A method for determining the content of ingredients in Zishen Jianpi Huayu tablets

Abstract

The invention discloses a method for determining the content of components in Zishen Jianpi Huayu Tablets, which belongs to the technical field of drug detection, and comprises the following steps: step one, preparation of a test solution; step two, preparation of a reference solution; step three: determination of the content of each component in Zishen Jianpi Huayu Tablets by UPLC-IMS-Q-TOF-MS. The invention has the beneficial effects of providing a method for determining the content of multiple components of Zishen Jianpi Huayu Tablets based on UPLC-IMS-Q-TOF-MS, which is efficient and simple, has good repeatability, and can provide a reference basis for the material basis of Zishen Jianpi Huayu Tablets and comprehensive quality control.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, and in particular to a method for determining the content of components in a Zishen Jianpi Huayu tablet. Background Art

[0002] Zishen Jianpi Huayu Tablets are a traditional Chinese medicine compound preparation composed of 12 kinds of traditional Chinese medicines, including wine cornus fruit, astragalus, Panax notoginseng, vinegar frankincense, rhubarb, raw rehmannia root, radix rutaecarpa, cassia bark, kudzu vine, fried silkworm, millettia reticulata, and hawthorn. It has the effects of nourishing the kidney and spleen, nourishing yin and qi, and removing blood stasis and unblocking meridians. It is mainly used in the clinic to treat diabetic retinopathy. Modern research shows that the active ingredients in this traditional Chinese medicine compound preparation are diverse, mainly including saponins, flavonoids, terpenes, etc.

[0003] At present, the quality standard research of Zishen Jianpi Huayu Tablets is only the qualitative identification by thin layer chromatography and the quantitative research by liquid chromatography on its single ingredient puerarin, but no report on the quantitative analysis of other active ingredients has been found. Therefore, it is urgent to establish an analytical method to conduct rapid and accurate quantitative analysis of the active ingredients of Zishen Jianpi Huayu Tablets, in order to provide data support for the establishment of quality control methods and the improvement of quality standards of Zishen Jianpi Huayu Tablets. Summary of the invention

[0004] In order to overcome the problem that the quality standard research of Zishen Jianpi Huayu Tablets in the prior art is only for qualitative identification by thin layer chromatography and quantitative research on its single component puerarin by liquid chromatography, while no quantitative analysis of other active ingredients has been reported, the present invention provides a method for determining the content of ingredients in Zishen Jianpi Huayu Tablets, comprising the following steps:

[0005] Step 1, preparation of the test solution, dissolving the Zishen Jianpi Huayu tablets in 75% methanol and subjecting the mixture to ultrasonic mixing, centrifugation and microporous membrane filtration to obtain the test solution;

[0006] Step 2, preparing a reference solution, dissolving liquiritigenin, astragaloside IV, aloe-emodin, chlorogenic acid, rhein, rehmannia glutinosa D, calycosin glucoside, ginsenoside Re, notoginseng saponin R1, loganin, 11-oxo-β-boswellic acid, puerarin, morroniside, ginsenoside Rg1, and ginsenoside Rb1 as reference substances in 75% methanol to obtain reference solutions and prepare a mixed reference solution;

[0007] Step 3: Use UPLC-IMS-Q-TOF-MS to measure the chromatograms of the reference solution and the test solution under the same detection conditions, and calculate the content of the components in the test solution of Zishen Jianpi Huayu Tablets corresponding to the reference solution based on the concentration of the reference solution, the peak area of ​​the reference solution in the chromatogram, and the peak area of ​​the components in the test solution corresponding to the reference solution in the chromatogram.

[0008] Preferably, the ultrasonic mixing time is 30-60 min, the pore size of the microporous filter membrane is 0.22 μm, and the concentration of the test solution is 0.5 mg / mL.

[0009] Preferably, the concentration of the reference solution of each component is 1 mg / mL, and the reference solution of each component with a concentration of 1 mg / mL is used to prepare a mixed reference solution.

[0010] Preferably, the chromatographic conditions of UPLC-IMS-Q-TOF-MS include: chromatographic column: Waters ACQUITYUPLC BEHC18 (2.1 mm×100 mm, 1.7 μm); mobile phase: A is 0.1% formic acid aqueous solution; B is acetonitrile; elution gradient: 0-0.5 min, 5-5% B phase; 0.5-1.0 min, 2-40% B phase; 1.0-4.5 min, 40-90% B phase; 4.5-6.0 min, 90-90% B phase; 6.0-6.1 min, 90-5% B phase; 6.1-8.0 min, 5-5% B phase; flow rate: 0.4 mL / min; column temperature: 40°C; injector temperature: 8°C; injection volume: 2 μL.

