A gradient chromatographic analysis system and method for simultaneous quantification of multiple flavonoid components

Abstract

The application discloses a gradient chromatographic analysis system and method for synchronously quantifying multiple flavone components, and relates to the technical field of gradient chromatographic analysis.The gradient chromatographic analysis system for synchronously quantifying multiple flavone components comprises an organic phase change monitoring module, a chromatographic peak identification monitoring module and a quantitative accuracy monitoring and analyzing module.The application analyzes the interference degree of the change of the organic phase, judges whether to perform chromatographic peak identification accuracy verification based on the analysis result, performs quantitative accuracy analysis of the multiple flavone components for evaluating the separation of the multiple flavone components after the chromatographic peak identification accuracy verification and at the end of the verification, and thus improves the accuracy of the synchronous quantification data of the multiple flavone components based on the gradient chromatography, and solves the problem of low accuracy of the synchronous quantification data of the multiple flavone components based on the gradient chromatography in the prior art.

Description

Technical Field

[0001] This invention relates to the field of gradient chromatography analysis technology, and in particular to a gradient chromatography analysis system and method for simultaneous quantitative analysis of polyflavonoid components. Background Technology

[0002] To further improve the quality standards of flavonoid-containing preparations (such as Guanxinkang tablets) and enhance product quality control, it is necessary to determine the content of active ingredients. Existing methods first involve sample pretreatment, typically using organic solvents (i.e., organic phases, such as methanol or ethanol) to ultrasonically extract solid samples (e.g., Guanxinkang tablets), or to dilute and extract liquid samples, followed by filtration through a microporous membrane to obtain a clear test solution. Simultaneously, each flavonoid reference standard is accurately weighed and prepared into a series of mixed reference standard solutions of known concentrations using appropriate solvents. Next, chromatographic conditions were optimized and established. A reversed-phase C18 column was selected, with methanol-water or acetonitrile-water (often with the addition of a small amount of acid such as phosphoric acid or acetic acid to improve peak shape) as the mobile phase. A non-linear gradient elution program was set: a low proportion of organic phase was used initially, followed by a gradual or segmental linear increase in the proportion of organic phase to a higher level to achieve effective separation of complex flavonoid mixtures. Column temperature was controlled by the column oven, and flow rate was controlled by the infusion pump. A DAD (Diode Array Detector) was used for full-wavelength scanning and peak purity identification. Then, method validation and sample determination were performed. Blank solvent, a series of mixed reference solutions, and the test solution were injected sequentially. Qualitative analysis was conducted using the retention time of the reference solution chromatograms, and a standard curve was established by linear regression of peak area against concentration. Finally, based on the peak area of ​​each flavonoid in the test solution, the accurate content of each flavonoid in the original sample was calculated by substituting it into the corresponding standard curve equation.

[0003] For example, Chinese invention patent CN103760278B discloses a high-performance liquid chromatography method for the simultaneous quantitative detection of six flavonoid components in Polygonum hydropiper. The method includes: using a Waters column, selecting methanol and 0.4% formic acid aqueous solution as the mobile phase, performing gradient elution, with a flow rate of 1 ml / min, and dividing the elution into three gradients. The volume ratios of methanol and 0.4% formic acid aqueous solution in the three gradients are 45:55, 55:45, and 70:30, respectively. The eluents are then detected by ultraviolet light at 359 nm to determine the concentration of each component.

[0004] The above-mentioned technology has at least the following technical problems:

[0005] In the simultaneous quantitative detection of polyflavonoid components, the continuous change in the organic phase ratio during gradient elution and insufficient column equilibration time lead to incomplete stabilization of the mobile phase composition and the chromatographic column, resulting in intensified baseline drift. The background signal intensity generated by the drift may approach or exceed the signal intensity of low-content peaks, potentially masking the chromatographic peaks of low-content components. This can lead to missed detection of target peaks (such as the chromatographic peaks corresponding to the main flavonoid components hyperoside, rutin, and quercetin), thus interfering with the accurate identification of chromatographic peaks. Existing technologies typically prioritize separation efficiency, often shortening column equilibration time or using steep gradient slopes, without allowing sufficient time for the mobile phase composition and the chromatographic column to reach stable equilibrium. This exacerbates baseline drift, potentially causing baseline fluctuations to overlap with target peak signals and blurring peak boundary determination. Consequently, the DAD detector cannot accurately distinguish between baseline drift signals and target peak spectral information during full-wavelength scanning and peak purity identification. The stray light signal introduced by baseline drift overlaps with the target peak spectrum, undermining the basis for peak purity identification and resulting in low accuracy of data for simultaneous quantitative analysis of polyflavonoid components based on gradient chromatography. Summary of the Invention

