Double-standard HPLC (High Performance Liquid Chromatography) quality control method for total flavonoids and rutin of

Abstract

The invention discloses a double-standard HPLC (High Performance Liquid Chromatography) quality control method for total flavonoids and rutin of polygonum capitatum, which relates to the technical field of quality control of traditional Chinese medicines and natural medicines, and comprises the following steps: preparing a mixed solution of a polygonum capitatum sample to be detected and a rutin standard substance, synchronously injecting and analyzing in single chromatographic operation, and carrying out gradient elution by adopting a C18 chromatographic column. Establishing a standard curve according to a rutin standard substance, and calculating the total flavonoid content based on rutin; and comparing the retention time of the sample characteristic peak with a preset fingerprint spectrum, confirming the peak attribution, distinguishing a rutin peak from other flavone peaks, summarizing the rutin peak area, substituting the rutin peak area into the standard curve again, and calculating the actual content of the rutin monomer. According to the method, two independent results of the total flavone converted content and the rutin monomer absolute content are synchronously and accurately obtained in one-time analysis, and the analysis efficiency and the specificity of the results are improved.

Description

Technical Field

[0001] This invention belongs to the field of quality control technology for traditional Chinese medicine and natural drugs, specifically a dual-standard HPLC quality control method for total flavonoids and rutin from Polygonum cuspidatum. Background Technology

[0002] In the field of quality analysis of traditional Chinese medicine and natural products, high-performance liquid chromatography (HPLC) is commonly used to determine the content of specific components. For multi-component samples such as total flavonoids from *Polygonum cuspidatum*, conventional methods typically use rutin as a reference standard, determining its content through the external standard method and converting it to total flavonoid content. This method only provides total amount information based on the reference standard and cannot reflect the true content of rutin monomers in the sample. To obtain the total flavonoid and monomer contents separately, two independent sample analysis and data processing procedures are required, which are cumbersome and inefficient.

[0003] Current techniques typically analyze sample and reference solutions separately. These two analyses are performed at different times and under different chromatographic conditions. Fluctuations in instrument status, environment, and mobile phase can cause shifts in retention time and peak response. This asynchronous data acquisition method introduces systematic errors, reduces the comparability of sample and reference data, and affects the accuracy of quantitative results.

[0004] Conventional calculation methods sum the peak areas of all flavonoid components in the sample chromatogram and substitute them into the rutin standard curve to calculate the total flavonoid content. This approach ignores the differences in response factors among different flavonoid components, leading to theoretical deviations in the total calculation results. Furthermore, this method lacks effective means to identify and separate the characteristic peaks of rutin from mixed chromatographic signals, making it impossible to accurately calculate the true content of rutin itself while obtaining the converted total flavonoid content. Current technologies struggle to achieve simultaneous and accurate dual quantification of total flavonoids and specific monomeric components in a single analysis. Summary of the Invention

[0005] This invention aims to solve at least one of the technical problems existing in the prior art;

[0006] Therefore, this invention proposes a dual-standard HPLC quality control method for total flavonoids and rutin from Polygonum tinctorium, comprising:

[0007] Prepare a mixed solution containing the total flavonoids of Polygonum hydropiper and rutin standard;

[0008] The analytical conditions for high performance liquid chromatography were set, using a C18 column and acetonitrile and phosphoric acid aqueous solution as the mobile phase, and gradient elution was performed.

[0009] The mixed solution was injected into the high performance liquid chromatograph, the chromatographic separation program was started, and the chromatographic elution signals of the test sample of total flavonoids of Polygonum tinctorium and rutin standard were recorded simultaneously.

[0010] The retention time and peak area data of the characteristic peaks of total flavonoids from Polygonum tinctorium and the characteristic peaks of rutin standard were extracted from the chromatographic elution signal.

[0011] Based on the peak area data of the characteristic peaks of the rutin standard, a standard curve for rutin content was established;

[0012] Substitute the peak area data of the characteristic peak of total flavonoids in Polygonum hydropiper into the standard curve to calculate the total flavonoid content of Polygonum hydropiper, calculated as rutin.

[0013] Meanwhile, the retention time of the characteristic peaks of total flavonoids from Polygonum hydropiper is matched with the preset fingerprint spectrum of total flavonoids from Polygonum hydropiper to confirm the assignment of the characteristic peaks.

[0014] Based on the results of the confirmation of the characteristic peak attribution, the characteristic peaks of the total flavonoids of the Polygonum tinctorium are identified, and the peaks belonging to rutin are distinguished from the peaks of other flavonoid components.

[0015] Summarize the peak area data of the peaks attributed to rutin, and substitute them back into the standard curve to calculate the actual content of rutin monomer in the sample.

[0016] Finally, the results of total flavonoid content of Polygonum tinctorium calculated as rutin and the actual content of rutin monomers in the sample were output in parallel to complete the dual-standard quality control.

[0017] Furthermore, the preparation of the mixed solution containing the test sample of total flavonoids from Polygonum hydropiper and rutin standard specifically includes:

[0018] Accurately weigh the dried and pulverized Polygonum tinctorium plant material, add a 60% (v / v) methanol aqueous solution, and perform ultrasonic-assisted extraction.

[0019] After extraction, the solution was cooled and filtered, and the crude extract of total flavonoids from Polygonum tinctorium was collected.

[0020] The crude extract of total flavonoids from Polygonum hydropiper was concentrated under reduced pressure to obtain a extract of total flavonoids from Polygonum hydropiper.

[0021] Accurately weigh the extract of total flavonoids from Polygonum hydropiper, dissolve it in methanol and make up to volume to prepare a sample solution of total flavonoids from Polygonum hydropiper to be tested;

[0022] In addition, accurately weigh the rutin standard that has been identified as having purity, dissolve it in methanol and dilute to volume to prepare a rutin standard stock solution;

[0023] The sample solution containing total flavonoids from Polygonum hydropiper was mixed with the rutin standard stock solution in a predetermined volume ratio to obtain the mixed solution.

[0024] Furthermore, the analytical conditions for setting the high-performance liquid chromatography (HPLC) are as follows: using a C18 column, with acetonitrile and phosphoric acid aqueous solution as the mobile phase, and performing gradient elution, specifically including:

[0025] A C18 reversed-phase column with a particle size of 5 micrometers, a column length of 250 mm, and an inner diameter of 4.6 mm was selected.

[0026] The column oven temperature of the C18 reversed-phase chromatographic column is set to 30 degrees Celsius;

[0027] Mobile phase A was prepared as 1% (v / v) aqueous phosphoric acid solution, and mobile phase B was prepared as chromatographic grade acetonitrile.

[0028] The flow rate was set to 1 ml per minute, the detection wavelength to 360 nm, and the injection volume to 10 μL.