[0011] Preferably, the mass spectrometry conditions of UPLC-IMS-Q-TOF-MS include: using an electrospray ion source, a negative ion mode scanning mode, using leucine enkephalin as a calibration solution, performing real-time calibration, a negative ion m / z of 554.2620, and MSE full-time full-information data acquisition;

[0012] Mass spectrometry parameter settings: capillary voltage is 0.8 kV; cone voltage is 30 V; capillary temperature is 120 °C; auxiliary gas heating temperature is 450 °C; cone gas flow rate is 50 L / h; desolvation gas flow rate is 900 L / h; primary collision energy is 6 eV; secondary collision energy is 20-45 eV; scanning range is 50-1500 m / z.

[0013] Preferably, step three includes a standard curve and a linear relationship investigation: the mixed reference solution is gradiently diluted to 1.25, 1.67, 2, 2.5, 3.33, 5, 10, 20, 40, 80, and 160 times, and 2-5 μL is accurately transferred for UPLC-IMS-Q-TOF-MS detection, and the concentration of the mixed reference solution after the gradient dilution is used as the abscissa (x), and the integral value of the chromatographic peak area is used as the ordinate (y). A standard curve is drawn, and the regression equation and linear range of each reference substance are calculated.

[0014] Preferably, step three includes a precision test: taking the same sample solution and injecting it continuously for 6 times under the same detection conditions, measuring the peak areas of 15 reference substances and calculating the RSD value of the peak area.

[0015] Preferably, step three includes a stability test: taking a portion of the test solution, placing it at room temperature for 0, 2, 4, 8, 12, and 24 hours, injecting and testing it under the same test conditions, recording the peak areas of 15 reference substances, and calculating the RSD value of the peak area.

[0016] Preferably, step three includes a repeatability test: taking 6 samples of the same batch number of Zishen Jianpi Huayu Tablets to prepare the test solution in parallel, injecting and testing under the same detection conditions, recording the peak areas of 15 reference substances, and calculating the RSD value of the peak area.

[0017] Preferably, step three includes a sample recovery test: 6 samples of the same batch number of Zishen Jianpi Huayu Tablets as the repeatability test are taken to prepare a test solution with a concentration of 0.5 mg / mL, and each reference solution is added at a ratio of 1:1 and then injected and tested under the same test conditions, and the peak area of ​​each component is recorded and calculated according to To calculate the recovery rate.

[0018] Beneficial effects:

[0019] The beneficial effects produced by the technical solution of the present invention are as follows:

[0020] The invention adopts ultra high performance liquid chromatography-ion mobility-quadrupole time-of-flight mass spectrometry (UPLC-IMS-Q-TOF-MS) to establish a quantitative analysis method for identifying 15 active ingredients of Zishen Jianpi Huayu Tablets, and calculates the contents of the 15 active ingredients in Zishen Jianpi Huayu Tablets. The determination method is efficient and simple, has good repeatability, and can provide a reference basis for the pharmacological material basis and comprehensive quality control of Zishen Jianpi Huayu Tablets. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the present invention will be clearly and completely described in conjunction with the embodiments below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Therefore, the following detailed description of the embodiments of the present invention provided is not intended to limit the scope of the claimed invention, but merely represents the selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0022] This embodiment uses ultra-high performance liquid chromatography-ion mobility-quadrupole time-of-flight mass spectrometry (UPLC-IMS-Q-TOF-MS) to establish a quantitative analysis method for the 15 active ingredients of Zishen Jianpi Huayu Tablets. The specific implementation method is as follows:

[0023] A method for determining the content of ingredients in a Zishen Jianpi Huayu tablet comprises the following steps:

[0024] Step 1, preparation of the test solution, dissolving the Zishen Jianpi Huayu tablets in 75% methanol and subjecting the mixture to ultrasonic mixing, centrifugation and microporous membrane filtration to obtain the test solution;

