the results of the external standard method and the one-measurement multiple evaluation method for the content calculation of the four components of andrographolide, deoxyandrographolide, dehydroandrographolide, and andrographolide, and the detection sensitivity of the two detection methods was consistent, indicating that there was no significant difference in the results of the four components in Andrographis paniculata detected by the external standard method and the one-measurement multiple evaluation method. The method for establishing the quality control of Andrographis paniculata using the one-measurement multiple evaluation method is established, and the quality control of Andrographis paniculata medicinal materials can be carried out by determining the components of andrographolide, deoxyandrographolide, dehydroandrographolide, and andrographolide in Andrographis paniculata by the one-measurement multiple evaluation method.

[0054] Table 5 Comparison of the results of detecting the contents of 9 index components by one-test-multiple-evaluation method and external standard method (n=3)

[0055]

[0056] Data processing of this method: Agilent mass spectrometry data and methodological investigation data were processed using Mass Hunter workstation. Shimadzu mass spectrometry data and methodological investigation data were processed using LabSolution workstation RSD and sample content were calculated using Excel 2021 software.

Claims

1. A method for establishing quality control of Andrographis paniculata using a one-test-multiple-evaluation method, characterized in that: The method comprises the following steps: (1) Preparation of test solution: Accurately weigh the homogenized sample of crushed Andrographis paniculata, place it in a volumetric flask, add 80% methanol to dilute to the mark, shake well, vortex mix, ultrasonically extract, centrifuge, take the supernatant and place it in another volumetric flask, add 80% methanol to dilute to the mark, shake well, vortex mix, take the supernatant and filter it through a 0.22 μm filter membrane into a sample injection vial to obtain the test solution; (2) Preparation of reference substance solution: Accurately weigh reference substances forsythiaside, dehydroandrographolide succinate half ester, deoxyandrographolide, oleanolic acid, caffeic acid, andrographolide, dehydroandrographolide, andrographolide sodium bisulfite into a volumetric flask, dissolve and dilute to scale with 80% methanol, shake well to obtain reference substance stock solution; Accurately weigh 1 mL of reference substance stock solution into a 50 mL volumetric flask, dissolve and dilute to scale with 80% methanol, shake well to obtain reference substance solution; (3) Determine the chromatographic and mass spectrometric conditions; (4) Determine the main components of the test sample: Place the test sample solution and the reference solution in an Agilent ultra-high performance liquid chromatography-triple quadrupole mass spectrometer for determination under the same analytical conditions. If the test sample solution shows chromatographic peaks of quantitative ion pairs and qualitative ion pairs, the retention time of the characteristic ion peak of the measured component is consistent with the retention time of the reference solution, and the relative abundance ratio of the selected qualitative ion does not exceed the maximum deviation of the relative abundance ratio of the qualitative ion of the reference solution of equivalent concentration, then the corresponding component can be determined to be present in the sample; andrographolide, deoxyandrographolide, dehydroandrographolide and andrographolide were found to be present in the test sample solution; (5) Establish a standard curve: Take 5 portions of reference solution with different volumes and increasing in volume, place them in volumetric flasks of the same volume, add 80% methanol water to dilute to the mark, shake well, and obtain 5 portions of reference solution with the same volume and different concentrations. Perform injection analysis according to the chromatographic conditions of step (3), and draw standard curves for the four components of andrographolide, deoxyandrographolide, dehydroandrographolide, and andrographolide, with the peak area Y as the ordinate and the mass concentration X, μg / mL as the abscissa; (6) Methodological investigation using external standard method: a. Instrument precision test: Take the reference solution and place it in a volumetric flask, add 80% methanol water to dilute to the scale, shake well, and obtain the corresponding reference solution to be tested; accurately pipette 5 μL of each reference solution to be tested, repeat the injection 6 times according to the conditions of step (3), and calculate the relative standard deviation (RSD) of the peak area of ​​andrographolide, deoxyandrographolide, dehydroandrographolide, and andrographolide reference solution; b. Repeatability study: Take the same batch of Andrographis paniculata medicinal materials and prepare 6 test samples in parallel. Prepare the test sample solution according to step (1), and perform the test according to the conditions of step (3). Calculate the RSD value of the content of andrographolide, deoxyandrographolide, dehydroandrographolide, and andrographolide in each test sample solution; c. Recovery rate investigation: accurately weigh 9 portions of the test sample with the measured content, 0.05 g each, prepare the test sample solution according to step (1), divide it into 3 groups, 3 portions in each group, accurately add the same volume of reference stock solution to the 3 portions in each group, and increase the volume of reference stock solution added to each group. Perform the test according to the conditions of step (3), and calculate the average recovery rate and RSD; (7) Determine the internal reference and relative correction factor for the one-measurement-multiple-evaluation method: Select a component with a moderate peak area as the internal reference. In this method, andrographolide is selected as the internal reference; The relative correction factor calculation formula is: fs / i=fs / fi=(Wi×As) / (Ws×Ai), where: fs / i is the relative correction factor between the internal reference and other components; As is the peak area of ​​the internal reference; Ws is the concentration of the andrographolide internal reference; Ai is the peak area of ​​other components; Wi is the concentration of other components; the content of each component in the test sample is calculated as follows: Wi= fs / i×Ws×Ai / As, where Ai is the peak area of ​​the component to be tested, Wi is the concentration of the component to be tested, As is the peak area of ​​the internal reference, Ws is the concentration of the internal reference, and fs / i is the relative correction factor of the internal reference to the component to be tested; (8) Investigating the durability of the relative correction factor and the positioning of the chromatographic peak: In order to investigate the durability of the relative correction factor for instruments and chromatographic columns from different manufacturers, three chromatographic columns from different manufacturers and two instruments of different models were selected to investigate the durability of the correction factor; (9) Comparative verification of the content of the determined samples: Accurately weigh different batches of Andrographis paniculata samples, prepare the test sample solution according to step (1), and prepare three replicates for each batch. Perform sample injection analysis according to the conditions of step (3). The external standard method uses a linear regression equation to calculate the content, and the one-measurement multiple evaluation method uses the average value to calculate the content. After determination and analysis, there is no significant difference between the results of the external standard method and the one-measurement multiple evaluation method for the content calculation of the four components of andrographolide, deoxyandrographolide, dehydroandrographolide, and andrographolide, and the detection sensitivity of the two detection methods is consistent, indicating that there is no significant difference in the results of the four components in Andrographis paniculata detected by the external standard method and the one-measurement multiple evaluation method. This method is established to establish the quality control method of Andrographis paniculata using the one-measurement multiple evaluation method. The quality control of Andrographis paniculata can be carried out by determining the components of andrographolide, deoxyandrographolide, dehydroandrographolide, and andrographolide in Andrographis paniculata by the one-measurement multiple evaluation method.