[0029] Establish a gradient elution program: from 0 to 5 minutes, mobile phase B is linearly changed from 15% to 20%; from 5 to 20 minutes, mobile phase B is linearly changed from 20% to 35%; from 20 to 25 minutes, mobile phase B is maintained at 35%; from 25 to 26 minutes, mobile phase B is linearly changed from 35% to 15%; from 26 to 30 minutes, mobile phase B is maintained at 15% for column equilibration.

[0030] Further, the step of injecting the mixed solution into the high-performance liquid chromatograph, starting the chromatographic separation program, and simultaneously recording the chromatographic elution signals of the total flavonoids of Polygonum tinctorium sample and the rutin standard specifically includes:

[0031] Accurately aspirate 10 μL of the mixture using a microsyringe and inject it into the system via the autosampler or manual injection valve of the high-performance liquid chromatograph.

[0032] The gradient elution program is initiated, and the mixed solution enters the C18 reversed-phase chromatographic column along with the mobile phase for separation.

[0033] At the detection wavelength of 360 nm, the light absorption intensity of the chromatographic column effluent was continuously monitored using an ultraviolet detector;

[0034] The ultraviolet detector converts the light absorption intensity signal into an electrical signal, which is collected and recorded by the chromatography workstation as a chromatogram that changes over time.

[0035] The chromatographic elution signal is the complete chromatogram data, which includes the time axis and the response intensity axis.

[0036] Furthermore, the extraction of the characteristic peaks of total flavonoids from *Polygonum hydropiper* and the characteristic peaks of rutin standard from the chromatographic elution signal specifically includes:

[0037] Open the recorded chromatogram in the chromatography workstation;

[0038] Baseline correction and smoothing are performed on the chromatograms to identify all chromatographic peaks that reach the preset signal-to-noise ratio;

[0039] Based on the retention time of the pure rutin standard obtained from pre-injection determination, the characteristic peak of the rutin standard is located in the chromatogram of the mixed solution;

[0040] Based on the known chromatographic behavior of total flavonoids from Polygonum hydropiper, other characteristic chromatographic peak groups belonging to total flavonoids from Polygonum hydropiper are identified within a certain time window before and after the characteristic peak of the rutin standard.

[0041] The exact retention time and peak area values ​​of each characteristic peak in the chromatographic peak group of the rutin standard and the total flavonoids of Polygonum tinctorium were obtained by integrating the values ​​of the chromatographic workstation.

[0042] Furthermore, the step of establishing a standard curve for rutin content based on the peak area data of the characteristic peaks of the rutin standard specifically includes:

[0043] Using the aforementioned rutin standard stock solution, prepare at least five rutin standard working solutions with different concentration gradients;

[0044] Each concentration of rutin standard working solution was injected into the high-performance liquid chromatograph and analyzed under the analytical conditions.

[0045] Record the peak area of ​​rutin corresponding to each concentration of rutin standard working solution;

[0046] A scatter plot was drawn with the concentration of the rutin standard on the x-axis and the corresponding rutin chromatographic peak area on the y-axis.

[0047] The least squares method was used to perform linear regression fitting on the scatter points to obtain a linear equation relating rutin content and peak area. This linear equation is the standard curve, and its correlation coefficient was recorded.

[0048] Further, the step of substituting the peak area data of the characteristic peak of total flavonoids in *Polygonum hydropiper* into the standard curve to calculate the total flavonoid content of *Polygonum hydropiper* calculated as rutin specifically includes:

[0049] The total peak area of ​​total flavonoids in the total chromatographic peak group of the above-mentioned total flavonoids in Polygonum tinctorium is obtained by summing up the peak area data of all characteristic peaks.

[0050] Substitute the total peak area value of the total flavonoids of the Polygonum hydropiper into the linear equation corresponding to the standard curve;

[0051] Based on the linear equation, the rutin equivalent concentration corresponding to the total peak area of ​​the total flavonoids in the Polygonum hydropiper was calculated in reverse.

[0052] Based on the dilution factor and sample weight during the preparation of the total flavonoid test sample solution of Polygonum tinctorium, the percentage of total flavonoids (calculated as rutin) in the original Polygonum tinctorium plant raw material was calculated.

[0053] Furthermore, simultaneously, the retention times of the characteristic peaks of total flavonoids from *Polygonum hydropiper* are matched with a preset fingerprint spectrum of total flavonoids from *Polygonum hydropiper* to confirm the attribution of the characteristic peaks, specifically including:

[0054] The pre-established standard fingerprint spectrum of total flavonoids from Polygonum hydropiper was called, which was derived from the chromatographic analysis results of multiple batches of qualified Polygonum hydropiper extracts.

[0055] The retention time of each peak in the characteristic chromatographic peak group of total flavonoids from Polygonum hydropiper is compared one by one with the retention time of all characteristic peaks in the standard fingerprint spectrum of total flavonoids from Polygonum hydropiper.

[0056] Calculate the relative difference between the current chromatographic peak retention time and the reference peak retention time in the standard fingerprint chromatogram;

[0057] If the relative difference is within the preset allowable deviation range, the current chromatographic peak is determined to be consistent with the reference peak in the standard fingerprint spectrum.

[0058] The attribution of all characteristic peaks of total flavonoids from Polygonum hydropiper was determined, and a confirmation list of characteristic peak attributions was generated.

[0059] Furthermore, based on the confirmation of the characteristic peak attribution, the characteristic peaks of the total flavonoids in *Polygonum hydropiper* are identified, distinguishing the peaks belonging to rutin from those belonging to other flavonoid components, specifically including:

[0060] Consult the characteristic peak attribution confirmation list and find the characteristic peaks that are confirmed to belong to rutin.

[0061] Locate the characteristic peaks belonging to rutin in the chromatogram and read their peak area data, recording them as the peak area of ​​rutin monomers;

[0062] All characteristic peaks in the total flavonoids characteristic chromatographic peak group of the above-mentioned Polygonum hydropiper, except for the characteristic peaks belonging to rutin, are classified as other flavonoid component peaks;

[0063] The peak area data of the other flavonoid components are summed to obtain the total peak area of ​​the other flavonoid components;

[0064] The peak area of ​​the rutin monomer and the total peak area of ​​the other flavonoid components together constitute the detailed composition information of the total flavonoids of Polygonum tinctorium.

[0065] Furthermore, the peak area data of the peaks attributed to rutin are summarized and substituted back into the standard curve to calculate the actual content of rutin monomers in the sample, specifically including:

[0066] Use the peak area data of the rutin monomers read from the chromatogram;

[0067] Substitute the peak area value of the rutin monomer into the linear equation corresponding to the standard curve;

[0068] The concentration of rutin monomer in the mixed solution was calculated based on the linear equation.