[0025] Step 2, preparing a reference solution, dissolving liquiritigenin, astragaloside IV, aloe-emodin, chlorogenic acid, rhein, rehmannia glutinosa D, calycosin glucoside, ginsenoside Re, notoginseng saponin R1, loganin, 11-oxo-β-boswellic acid, puerarin, morroniside, ginsenoside Rg1, and ginsenoside Rb1 as reference substances in 75% methanol to obtain reference solutions and prepare a mixed reference solution;

[0026] Step 3: Use UPLC-IMS-Q-TOF-MS to measure the chromatograms of the reference solution and the test solution under the same detection conditions, and calculate the content of the components in the test solution of Zishen Jianpi Huayu Tablets corresponding to the reference solution based on the concentration of the reference solution, the peak area of ​​the reference solution in the chromatogram, and the peak area of ​​the components in the test solution corresponding to the reference solution in the chromatogram.

[0027] Specifically, the ultrasonic mixing time is 30-60 min, the pore size of the microporous filter membrane is 0.22 μm, and the concentration of the test solution is 0.5 mg / mL.

[0028] Specifically, the concentration of the reference solution of each component is 1 mg / mL, and the reference solution of each component with a concentration of 1 mg / mL is used to prepare a mixed reference solution.

[0029] Specifically, the chromatographic conditions of UPLC-IMS-Q-TOF-MS include: chromatographic column: Waters ACQUITYUPLC BEHC18 (2.1 mm×100 mm, 1.7 μm); mobile phase: A is 0.1% formic acid aqueous solution; B is acetonitrile; elution gradient: 0-0.5 min, 5-5% B phase; 0.5-1.0 min, 2-40% B phase; 1.0-4.5 min, 40-90% B phase; 4.5-6.0 min, 90-90% B phase; 6.0-6.1 min, 90-5% B phase; 6.1-8.0 min, 5-5% B phase; flow rate: 0.4 mL / min; column temperature: 40°C; injector temperature: 8°C; injection volume: 2 μL.

[0030] Specifically, the mass spectrometry conditions of UPLC-IMS-Q-TOF-MS include: using an electrospray ion source, a negative ion mode scanning mode, using leucine enkephalin as a calibration solution, performing real-time calibration, a negative ion m / z of 554.2620, and MSE full-time full-information data acquisition;

[0031] Mass spectrometry parameter settings: capillary voltage is 0.8 kV; cone voltage is 30 V; capillary temperature is 120 °C; auxiliary gas heating temperature is 450 °C; cone gas flow rate is 50 L / h; desolvation gas flow rate is 900 L / h; primary collision energy is 6 eV; secondary collision energy is 20-45 eV; scanning range is 50-1500 m / z.

[0032] Specifically, step three includes the investigation of the standard curve and the linear relationship: the mixed reference solution is gradiently diluted to 1.25, 1.67, 2, 2.5, 3.33, 5, 10, 20, 40, 80, and 160 times, and 2-5 μL is accurately transferred for UPLC-IMS-Q-TOF-MS detection. The concentration of the mixed reference solution after the gradient dilution is used as the horizontal axis (x), and the integral value of the chromatographic peak area is used as the vertical axis (y). The standard curve is drawn, and the regression equation and linear range of each reference substance are calculated.

[0033] Specifically, step three includes a precision test: taking the same sample solution and injecting it continuously for 6 times under the same detection conditions, measuring the peak areas of 15 reference substances and calculating the RSD values ​​of the peak areas.

[0034] Specifically, step three includes a stability test: taking a portion of the test solution, placing it at room temperature for 0, 2, 4, 8, 12, and 24 hours, injecting and testing it under the same test conditions, recording the peak areas of 15 reference substances, and calculating the RSD value of the peak area.

[0035] Specifically, step three includes a repeatability test: 6 samples of the same batch number of Zishen Jianpi Huayu Tablets are taken to prepare the test solution in parallel, the samples are injected and tested under the same detection conditions, the peak areas of 15 reference substances are recorded, and the RSD value of the peak area is calculated.

[0036] Specifically, step three includes a sample recovery test: 6 samples of the same batch number of Zishen Jianpi Huayu Tablets as the repeatability test are taken to prepare a test solution with a concentration of 0.5 mg / mL, and each reference solution is added at a ratio of 1:1 to prepare a sample recovery test solution. The sample is injected, tested and analyzed under the same test conditions. To calculate the recovery rate.

[0037] The method for determining the content of chemical components in the Zishen Jianpi Huayu Tablets in this embodiment is further reviewed below through examples.