2. The method for establishing quality control of Andrographis paniculata by using a one-test-multiple-evaluation method as claimed in claim 1, characterized in that: The chromatographic conditions and mass spectrometry conditions are: a. Chromatographic conditions: Chromatographic column: ACQUITY UPLC BEH C18 (50 mm×2.1 mm, 1.8 mm), column temperature of 35 ℃, mobile phase system: A: aqueous solution, B: methanol + acetonitrile (7+3); flow rate 0.3 mL / min; gradient elution conditions: 0~0.5 min, 45% A; 0.5~1 min, 45%~100% A; 1.0~3.0 min, 100% A; 3.0~5.0 min,45% A, injection volume 5 μL; b. Mass spectrometry conditions: ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry was performed in electrospray negative ionization mode (ESI-), multiple reaction monitoring (MRM) mode; Agilent 6460 HPLC-tandem mass spectrometer (Agilent Technologies, Inc., USA), nebulizer pressure: 30 PSI; dryer temperature: 300 °C; dryer flow rate: 8 L / min; capillary total voltage: 4000 V, collision gas was high-purity nitrogen, Shimadzu LCMS-8050 HPLC-tandem mass spectrometer, nebulizer flow rate 2 L / min, heater flow rate 10 L / min, interface temperature 250 °C, desolvation temperature 444 °C, DL temperature 200 °C, heating module temperature 200 °C, dryer flow rate 10 L / min, collision gas was high-purity argon.

3. A method for establishing quality control of Andrographis paniculata using a one-test-multiple-evaluation method as claimed in claim 2, characterized in that: The maximum deviation between the relative abundance ratio of the selected qualifier ion and the relative abundance ratio of the qualifier ion of the reference solution of equivalent concentration is: when the relative ion abundance is greater than 50, the allowable relative deviation is ±20%; when the relative ion abundance is greater than 20-50, the allowable relative deviation is ±25%; when the relative ion abundance is greater than 10-20, the allowable relative deviation is ±30%; when the relative ion abundance is ≤10, the allowable relative deviation is ±50%.