[0006] To address the low accuracy of existing gradient chromatography-based methods for simultaneous quantification of polyflavonoid components, this invention provides a gradient chromatography analysis system and method for the simultaneous quantification of polyflavonoid components. The technical solution is as follows:

[0007] On one hand, a gradient chromatographic analysis system for simultaneous quantitative analysis of polyflavonoid components is provided, including: an organic phase change monitoring module, a chromatographic peak identification monitoring module, and a quantitative accuracy monitoring and analysis module. The organic phase change monitoring module is used to analyze the interference level of organic phase changes during the gradient chromatographic analysis of polyflavonoid components, obtaining analytical results reflecting the changes in the proportion of organic phase during gradient elution. This determines whether to perform chromatographic peak identification accuracy verification to assess the accuracy of peak identification. If so, the corresponding judgment result is transmitted to the chromatographic peak identification monitoring module; otherwise, a mobile phase stability qualification test is performed to assess the stability of the mobile phase during gradient elution. The chromatographic peak identification monitoring module is used to perform chromatographic peak identification accuracy verification to assess the accuracy of peak identification. After verification, the qualified verification result is transmitted to the quantitative accuracy monitoring and analysis module. The quantitative accuracy monitoring and analysis module is used to perform quantitative accuracy analysis of polyflavonoid components based on the received qualified verification result, used to analyze the degree of separation qualification of polyflavonoid components.

[0008] On the other hand, a gradient chromatographic analysis method for simultaneous quantitative analysis of polyflavonoid components is provided, applied to a gradient chromatographic analysis system for simultaneous quantitative analysis of polyflavonoid components, including the following steps: S1, during the gradient chromatographic analysis of polyflavonoid components, the degree of interference of changes in the organic phase is analyzed, and the analytical results reflecting the changes in the proportion of the organic phase in gradient elution are obtained, thereby determining whether to perform accurate peak identification verification to assess the accuracy of peak identification. If so, the corresponding judgment result is transmitted to S2; otherwise, the mobile phase stability qualification test is performed to assess the stability of the mobile phase in gradient elution; S2, accurate peak identification verification is performed to assess the accuracy of peak identification. After the verification is completed, the qualified verification result is transmitted to S3; S3, based on the received qualified verification result, accurate quantitative analysis of polyflavonoid components is performed to analyze the degree of separation qualification of polyflavonoid components.

[0009] The beneficial effects of the technical solutions provided by the embodiments of the present invention include at least the following:

[0010] 1. By analyzing the degree of interference in changes in the organic phase, analytical results reflecting changes in the proportion of the organic phase during gradient elution are obtained. Based on these results, it is determined whether to perform accurate peak identification verification. This helps to quantitatively predict and classify the baseline drift risk caused by fluctuations in the organic phase proportion, identify interference sources that may mask low-content peaks in advance, and avoid ineffective peak identification verification processes. After accurate peak identification verification, accurate quantitative analysis of polyflavonoid components is performed to evaluate the separation of polyflavonoid components. This helps to accurately control the identification and separation effect of target peaks, ensuring that target peaks (such as hyperoside and rutin) are not masked by baseline drift, and verifying the reliability of the basic data for quantitative calculation through indicators such as resolution. This improves the accuracy of simultaneous quantitative data of polyflavonoid components based on gradient chromatography, solving the problem of low accuracy in simultaneous quantitative data of polyflavonoid components based on gradient chromatography in existing technologies.

[0011] 2. By analyzing the degree of interference of organic phase changes, the characteristic value of organic phase change interference is obtained. When the characteristic value of organic phase change interference is greater than the predefined characteristic value, the mobile phase stability qualification test is triggered; otherwise, the accurate verification of chromatographic peak identification is triggered. Compared with the existing technology that only focuses on separation efficiency and ignores mobile phase stability, this method helps to achieve graded processing based on the degree of organic phase interference, prioritize the solution of baseline drift caused by mobile phase instability, and thus improve the anti-interference ability and early stability of the gradient elution system, and reduce subsequent quantitative deviations caused by mobile phase instability.

[0012] 3. By accurately verifying the chromatographic peak masking value through chromatographic peak identification, the quantitative analysis of polyflavonoid components is initiated when the chromatographic peak masking value is not greater than the predefined chromatographic peak masking value; otherwise, the chromatographic peak masking optimization operation is initiated. Compared with the existing technology, which cannot quantify the degree of peak masking and thus cause low-content peaks to be missed, this method helps to quantitatively assess and target the risk of baseline noise masking the target peak, accurately determine whether it is necessary to reduce baseline noise by adjusting the flow rate, column temperature, or extending the equilibration time, and ensure that the low-content peak signal is clearly distinguishable. This ensures the integrity and accuracy of the quantitative results of polyflavonoid components (especially trace components) and avoids the underestimation of content caused by missed detection.