[0069] By combining the mixing ratio of the sample solution of total flavonoids from Polygonum hydropiper to the rutin standard stock solution during the preparation of the mixed solution, the actual concentration of rutin monomer in the original sample solution of total flavonoids from Polygonum hydropiper can be deduced.

[0070] By further combining the dissolution volume of the extract and the sample weight of the raw material, the percentage content of rutin monomer in the original Polygonum tinctorium plant raw material was calculated, and the percentage content of rutin monomer was output in parallel with the total flavonoid content of Polygonum tinctorium calculated as rutin.

[0071] Compared with the prior art, the beneficial effects of the present invention are:

[0072] A technique involving the preparation of a mixed solution of the analyte and rutin standard, and simultaneous injection and analysis within a single chromatographic run, ensures that the target analyte and internal reference are under identical instantaneous chromatographic conditions. This eliminates systematic errors introduced by multiple independent injections due to instrument drift, fluctuations in mobile phase ratios, and environmental differences. The acquisition of retention time and peak area data achieves synchronicity and consistency, enhancing the reliability of direct comparisons between different signals. Subsequent peak attribution comparisons based on retention time and quantitative calculations based on peak area are all based on a unified reference standard, improving data reliability. The streamlined analytical process reduces operational steps and instrument runtime.

[0073] Based on the obtained synchronous chromatographic data, the total flavonoid content, expressed as rutin, was calculated according to the rutin standard curve. The retention times of the sample's characteristic peaks were pattern matched with a pre-defined fingerprint spectrum to qualitatively assign the chromatographic peaks. This step clearly distinguishes the chromatographic signal from rutin monomers from the responses of other flavonoid components. Based on this qualitative result, the peak areas of all chromatographic peaks attributed to rutin were specifically extracted and summarized, and then substituted back into the same rutin standard curve for calculation. This process accurately separates the response signal generated solely by rutin from the total mixed response value containing different response factors. The method ultimately outputs two independent quantitative results in parallel in a single analysis: the overall response-converted content of each flavonoid component with rutin as a reference; and the absolute content of the rutin monomer component itself. This overcomes the theoretical bias caused by the simple generalization of mixed responses in conventional methods, providing a dual quantitative dimension for the quality evaluation of complex mixtures. Attached Figure Description

[0074] Figure 1 This is a flowchart illustrating the steps of the HPLC quality control method for dual standardization of total flavonoids and rutin from Polygonum tinctorium as described in this invention.

[0075] Figure 2 Flowchart for the preparation of mixed solutions;

[0076] Figure 3 This is a flowchart of chromatographic separation and signal recording;

[0077] Figure 4 This is a standard curve of rutin standard concentration versus peak area.

[0078] Figure 5 The image shows the HPLC chromatogram of a mixed sample of total flavonoids from Polygonum hydropiper and rutin. Detailed Implementation

[0079] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0080] See Figure 1A mixed solution containing total flavonoids from *Polygonum hydropiper* (a sample to be tested) and rutin standard was prepared. The analytical conditions for high-performance liquid chromatography (HPLC) were set, using a C18 column and gradient elution with acetonitrile and phosphoric acid aqueous solution as the mobile phase. The prepared mixed solution was injected into the HPLC system, and the chromatographic separation program was started, simultaneously recording the chromatographic elution signals of the *Polygonum hydropiper* sample and rutin standard. The retention times and peak areas of the characteristic peaks of *Polygonum hydropiper* and rutin standard were extracted from the recorded chromatographic elution signals. Based on the peak area data of the rutin standard characteristic peak, a standard curve for rutin content was established. The peak area data of the *Polygonum hydropiper* characteristic peak were substituted into this standard curve to calculate the total flavonoid content of *Polygonum hydropiper*, expressed as rutin. Simultaneously, the retention times of the *Polygonum hydropiper* characteristic peaks were pattern matched with a pre-defined fingerprint chromatogram of *Polygonum hydropiper* to confirm the characteristic peak assignment. Based on the confirmation of characteristic peak attribution, the characteristic peaks of total flavonoids in *Polygonum cuspidatum* were identified, distinguishing peaks belonging to rutin from those belonging to other flavonoid components. The peak area data of the peaks belonging to rutin were summarized and substituted back into the standard curve to calculate the actual content of rutin monomers in the sample. Finally, the results of the total flavonoid content of *Polygonum cuspidatum* calculated as rutin and the actual content of rutin monomers in the sample were output in parallel, thus completing the dual-standard quality control.

[0081] See Figure 2 In one embodiment of the present invention, dried and pulverized Polygonum hydropiper plant material is accurately weighed, and a 60% (v / v) methanol aqueous solution is added for ultrasonic-assisted extraction. After extraction, the mixture is cooled and filtered, and the crude extract of total flavonoids from Polygonum hydropiper is collected. The crude extract of total flavonoids from Polygonum hydropiper is concentrated under reduced pressure to obtain a extract of total flavonoids from Polygonum hydropiper. The extract of total flavonoids from Polygonum hydropiper is accurately weighed, dissolved in methanol and diluted to volume to prepare a sample solution of total flavonoids from Polygonum hydropiper to be tested. In addition, a rutin standard with identified purity is accurately weighed, dissolved in methanol and diluted to volume to prepare a rutin standard stock solution. The sample solution of total flavonoids from Polygonum hydropiper to be tested and the rutin standard stock solution are mixed evenly according to a preset volume ratio to obtain a mixed solution. For example, accurately weigh 5.00 grams of dried and pulverized Polygonum hydropiper plant material, add 50 ml of 60% methanol aqueous solution, and perform ultrasonic-assisted extraction for 30 minutes. After cooling and filtration, concentrate the crude extract of total flavonoids from Polygonum hydropiper under reduced pressure at 40 degrees Celsius to a paste state. Accurately weigh 0.05 grams of the extract of total flavonoids from Polygonum hydropiper, dissolve it in methanol, and dilute to a volumetric flask of 25 ml to prepare the sample solution of total flavonoids from Polygonum hydropiper to be tested.

[0082] In some embodiments, 10.00 g of dried and pulverized Polygonum hydropiper plant material is accurately weighed and added to 100 mL of a 60% methanol aqueous solution. After ultrasonic-assisted extraction, the crude extract of total flavonoids from Polygonum hydropiper is filtered and concentrated under reduced pressure to obtain a extract of total flavonoids from Polygonum hydropiper. 0.10 g of the extract of total flavonoids from Polygonum hydropiper is accurately weighed, dissolved in methanol, and diluted to a 50 mL volumetric flask to prepare the sample solution of total flavonoids from Polygonum hydropiper. Optionally, the ultrasonic-assisted extraction conditions are set to a power of 200 W, a frequency of 40 kHz, an extraction time of 30 min, and a reduced pressure concentration temperature controlled at 40°C until the volume of the crude extract of total flavonoids from Polygonum hydropiper is reduced to one-tenth of its original volume to obtain the extract of total flavonoids from Polygonum hydropiper.