4. A method for quality control of Andrographis paniculata, characterized in that: The method comprises the following steps: (1) Preparation of test solution: Accurately weigh the homogenized sample of crushed Andrographis paniculata, place it in a volumetric flask, add 80% methanol to dilute to the mark, shake well, vortex mix, ultrasonically extract, centrifuge, take the supernatant and place it in another volumetric flask, add 80% methanol to dilute to the mark, shake well, vortex mix, take the supernatant and filter it through a 0.22 μm filter membrane into a sample injection vial to obtain the test solution; (2) Preparation of reference substance solution: Accurately weigh the reference substance andrographolide into a volumetric flask, dissolve it with 80% methanol and dilute it to the mark, shake well to obtain the reference substance stock solution; accurately weigh 1 mL of the reference substance stock solution into a 50 mL volumetric flask, dissolve it with 80% methanol and dilute it to the mark, shake well to obtain the reference substance solution; (3) Determine the chromatographic conditions and mass spectrometry conditions; a. Chromatographic conditions: Chromatographic column: ACQUITY UPLC BEH C18 (50 mm×2.1 mm, 1.8 mm), column temperature of 35 °C, mobile phase system: A: aqueous solution, B: methanol + acetonitrile (7+3); flow rate of 0.3 mL / min; gradient elution conditions: 0~0.5 min, 45% A; 0.5~1 min, 45%~100% A; 1.0~3.0min, 100% A; 3.0~5.0 min, 45% A, injection volume 5 μL; b. Mass spectrometry conditions: Ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry was performed in electrospray negative ionization mode (ESI-), multiple reaction monitoring (MRM) mode; Agilent 6460 high performance liquid chromatography-tandem mass spectrometer (Agilent Technologies, USA), nebulizer pressure: 30PSI; dryer temperature: 300℃; dryer flow rate: 8 L / min; capillary total voltage: 4000 V, collision gas was high purity nitrogen, Shimadzu LCMS-8050 high performance liquid chromatography-tandem mass spectrometer nebulizer flow rate 2 L / min, heater flow rate 10 L / min, interface temperature 250℃, desolvation temperature 444℃, DL temperature 200℃, heating module temperature 200℃, dryer flow rate 10 L / min, collision gas was high purity argon; (4) Determine the main components of the test sample: Place the test sample solution and the reference solution in an Agilent ultra-high performance liquid chromatography-triple quadrupole mass spectrometer under the same analytical conditions for determination. If the test sample solution shows chromatographic peaks of quantitative ion pairs and qualitative ion pairs, the retention time of the characteristic ion peak of the measured component is consistent with the retention time of the reference solution, and the relative abundance ratio of the selected qualitative ion does not exceed the maximum deviation of the relative abundance ratio of the qualitative ion of the reference solution of equivalent concentration, then the corresponding component can be determined to be present in the sample; the main components present in the test sample solution are determined; (5) Establishing a standard curve: Take 5 portions of reference solution with different volumes and increasing in volume, place them in volumetric flasks of the same volume, add 80% methanol water to dilute to the mark, shake well, and obtain 5 portions of reference solution with the same volume and different concentrations; perform injection analysis according to the chromatographic conditions of step (3), and draw a standard curve of andrographolide reference solution with peak area Y as the ordinate and mass concentration X, μg / mL as the abscissa; (6) Determine the calculation formula for the content of the test sample in the one-test-multiple-evaluation method: The internal reference substance in this method is andrographolide. The calculation formula for the content of each component in the test sample in this method is as follows: Wi = fs / i × Ws × Ai / As, where Ai is the peak area of ​​the component to be tested, Wi is the concentration of the component to be tested, As is the peak area of ​​the internal reference substance, Ws is the concentration of the internal reference substance, and fs / i is the relative correction factor of the internal reference substance to the component to be tested; (7) Comparative verification of sample content: Accurately weigh the Andrographis paniculata sample to be measured, prepare the test solution according to step (1), and prepare three parallel samples. Perform sample injection analysis according to the conditions of step (3) to measure the peak area of ​​the component to be measured. Fill the peak area of ​​the component to be measured, the peak area of ​​the internal reference substance, the concentration of the internal reference substance, and the relative correction factor of the internal reference substance to the component to be measured into the formula to obtain the concentration content of the component to be measured. This can be used to evaluate the quality of the Andrographis paniculata sample to be measured.

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