[0038] Example 1

[0039] 1. Instrument

[0040] UPLC ultra-high performance liquid chromatograph (Waters Corporation, Milford, MA, USA); VionTM IMS-QTOF ion mobility quadrupole time-of-flight mass spectrometer (Waters Corporation, Milford, MA, USA); AS-20500AT ultrasonic cleaner (Tianjin Aotesense Instrument Co., Ltd., Tianjin, China); Quintix35-1CN one-hundred-thousandth balance (Sartorius Scientific Instrument (Beijing) Co., Ltd., Beijing, China); BCE224-1CCN one-ten-thousandth balance (Sartorius Scientific Instrument (Beijing) Co., Ltd., Beijing, China); Vortex Genius3 vortex mixer (Aika (Guangzhou) Instrument Equipment Co., Ltd., Guangzhou, China); ST8R high-speed refrigerated centrifuge (Thermo Fisher Scientific, Waltham, MA,, USA).

[0041] 2. Drug testing

[0042] Acetonitrile and formic acid (Merck KGaA, Darmstadt, Germany) were all chromatography / mass spectrometry grade; deionized water was IQ 7000 system (Merck KGaA, Darmstadt, Germany) was used for purification. Six different batches of Zishen Jianpi Huayu Tablets were produced and provided by the First Affiliated Hospital of Guangzhou University of Chinese Medicine (batch numbers: 20210401, 20220701, 20230401, 20230901, 20240401, 20240801). The names, molecular formulas, and manufacturers of the 15 reference substances are shown in Table 1.

[0043] Table 1 Reference material information

[0044]

[0045]

[0046]

[0047] 3. Preparation of test solution

[0048] After grinding the Zishen Jianpi Huayu Tablets into powder, weigh 1g and dissolve it in 10mL of 75% methanol aqueous solution. After 30min of ultrasonic mixing, take 2mL of the Zishen Jianpi Huayu Tablets solution for centrifugation. Take 1mL of the supernatant after centrifugation and filter it through a 0.22μm microporous filter membrane to obtain an extraction solution with a concentration of 100mg / mL. Dilute the 100mg / mL extraction solution with 75% methanol aqueous solution to obtain a 0.5mg / mL test solution. The test solution is stored in an environment of 2-8°C before use.

[0049] 4. Preparation of reference solution

[0050] Weigh 2 mg of glycyrrhizin, astragaloside IV, aloe-emodin, chlorogenic acid, rhein, rehmannia glutinosa D, calycosin glucoside, ginsenoside Re, notoginseng saponin R1, loganin, 11-oxo-β-boswellic acid, puerarin, morroniside, ginsenoside Rg1, and ginsenoside Rb1 as reference substances, and dissolve them in 2 ml of 75% methanol aqueous solution to obtain reference substance solutions of each component with a concentration of 1 mg / mL, and use the reference substance solutions of each component with a concentration of 1 mg / mL to prepare a mixed reference substance solution, and each control group solution is stored at 2-8°C before use.

[0051] 5. Sample testing

[0052] The chromatograms of the reference solution and the test solution were measured respectively by UPLC-IMS-Q-TOF-MS under the same detection conditions.

[0053] Chromatographic conditions: Chromatographic column: Waters ACQUITY UPLC BEH C18 (2.1 mm×100 mm, 1.7 μm); mobile phase: A is 0.1% formic acid aqueous solution; B is acetonitrile; elution gradient: 0-0.5 min, 5-5% B phase; 0.5-1.0 min, 2-40% B phase; 1.0-4.5 min, 40-90% B phase; 4.5-6.0 min, 90-90% B phase; 6.0-6.1 min, 90-5% B phase; 6.1-8.0 min, 5-5% B phase; flow rate: 0.4 mL / min; column temperature: 40°C; injector temperature: 8°C; injection volume: 2 μL.

[0054] The mass spectrometry conditions included: using an electrospray ion source, a negative ion mode scanning mode, using leucine enkephalin as a calibration solution, real-time calibration, a negative ion m / z of 554.2620, and MSE full-time full-information data acquisition;

[0055] Mass spectrometry parameter settings: capillary voltage is 0.8 kV; cone voltage is 30 V; capillary temperature is 120 °C; auxiliary gas heating temperature is 450 °C; cone gas flow rate is 50 L / h; desolvation gas flow rate is 900 L / h; primary collision energy is 6 eV; secondary collision energy is 20-45 eV; scanning range is 50-1500 m / z.