[0083] In practice, 10.00 mg of rutin standard, whose purity has been determined, is accurately weighed, dissolved in methanol, and diluted to a volumetric flask with a concentration of 1.00 mg / mL to prepare a rutin standard stock solution. It can be understood that when mixing the sample solution of total flavonoids from *Polygonum cuspidatum* and the rutin standard stock solution according to a predetermined volume ratio, the predetermined volume ratio is determined by the formula:

[0084]

[0085] in: Indicates the volume ratio of the mixture. This indicates the volume of the sample solution containing total flavonoids from Polygonum hydropiper. This indicates the volume of the rutin standard stock solution. For example, a preset volume ratio. The value is 1, which corresponds to a volume of 1 mL of the sample solution of total flavonoids from Polygonum tinctorium and a volume of 1 mL of the rutin standard stock solution. Both are transferred into the same container and shaken with a vortex mixer for 30 seconds to obtain a mixed solution.

[0086] In some embodiments, a preset volume ratio The value is 2, corresponding to a volume of 2 mL for the total flavonoids sample solution from *Polygonum cuspidatum* and a volume of 1 mL for the rutin standard stock solution. These are then mixed to obtain the final solution. Optionally, a preset volume ratio can be used. The volume ratio was 0.5, corresponding to a 0.5 mL volume for the total flavonoids sample solution from *Polygonum hydropiper* and a 1 mL volume for the rutin standard stock solution. Mixing was performed at room temperature to ensure homogeneity. In practice, data comparison showed that when the *Polygonum hydropiper* sample weight was 5.00 g and the final volume was 25 mL, the concentration of the prepared *Polygonum hydropiper* total flavonoids sample solution was proportional to the concentration when the sample weight was 10.00 g and the final volume was 50 mL. It can be understood that the mixing volume ratio of the *Polygonum hydropiper* total flavonoids sample solution to the rutin standard stock solution affects the signal intensity in subsequent chromatographic analysis. The selection was based on the estimated concentration of the target component in the sample solution of total flavonoids from Polygonum hydropiper and the concentration of the rutin standard stock solution.

[0087] In one embodiment of the present invention, a C18 reversed-phase chromatographic column with a particle size of 5 micrometers, a column length of 250 mm, and an inner diameter of 4.6 mm is selected, such as an Agilent ZORBAX Eclipse Plus C18 column. The column oven temperature of the C18 reversed-phase chromatographic column is set to 30 degrees Celsius. Mobile phase A is prepared as a 1% (v / v) aqueous solution of phosphoric acid, which is prepared by adding 10 mL of phosphoric acid to 990 mL of ultrapure water and mixing thoroughly. Mobile phase B is prepared as chromatographically pure acetonitrile. The high-performance liquid chromatograph (HPLC) was operated at a flow rate of 1 mL / min, a detection wavelength of 360 nm, and an injection volume of 10 μL. A gradient elution program was established, set as follows: 0-5 minutes, mobile phase B linearly changed from 15% to 20%; 5-20 minutes, mobile phase B linearly changed from 20% to 35%; 20-25 minutes, mobile phase B was maintained at 35%; 25-26 minutes, mobile phase B linearly changed from 35% to 15%; 26-30 minutes, mobile phase B was maintained at 15% for column equilibration. In practice, the pH of a 1% phosphoric acid aqueous solution was determined to be 2.1, the purity of chromatographic acetonitrile was 99.9%, and the HPLC detector was a diode array detector with a spectral bandwidth of 4 nm and a sampling frequency of 10 Hz.

[0088] In some embodiments, the C18 reversed-phase column is a Waters Symmetry C18, the column oven temperature is set to 30 degrees Celsius, mobile phase A uses 0.1% phosphoric acid aqueous solution, mobile phase B uses chromatographic grade acetonitrile, the flow rate is set to 1.0 mL / min, the detection wavelength is set to 360 nm, and the injection volume is set to 10 μL. Optionally, the gradient elution program is set as follows: 0 to 8 minutes, mobile phase B linearly changes from 10% to 25%; 8 to 22 minutes, mobile phase B linearly changes from 25% to 40%; 22 to 28 minutes, mobile phase B is maintained at 40%; 28 to 30 minutes, mobile phase B linearly changes from 40% to 10%; 30 to 35 minutes, mobile phase B is maintained at 10% for column equilibration. The linear change in the mobile phase ratio follows the formula:

[0089]

[0090] in: Indicates time The volume percentage of mobile phase B at that time. This represents the initial volume percentage of the mobile phase B at the start time of the gradient segment. Represents the gradient slope. This indicates the time taken from the starting point of the gradient segment.

[0091] It is understandable that the gradient slope The value is calculated by dividing the target percentage change of mobile phase B within the gradient segment by the time span of that gradient segment. For example, in the 0 to 5 minute gradient segment, mobile phase B changes from 15% to 20%, and the gradient slope of that segment... The calculated value is 1% per minute. In some embodiments, the gradient slope of the gradient segment from 0 to 5 minutes in the gradient elution procedure is... The gradient slope is 1% per minute, for gradient segments from 5 to 20 minutes. The gradient slope is 1% per minute for the 25- to 26-minute gradient segment. The elution rate is -20% per minute. Optionally, the column oven temperature can be set from 25°C to 40°C, and the detection wavelength from 350 nm to 370 nm. For different column brands, the time points and the proportion range of mobile phase B in the gradient elution program can be adaptively adjusted. It can be understood that the gradient elution program is designed to effectively separate the components in the total flavonoids of *Polygonum hydropiper* from the rutin standard on the chromatogram. The proportion of mobile phase B is initially lower to retain more polar components, then the proportion is increased to elute less polar components, and finally the initial proportion is restored to achieve column equilibration, preparing for the next injection analysis.

[0092] See Figure 3In one embodiment of the present invention, a microsyringe is used to accurately aspirate 10 μL of the mixed solution, which is then injected into the system via the autosampler or manual injection valve of a high-performance liquid chromatograph (HPLC). A gradient elution program is initiated, and the mixed solution, along with the mobile phase, enters a C18 reversed-phase column for separation. At a detection wavelength of 360 nm, an ultraviolet (UV) detector continuously monitors the light absorption intensity of the column effluent. The UV detector converts the light absorption intensity signal into an electrical signal, which is then collected and recorded by a chromatography workstation as a chromatogram over time. This chromatographic effluent signal constitutes complete chromatogram data, including both the time axis and the response intensity axis. The system opens the recorded chromatogram, performs baseline correction and smoothing, identifies all chromatographic peaks reaching the preset signal-to-noise ratio, and locates the characteristic peak of the rutin standard in the chromatogram of the mixed solution based on the retention time of the pure rutin standard obtained from pre-injection determination. Based on the known chromatographic behavior of total flavonoids from *Polygonum hydropiper*, other characteristic chromatographic peak groups belonging to *Polygonum hydropiper* are identified within a certain time window before and after the characteristic peak of the rutin standard. For each characteristic peak in both the rutin standard and the *Polygonum hydropiper* characteristic peak groups, the exact retention time and peak area values ​​are obtained through the integration function of the chromatography workstation. For example, the autosampler accurately draws 10.0 μL of the mixed solution into the quantitative loop of the injection valve, the gradient elution program is immediately started, the mobile phase carries the sample into the chromatographic column, the UV detector monitors at a wavelength of 360 nm, and the chromatography workstation acquires signals at a rate of 2 data points per second, generating a two-dimensional data array containing time and absorbance responses, i.e., the chromatogram.

[0093] In some embodiments, manual injection involves accurately aspirating 10.0 μL of the mixed solution using a 25 μL syringe, manually rotating the injection valve to the Load position to introduce the sample into the quantitative loop, and then switching to the Inject position to inject the sample into the mobile phase flow path. Optionally, a preset signal-to-noise ratio threshold of 3 is set, and the baseline correction function of the chromatography workstation employs an exponential smoothing algorithm with a smoothing window width of 9 data points to identify all chromatographic peaks with a signal-to-noise ratio greater than 3. In a specific implementation, a pure rutin standard solution is pre-injected and measured to obtain a retention time of 12.5 minutes for the characteristic peak of the rutin standard. In the chromatogram of the mixed solution, a time window of ±0.5 minutes is set with 12.5 minutes as the center. The chromatographic peak identified within this window is then identified as the characteristic peak of the rutin standard in the mixed solution. It is understandable that the known chromatographic behavior of total flavonoids from *Polygonum hydropiper* is characterized by the appearance of multiple chromatographic peaks before and after the characteristic peak of rutin. A time range of 8 to 20 minutes is set as the window for identifying other characteristic chromatographic peak groups of total flavonoids from *Polygonum hydropiper*. Within this time window, in addition to the already identified characteristic peak of rutin standard, the chromatography workstation identifies five other chromatographic peaks that reach the preset signal-to-noise ratio through peak detection algorithms. These five chromatographic peaks constitute other characteristic chromatographic peak groups of total flavonoids from *Polygonum hydropiper*.

[0094] In some embodiments, the retention time of the characteristic peak of the rutin standard, pre-injected and determined, is 13.2 minutes. A chromatographic peak is identified near 13.2 minutes in the mixed solution chromatogram; this peak is confirmed as the characteristic peak of the rutin standard. The identification time window for other characteristic chromatographic peaks of total flavonoids from *Polygonum cuspidatum* is set to 9 to 22 minutes. Optionally, the integration function of the chromatography workstation uses a vertical segmentation method to determine the start and end points of each chromatographic peak. For the characteristic peak of the rutin standard, integration yields a retention time of 12.51 minutes and a peak area of ​​152,437 microvolts. For the five characteristic peaks in the characteristic chromatographic peak group of total flavonoids from *Polygonum cuspidatum*, integration yields retention time and peak area data respectively. For example, the retention time of the first characteristic peak is 9.85 minutes, and the peak area is 32,415 microvolts. The process of extracting peak area data from the chromatographic elution signal involves numerical integration of the area under the curve for each chromatographic peak. From the formula:

[0095]

[0096] in: This represents the peak area of ​​the chromatographic peak. Indicates the start time of the chromatographic peak. Indicates the termination time of the chromatographic peak. This represents the raw signal intensity value of the chromatogram at time t. This represents the baseline signal strength value at time t. This is understandable. and The peak detection algorithm of the chromatography workstation can automatically determine the peak based on changes in the first or second derivative of the signal, or it can be manually set by the operator. It is a discrete numerical sequence of real-time voltage signals acquired by an ultraviolet detector after analog-to-digital conversion. It is a baseline function obtained by connecting the start and end points of chromatographic peaks or by fitting a more complex baseline model.

[0097] In one embodiment of the present invention, at least five rutin standard working solutions with different concentration gradients are prepared using a rutin standard stock solution. For example, an appropriate volume is precisely measured from the rutin standard stock solution and diluted stepwise with methanol to prepare rutin standard working solutions with concentrations of 5.00 μg / mL, 10.00 μg / mL, 20.00 μg / mL, 40.00 μg / mL, and 80.00 μg / mL, respectively. Each concentration of rutin standard working solution is injected into a high-performance liquid chromatograph and analyzed under the stated analytical conditions. The peak area of ​​rutin corresponding to each concentration of rutin standard working solution is recorded. A scatter plot is drawn with the concentration of rutin standard as the abscissa and the corresponding rutin peak area as the ordinate. The scatter plot is fitted with a linear regression using the least squares method to obtain a linear equation relating rutin content to peak area. This linear equation is the standard curve, and its correlation coefficient is recorded. The peak areas of all characteristic peaks in the characteristic chromatographic peak group of total flavonoids in *Polygonum hydropiper* are summed to obtain the total peak area of ​​total flavonoids. This total peak area is then substituted into the linear equation corresponding to the standard curve. Based on this linear equation, the rutin equivalent concentration corresponding to the total peak area of ​​total flavonoids is calculated in reverse. Combining this with the dilution factor and sample weight during the preparation of the *Polygonum hydropiper* sample solution, the percentage of total flavonoids (calculated as rutin) in the original *Polygonum hydropiper* plant material is calculated. For example, if the characteristic chromatographic peak group of total flavonoids in *Polygonum hydropiper* contains five characteristic peaks with peak areas of 32415 μV / s, 28547 μV / s, 152437 μV / s, 8912 μV / s, and 6543 μV / s, the total peak area of ​​total flavonoids in *Polygonum hydropiper* is the sum of these values.

[0098] In some embodiments, the concentration gradient of the rutin standard working solution is set to 2.50 μg / mL, 5.00 μg / mL, 10.00 μg / mL, 20.00 μg / mL, and 40.00 μg / mL, with each concentration analyzed three times, and the average peak area of ​​rutin is recorded. Optionally, a linear equation is obtained by least squares linear regression fitting. ,in This represents the peak area of ​​rutin in chromatograms, measured in microvolt-seconds. The concentration of rutin standard is expressed in micrograms per milliliter, and the correlation coefficient of the linear equation is 0.9995. In practice, the total peak area is obtained by summing the peak areas of all characteristic peaks in the characteristic chromatographic peak group of total flavonoids from *Polygonum cuspidatum*. microvolt-seconds, this total peak area value Substitute into the linear equation The equivalent concentration of rutin was calculated. Micrograms per milliliter, see Table 1.