[0056] 6. Methodological validation and results

[0057] (1) Standard curve and linear relationship investigation

[0058] The mixed reference solution was diluted to 1.25, 1.67, 2, 2.5, 3.33, 5, 10, 20, 40, 80, 160 times and 2-5 μL was accurately pipetted for UPLC-IMS-Q-TOF-MS detection. The concentration of the mixed reference solution after gradient dilution was used as the abscissa (x) and the integral value of the chromatographic peak area was used as the ordinate (y). The standard curve was drawn and the regression equation and linear range of each reference substance were calculated. The detection limit and quantification limit were determined at the signal-to-noise ratio S / N=3 and S / N=10, respectively. The results are shown in Table 2.

[0059] Table 2 Regression equation, correlation coefficient, linear range, detection limit and quantification limit of each compound

[0060]

[0061]

[0062] (2) Precision test

[0063] The same sample solution was injected 6 times under the same detection conditions, the peak areas of 15 reference substances were measured and the RSD values ​​of the peak areas were calculated. The results are shown in Table 3. The RSD values ​​of the peak areas of the 15 components were in the range of 2.11% to 4.93%, indicating that the instrument had good precision.

[0064] Table 3 Precision test results of each compound

[0065]

[0066]

[0067] (3) Stability test

[0068] Take a portion of the test solution, place it at room temperature for 0, 2, 4, 8, 12, and 24 hours, then inject and test it under the same test conditions, record the peak areas of 15 reference substances, and calculate the RSD values ​​of the peak areas. As shown in Table 4, the RSD values ​​of the peak areas of the 15 components are 2.34% to 4.70%, indicating that the test solution is stable within 24 hours.

[0069] Table 4 Stability test results of each compound

[0070]

[0071]

[0072] (4) Repeatability test

[0073] Take 6 samples of Zishen Jianpi Huayu Tablets of the same batch number and prepare the test solution according to “3. Preparation of test solution”. Sampling and detection are carried out under the same detection conditions. The peak areas of 15 reference substances are recorded, and the RSD values ​​of the peak areas are calculated. The peak area RSD values ​​of each target compound are shown in Table 5. The RSD values ​​of the 15 compounds are all less than 5%, indicating that the extraction method and the detection method have good repeatability.

[0074] Table 5 Repeatability test results of each compound

[0075]

[0076]

[0077] (5) Sample recovery test

[0078] Take 6 samples of Zishen Jianpi Huayu Tablets with the same batch number as the repeatability test to prepare a test solution with a concentration of 0.5 mg / mL, and add each reference solution in a ratio of 1:1 to prepare a sample recovery test solution. The recovery rate was calculated, and the results are shown in Table 6. The average recovery rates of the 15 compounds were between 93.06% and 103.04%, and the RSD values ​​of the recovery rates were all less than 4.91%, indicating that the method had good accuracy.

[0079] Table 6 Sample recovery test results of each compound

[0080]

[0081]

[0082]

[0083] (6) Content determination results

[0084] According to the above verified method, the contents of 15 components in 6 different batches of Zishen Jianpi Huayu Tablets were determined, as shown in Table 7.

[0085] Table 7 Assay results

[0086]

[0087]

[0088] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for determining the content of ingredients in Zishen Jianpi Huayu Tablets, characterized in that: The following steps are involved: Step 1, preparation of a test solution, dissolving the Zishen Jianpi Huayu Tablets in a solvent and subjecting the mixture to ultrasonic mixing, centrifugation and microporous membrane filtration to obtain the test solution; Step 2, preparing a reference solution, dissolving glycyrrhizin, astragaloside IV, aloe-emodin, chlorogenic acid, rhein, rehmannia glutinosa D, calycosin glucoside, ginsenoside Re, notoginseng saponin R1, loganin, 11-oxo-β-boswellic acid, puerarin, morroniside, ginsenoside Rg1, and ginsenoside Rb1 as reference substances in a solvent to obtain the reference solution and prepare a mixed reference solution; Step 3: Use UPLC-IMS-Q-TOF-MS to respectively measure the chromatograms of the reference solution and the test solution under the same detection conditions, and calculate the content of the components corresponding to the reference solution in the test solution of Zishen Jianpi Huayu Tablets according to the concentration of the reference solution, the peak area of ​​the reference solution in the chromatogram, and the peak area of ​​the components corresponding to the reference solution in the test solution in the chromatogram.