[0099] Table 1: Correspondence between concentration and peak area of ​​rutin standard working solution

[0100] It is understandable that in the preparation of the test sample solution of total flavonoids from *Polygonum cuspidatum*, 0.05 g of the extract of total flavonoids from *Polygonum cuspidatum* was weighed and diluted to 25 mL with methanol. This test sample solution was further mixed with the rutin standard stock solution at a volume ratio of 1:1. After mixing, the sample was injected for analysis. The rutin equivalent concentration corresponding to the total peak area of ​​total flavonoids from *Polygonum cuspidatum* was measured from the chromatogram of the mixed solution to be 12.30 μg / mL. This concentration is the concentration of total flavonoids from *Polygonum cuspidatum* in the mixed solution, expressed as rutin. Combined with the mixing ratio, the concentration of total flavonoids from *Polygonum cuspidatum* in the original test sample solution, expressed as rutin, is... Micrograms per milliliter, where factor 2 arises from the dilution of the sample solution with an equal volume of standard solution during mixing. In some embodiments, the sample weight of *Polygonum cuspidatum* plant material is 5.00 g, which is extracted, concentrated, dissolved, and diluted to 25 mL to obtain the *Polygonum cuspidatum* total flavonoid test sample solution. The percentage of total flavonoids, calculated as rutin, in the original *Polygonum cuspidatum* plant material is... Through the formula:

[0101]

[0102] in: This indicates the percentage of total flavonoids in Polygonum tinctorium, calculated as rutin. This indicates the concentration of total flavonoids from *Polygonum hydropiper* in the test sample solution, expressed as rutin. This indicates the final volume of the sample solution containing total flavonoids from Polygonum hydropiper. This indicates the dilution factor from the original sample solution to the analytical solution. This indicates the sample quantity of Polygonum hydropiper plant material. It is the conversion factor from micrograms to grams.

[0103] See Figure 4 This is a standard curve of rutin standard concentration versus peak area. This curve establishes a linear relationship between rutin concentration and chromatographic peak area. The rutin content can then be inferred from the peak area of ​​the sample, making it a crucial step in the HPLC quality control method for dual-standard rutin and total flavonoids from *Polygonum hydropiper*. The measured points in the curve highly coincide with the fitted curve, demonstrating a good linear relationship between peak area and concentration within the range of 5.00–80.00 μg / mL, meeting the core methodological requirements of HPLC quantitative analysis. This curve can be used to subsequently calculate the total flavonoid content (calculated as rutin) in *Polygonum hydropiper* samples, as well as the actual content of rutin monomers. The content results obtained from this curve can be directly used to determine whether *Polygonum hydropiper* raw materials and extracts meet quality standards, serving as the core data source for the entire quality control process.

[0104] In one embodiment of the present invention, a pre-established standard fingerprint spectrum of total flavonoids from *Polygonum hydropiper* is invoked. This standard fingerprint spectrum is derived from the chromatographic analysis results of multiple batches of qualified *Polygonum hydropiper* extracts. The retention time of each peak in the currently measured characteristic chromatographic peak group of total flavonoids from *Polygonum hydropiper* is compared one by one with the retention times of all characteristic peaks in the standard fingerprint spectrum. The relative difference between the current chromatographic peak retention time and the retention time of the reference peak in the standard fingerprint spectrum is calculated. If the relative difference is within a preset allowable deviation range, it is determined that the current chromatographic peak and the reference peak in the standard fingerprint spectrum are consistent. The attribution determination of all characteristic peaks of total flavonoids from *Polygonum hydropiper* is completed, and a characteristic peak attribution confirmation list is generated. For example, the standard fingerprint chromatogram of total flavonoids from *Polygonum cuspidatum* contains six characteristic reference peaks with retention times of 9.80 min, 10.50 min, 12.50 min, 14.20 min, 16.80 min, and 18.50 min, respectively. The currently measured retention times of the characteristic chromatographic peak group of total flavonoids from *Polygonum cuspidatum* are 9.85 min, 10.52 min, 12.51 min, 14.25 min, 16.85 min, and 18.54 min, respectively. The relative difference is calculated using the formula:

[0105]

[0106] in: This indicates the percentage deviation from the relative retention time. Indicates the current chromatographic peak retention time. The retention time of the corresponding reference peak in the standard fingerprint spectrum is indicated. The preset allowable deviation range is ±2%. The calculated relative differences of each peak are 0.51%, 0.19%, 0.08%, 0.35%, 0.30%, and 0.22%, respectively, all within the allowable deviation range. Therefore, it is determined that all peaks are assigned to the same peak. The characteristic peak assignment confirmation list records the assignment information of each peak. For example, the first peak is assigned to quercetin-3-O-glucoside, and the third peak is assigned to rutin.

[0107] In some embodiments, the retention time of the reference peak in the standard fingerprint chromatogram of total flavonoids from *Polygonum cuspidatum* is determined based on the average value of chromatograms of ten batches of qualified medicinal material extracts, with a preset allowable deviation range of ±2.5%. The comparison between the currently measured retention time and the retention time of the reference peak in the standard fingerprint chromatogram is completed through the automatic matching function of the chromatography workstation. After successful matching, a characteristic peak attribution confirmation list is generated. Optionally, the characteristic peak attribution confirmation list is presented in tabular form, listing the current chromatographic peak number, retention time, standard fingerprint chromatogram reference peak number, reference retention time, relative difference, and attribution determination result. The characteristic peaks attribution confirmation list are consulted to identify those confirmed to belong to rutin. The characteristic peaks belonging to rutin are located in the chromatogram, and their peak area data is read and recorded as the rutin monomer peak area. All characteristic peaks in the characteristic chromatographic peak group of total flavonoids from *Polygonum cuspidatum*, excluding those belonging to rutin, are classified as other flavonoid component peaks. The peak area data of other flavonoid component peaks are accumulated to obtain the total peak area of ​​other flavonoid components. For example, the characteristic peak attribution confirmation list shows that the third peak is attributed to rutin. Locate this peak in the chromatogram and read its peak area as 152437 microvolts per second. Record this as the peak area of ​​the rutin monomer. The peak areas of the other five characteristic peaks are 32415 microvolts per second, 28547 microvolts per second, 8912 microvolts per second, 6543 microvolts per second, and 5210 microvolts per second, respectively. The total peak area of ​​the other flavonoid components is 32415 + 28547 + 8912 + 6543 + 5210 = 81627 microvolts per second.

[0108] In practice, the peak area data of rutin monomers read from the chromatogram is used. The peak area values ​​of rutin monomers are substituted into the linear equation corresponding to the standard curve. Based on the linear equation, the concentration of rutin monomers in the mixed solution is calculated. Combining this with the mixing ratio of the *Polygonum cuspidatum* total flavonoids test sample solution and the rutin standard stock solution during the preparation of the mixed solution, the actual concentration of rutin monomers in the original *Polygonum cuspidatum* total flavonoids test sample solution is deduced. Further, combining the dissolution volume of the extract and the sample weight, the percentage content of rutin monomers in the original *Polygonum cuspidatum* plant raw material is calculated. The percentage content result of rutin monomers is then output alongside the result of the total flavonoid content of *Polygonum cuspidatum* calculated as rutin. For example, the linear equation of the standard curve is... rutin single peak area Substitute the values ​​of microvolts per second into the equation to calculate the concentration of rutin monomer in the mixed solution. The volume ratio of the sample solution of total flavonoids from *Polygonum cuspidatum* to the rutin standard stock solution in the mixed solution is 1:1 (µg / mL). Therefore, the actual concentration of rutin monomer in the original sample solution of total flavonoids from *Polygonum cuspidatum* is... Micrograms per milliliter, where factor 2 is derived from the dilution of the sample solution by an equal volume of standard solution during mixing.

[0109] See Figure 5This is an HPLC chromatogram of a mixed sample of total flavonoids and rutin from *Polygonum hydropiper*, which has significant analytical value in dual-standard quality control methods. Comparison with standard fingerprint chromatograms can confirm the component attribution of each peak and distinguish rutin monomers from other flavonoids. By integrating the peak areas, the total flavonoid content (calculated as rutin) and the actual content of rutin monomers can be calculated. The chromatographic peaks are sharp, the baselines are stable, and the separation between different peaks is good, proving that this gradient elution program can effectively separate flavonoid components in *Polygonum hydropiper*, providing direct evidence of the reliability of the HPLC method. Comparison of the retention times of each peak with the standard fingerprint chromatogram of total flavonoids from *Polygonum hydropiper* confirms the attribution of characteristic peaks and clearly distinguishes rutin from other flavonoid components. This figure is the original data source for obtaining the areas of each characteristic peak; integration yields the peak areas of rutin monomers and total flavonoids.

[0110] The above embodiments are only used to illustrate the technical methods of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical methods of the present invention without departing from the spirit and scope of the technical methods of the present invention.

Claims

1. A dual-standard HPLC quality control method for total flavonoids and rutin from Polygonum hydropiper, characterized in that, include: Prepare a mixed solution containing the total flavonoids of Polygonum hydropiper and rutin standard; The analytical conditions for high performance liquid chromatography were set, using a C18 column and acetonitrile and phosphoric acid aqueous solution as the mobile phase, and gradient elution was performed. The mixed solution was injected into the high performance liquid chromatograph, the chromatographic separation program was started, and the chromatographic elution signals of the test sample of total flavonoids of Polygonum tinctorium and rutin standard were recorded simultaneously. The retention time and peak area data of the characteristic peaks of total flavonoids from Polygonum tinctorium and the characteristic peaks of rutin standard were extracted from the chromatographic elution signal. Based on the peak area data of the characteristic peaks of the rutin standard, a standard curve for rutin content was established; Substitute the peak area data of the characteristic peak of total flavonoids in Polygonum hydropiper into the standard curve to calculate the total flavonoid content of Polygonum hydropiper, calculated as rutin. Meanwhile, the retention time of the characteristic peaks of total flavonoids from Polygonum hydropiper is matched with the preset fingerprint spectrum of total flavonoids from Polygonum hydropiper to confirm the assignment of the characteristic peaks. Based on the results of the confirmation of the characteristic peak attribution, the characteristic peaks of the total flavonoids of the Polygonum tinctorium are identified, and the peaks belonging to rutin are distinguished from the peaks of other flavonoid components. Summarize the peak area data of the peaks attributed to rutin, and substitute them back into the standard curve to calculate the actual content of rutin monomer in the sample. Finally, the results of total flavonoid content of Polygonum tinctorium calculated as rutin and the actual content of rutin monomers in the sample were output in parallel to complete the dual-standard quality control.

2. The HPLC quality control method for dual standardization of total flavonoids and rutin from Polygonum hydropiper according to claim 1, characterized in that, The preparation of the mixed solution containing the test sample of total flavonoids from Polygonum hydropiper and rutin standard specifically includes: Accurately weigh the dried and pulverized Polygonum tinctorium plant material, add a 60% (v / v) methanol aqueous solution, and perform ultrasonic-assisted extraction. After extraction, the solution was cooled and filtered, and the crude extract of total flavonoids from Polygonum tinctorium was collected. The crude extract of total flavonoids from Polygonum hydropiper was concentrated under reduced pressure to obtain a extract of total flavonoids from Polygonum hydropiper. Accurately weigh the extract of total flavonoids from Polygonum hydropiper, dissolve it in methanol and make up to volume to prepare a sample solution of total flavonoids from Polygonum hydropiper to be tested; In addition, accurately weigh the rutin standard that has been identified as having purity, dissolve it in methanol and dilute to volume to prepare a rutin standard stock solution; The sample solution containing total flavonoids from Polygonum hydropiper was mixed with the rutin standard stock solution in a predetermined volume ratio to obtain the mixed solution.

3. The HPLC quality control method for dual standardization of total flavonoids and rutin from Polygonum tinctorium according to claim 2, characterized in that, The analytical conditions for setting the high-performance liquid chromatography (HPLC) are as follows: using a C18 column, with acetonitrile and phosphoric acid aqueous solution as the mobile phase, and performing gradient elution, specifically including: A C18 reversed-phase column with a particle size of 5 micrometers, a column length of 250 mm, and an inner diameter of 4.6 mm was selected. The column oven temperature of the C18 reversed-phase chromatographic column is set to 30 degrees Celsius; Mobile phase A was prepared as 1% (v / v) aqueous phosphoric acid solution, and mobile phase B was prepared as chromatographic grade acetonitrile. The flow rate was set to 1 ml per minute, the detection wavelength to 360 nm, and the injection volume to 10 μL. Establish a gradient elution program: from 0 to 5 minutes, mobile phase B is linearly changed from 15% to 20%; from 5 to 20 minutes, mobile phase B is linearly changed from 20% to 35%; from 20 to 25 minutes, mobile phase B is maintained at 35%; from 25 to 26 minutes, mobile phase B is linearly changed from 35% to 15%; from 26 to 30 minutes, mobile phase B is maintained at 15% for column equilibration.