2. A method for determining the content of chemical components in a Zishen Jianpi Huayu tablet according to claim 1, characterized in that: The ultrasonic mixing time is 30-60 min, the pore size of the microporous filter membrane is 0.22 μm, and the concentration of the test solution is 0.5 mg / mL.

3. A method for determining the content of chemical components in a Zishen Jianpi Huayu tablet according to claim 1, characterized in that: The concentration of the reference solution of each component is 1 mg / mL, and the reference solution of each component with a concentration of 1 mg / mL is used to prepare the mixed reference solution.

4. A method for determining the content of chemical components in a Zishen Jianpi Huayu tablet according to claim 1, characterized in that: The chromatographic conditions of UPLC-IMS-Q-TOF-MS include: chromatographic column: Waters ACQUITYUPLC BEH C18 (2.1 mm×100 mm, 1.7 μm); mobile phase: A is 0.1% formic acid aqueous solution; B is acetonitrile; elution gradient: 0-0.5 min, 5-5% B phase; 0.5-1.0 min, 5-40% B phase; 1.0-4.5 min, 40-90% B phase; 4.5-6.0 min, 90-90% B phase; 6.0-6.1 min, 90-5% B phase; 6.1-8.0 min, 5-5% B phase; flow rate: 0.4 mL / min; column temperature: 40°C; injector temperature: 8°C; injection volume: 2 μL.

5. A method for determining the content of chemical components in Zishen Jianpi Huayu Tablets according to claim 1, characterized in that: The mass spectrometry conditions of UPLC-IMS-Q-TOF-MS included: using an electrospray ion source, a negative ion mode scanning mode, using leucine enkephalin as a calibration solution, real-time calibration, a negative ion m / z of 554.2620, and MSE full-time full-information data acquisition; Mass spectrometry parameter settings: capillary voltage is 0.8 kV; cone voltage is 30 V; capillary temperature is 120 °C; auxiliary gas heating temperature is 450 °C; cone gas flow rate is 50 L / h; desolvation gas flow rate is 900 L / h; primary collision energy is 6 eV; secondary collision energy is 20-45 eV; scanning range is 50-1500 m / z.

6. A method for determining the content of chemical components in Zishen Jianpi Huayu Tablets according to claim 1, characterized in that: The step three includes a standard curve and a linear relationship investigation: the mixed reference solution is gradiently diluted to 1.25, 1.67, 2, 2.5, 3.33, 5, 10, 20, 40, 80, and 160 times, and 2-5 μL is precisely transferred for UPLC-IMS-Q-TOF-MS detection, and the concentration of the mixed reference solution after the gradient dilution is used as the horizontal axis (x), and the integral value of the chromatographic peak area is used as the vertical axis (y). A standard curve is drawn, and the regression equation and linear range of each reference substance are calculated.

7. A method for determining the content of chemical components in Zishen Jianpi Huayu Tablets according to claim 1, characterized in that: The step three includes a precision test: taking the same portion of the test solution and injecting it continuously for 6 times under the same detection conditions, measuring the peak areas of 15 of the reference substances and calculating the RSD value of the peak area.

8. A method for determining the content of chemical components in Zishen Jianpi Huayu Tablets according to claim 1, characterized in that: The step three includes a stability test: taking a portion of the test solution, placing it at room temperature for 0, 2, 4, 8, 12, and 24 hours, injecting and testing it under the same test conditions, recording the peak areas of 15 of the reference substances, and calculating the RSD value of the peak area.

9. A method for determining the content of chemical components in Zishen Jianpi Huayu Tablets according to claim 1, characterized in that: The step three includes a repeatability test: taking 6 samples of the same batch number of Zishen Jianpi Huayu Tablets to prepare the test solution in parallel, injecting and testing under the same detection conditions, recording the peak areas of 15 of the reference substances, and calculating the RSD value of the peak area.

10. A method for determining the content of chemical components in Zishen Jianpi Huayu Tablets according to claim 9, characterized in that: The step three includes a sample recovery test: 6 samples of the same batch number of the Zishen Jianpi Huayu Tablets as the repeatability test are taken to prepare a test solution with a concentration of 0.5 mg / mL, and each of the reference solutions is added at a ratio of 1:1, and then sampled and tested under the same test conditions, and the peak area of ​​each component is recorded and calculated according to the test results. To calculate the recovery rate.

Images