4. The HPLC quality control method for dual standardization of total flavonoids and rutin from Polygonum hydropiper according to claim 3, characterized in that, The step of injecting the mixed solution into the high-performance liquid chromatograph, starting the chromatographic separation program, and simultaneously recording the chromatographic elution signals of the total flavonoids of Polygonum tinctorium sample and the rutin standard specifically includes: Accurately aspirate 10 μL of the mixture using a microsyringe and inject it into the system via the autosampler or manual injection valve of the high-performance liquid chromatograph. The gradient elution program is initiated, and the mixed solution enters the C18 reversed-phase chromatographic column along with the mobile phase for separation. At the detection wavelength of 360 nm, the light absorption intensity of the chromatographic column effluent was continuously monitored using an ultraviolet detector; The ultraviolet detector converts the light absorption intensity signal into an electrical signal, which is collected and recorded by the chromatography workstation as a chromatogram that changes over time. The chromatographic elution signal is the complete chromatogram data, which includes the time axis and the response intensity axis.

5. The HPLC quality control method for dual standardization of total flavonoids and rutin from *Polygonum hydropiper* according to claim 4, characterized in that... The retention time and peak area data for extracting the characteristic peaks of total flavonoids from Polygonum hydropiper and the characteristic peaks of rutin standard from the chromatographic elution signal specifically include: Open the recorded chromatogram in the chromatography workstation; Baseline correction and smoothing are performed on the chromatograms to identify all chromatographic peaks that reach the preset signal-to-noise ratio; Based on the retention time of the pure rutin standard obtained from pre-injection determination, the characteristic peak of the rutin standard is located in the chromatogram of the mixed solution; Based on the known chromatographic behavior of total flavonoids from Polygonum hydropiper, other characteristic chromatographic peak groups belonging to total flavonoids from Polygonum hydropiper are identified within a certain time window before and after the characteristic peak of the rutin standard. The exact retention time and peak area values ​​of each characteristic peak in the chromatographic peak group of the rutin standard and the total flavonoids of Polygonum tinctorium were obtained by integrating the values ​​of the chromatographic workstation.

6. The HPLC quality control method for dual standardization of total flavonoids and rutin from *Polygonum hydropiper* according to claim 5, characterized in that... The step of establishing a standard curve for rutin content based on the peak area data of the characteristic peaks of the rutin standard specifically includes: Using the aforementioned rutin standard stock solution, prepare at least five rutin standard working solutions with different concentration gradients; Each concentration of rutin standard working solution was injected into the high-performance liquid chromatograph and analyzed under the analytical conditions. Record the peak area of ​​rutin corresponding to each concentration of rutin standard working solution; A scatter plot was drawn with the concentration of the rutin standard on the x-axis and the corresponding rutin chromatographic peak area on the y-axis. The least squares method was used to perform linear regression fitting on the scatter points to obtain a linear equation relating rutin content and peak area. This linear equation is the standard curve, and its correlation coefficient was recorded.

7. The HPLC quality control method for dual standardization of total flavonoids and rutin from Polygonum hydropiper according to claim 6, characterized in that, The step of substituting the peak area data of the characteristic peak of total flavonoids in Polygonum hydropiper into the standard curve to calculate the total flavonoid content in Polygonum hydropiper, calculated as rutin, specifically includes: The total peak area of ​​total flavonoids in the total chromatographic peak group of the above-mentioned total flavonoids in Polygonum tinctorium is obtained by summing up the peak area data of all characteristic peaks. Substitute the total peak area value of the total flavonoids of the Polygonum hydropiper into the linear equation corresponding to the standard curve; Based on the linear equation, the rutin equivalent concentration corresponding to the total peak area of ​​the total flavonoids in the Polygonum hydropiper was calculated in reverse. Based on the dilution factor and sample weight during the preparation of the total flavonoid test sample solution of Polygonum tinctorium, the percentage of total flavonoids (calculated as rutin) in the original Polygonum tinctorium plant raw material was calculated.

8. The HPLC quality control method for dual standardization of total flavonoids and rutin from *Polygonum hydropiper* according to claim 7, characterized in that, Simultaneously, the retention times of the characteristic peaks of total flavonoids from *Polygonum hydropiper* are matched with a preset fingerprint spectrum of total flavonoids from *Polygonum hydropiper* to confirm the attribution of the characteristic peaks, specifically including: The pre-established standard fingerprint spectrum of total flavonoids from Polygonum hydropiper was called, which was derived from the chromatographic analysis results of multiple batches of qualified Polygonum hydropiper extracts. The retention time of each peak in the characteristic chromatographic peak group of total flavonoids from Polygonum hydropiper is compared one by one with the retention time of all characteristic peaks in the standard fingerprint spectrum of total flavonoids from Polygonum hydropiper. Calculate the relative difference between the current chromatographic peak retention time and the reference peak retention time in the standard fingerprint chromatogram; If the relative difference is within the preset allowable deviation range, the current chromatographic peak is determined to be consistent with the reference peak in the standard fingerprint spectrum. The attribution of all characteristic peaks of total flavonoids from Polygonum hydropiper was determined, and a confirmation list of characteristic peak attributions was generated.

9. The HPLC quality control method for dual standardization of total flavonoids and rutin from *Polygonum cuspidatum* according to claim 8, characterized in that, Based on the confirmation of the characteristic peak attribution, the characteristic peaks of the total flavonoids in *Polygonum hydropiper* are identified, distinguishing the peaks belonging to rutin from those of other flavonoid components. Specifically, this includes: Consult the characteristic peak attribution confirmation list and find the characteristic peaks that are confirmed to belong to rutin. Locate the characteristic peaks belonging to rutin in the chromatogram and read their peak area data, recording them as the peak area of ​​rutin monomers; All characteristic peaks in the total flavonoids characteristic chromatographic peak group of the above-mentioned Polygonum hydropiper, except for the characteristic peaks belonging to rutin, are classified as other flavonoid component peaks; The peak area data of the other flavonoid components are summed to obtain the total peak area of ​​the other flavonoid components; The peak area of ​​the rutin monomer and the total peak area of ​​the other flavonoid components together constitute the detailed composition information of the total flavonoids of Polygonum tinctorium.

10. The HPLC quality control method for dual standardization of total flavonoids and rutin from Polygonum hydropiper according to claim 9, characterized in that, The peak area data of the peaks attributed to rutin are summarized and substituted back into the standard curve to calculate the actual content of rutin monomers in the sample, specifically including: Use the peak area data of the rutin monomers read from the chromatogram; Substitute the peak area value of the rutin monomer into the linear equation corresponding to the standard curve; The concentration of rutin monomer in the mixed solution was calculated based on the linear equation. By combining the mixing ratio of the sample solution of total flavonoids from Polygonum hydropiper to the rutin standard stock solution during the preparation of the mixed solution, the actual concentration of rutin monomer in the original sample solution of total flavonoids from Polygonum hydropiper can be deduced. By further combining the dissolution volume of the extract and the sample weight of the raw material, the percentage content of rutin monomer in the original Polygonum tinctorium plant raw material was calculated, and the percentage content of rutin monomer was output in parallel with the total flavonoid content of Polygonum tinctorium calculated as rutin.

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