[0013] 4. After repeated use of the chromatographic column, the column efficiency decreases, leading to reduced separation and increased overlap of adjacent flavonoid peaks. Based on this, the target peak overlap value is obtained through accurate verification of chromatographic peak identification. When the target peak overlap value is greater than the predefined target peak overlap value, the target peak overlap degree analysis is initiated; otherwise, the quantitative analysis of multiple flavonoid components is initiated. Compared with existing technologies that ignore the impact of column efficiency decrease on peak overlap and directly quantify, this helps to achieve dynamic monitoring and intervention of peak overlap interference caused by column efficiency decrease, ensuring clear target peak boundaries, thereby ensuring the separation accuracy of structurally similar flavonoid components (such as luteolin and hyperoside) and avoiding cross-interference of quantitative data caused by peak overlap. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 This is a schematic diagram of the structure of a gradient chromatography analysis system for simultaneous quantitative analysis of polyflavonoid components provided in an embodiment of the present invention;

[0016] Figure 2 This is an overall flowchart of a gradient chromatography analysis system for simultaneous quantitative analysis of polyflavonoid components provided in an embodiment of the present invention;

[0017] Figure 3 This is a schematic diagram illustrating the mobile phase stability test of a gradient chromatography analysis system for simultaneous quantitative analysis of polyflavonoid components provided in an embodiment of the present invention.

[0018] Figure 4 This is a linear relationship graph of rutin concentration and peak area in a gradient chromatographic analysis method for simultaneous quantitative analysis of polyflavonoid components provided in an embodiment of the present invention.

[0019] Figure 5This is a flowchart of a gradient chromatographic analysis for simultaneous quantitative determination of polyflavonoid components provided in an embodiment of the present invention. Detailed Implementation

[0020] The technical solution of the present invention will now be described with reference to the accompanying drawings.

[0021] In embodiments of the present invention, words such as "exemplarily," "for example," etc., are used to indicate that something is an example, illustration, or description. Any embodiment or design described as "exemplary" in the present invention should not be construed as being more preferred or advantageous than other embodiments or designs. Specifically, the use of the word "exemplary" is intended to present the concept in a concrete manner. Furthermore, in embodiments of the present invention, the meaning expressed by "and / or" can be both, or either one.

[0022] In the embodiments of this invention, the terms "image" and "picture" may sometimes be used interchangeably. It should be noted that, without emphasizing the distinction between them, they convey the same meaning. Similarly, the terms "of," "corresponding (relevant)," and "corresponding" may sometimes be used interchangeably. It should be noted that, without emphasizing the distinction between them, they convey the same meaning.

[0023] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0024] This invention provides a gradient chromatographic analysis system for the simultaneous quantitative determination of polyflavonoid components. For example... Figure 1 The schematic diagram shown illustrates the structure of a gradient chromatographic analysis system for simultaneous quantitative analysis of polyflavonoid components. The system includes an organic phase change monitoring module, a peak identification monitoring module, and a quantitative accuracy monitoring and analysis module. The organic phase change monitoring module analyzes the interference level of organic phase changes during gradient chromatographic analysis of polyflavonoid components, obtaining analytical results reflecting changes in the proportion of organic phase during gradient elution. Based on these results, it determines whether peak identification accuracy verification is required to assess the accuracy of peak identification. If verification is performed, the corresponding result is transmitted to the peak identification monitoring module; otherwise, a mobile phase stability test is performed to assess the stability of the mobile phase during gradient elution. By monitoring organic phase changes, it is possible to monitor the dynamic changes in the proportion of organic phase and the degree of interference in gradient elution in real time, predict mobile phase stability in advance, and avoid the risk of increased baseline drift.

[0025] The chromatographic peak identification and monitoring module is used to verify the accuracy of chromatographic peak identification based on the received judgment results in order to evaluate the accuracy of chromatographic peak identification. After the verification is completed, the qualified verification results are transmitted to the quantitative accuracy monitoring and analysis module. By performing chromatographic peak identification and monitoring, it is helpful to accurately verify the accuracy of chromatographic peak identification results, effectively distinguish baseline drift signals from target peak signals, and prevent the missed detection and misjudgment of chromatographic peaks of low-content flavonoid components.

[0026] The quantitative accurate monitoring and analysis module is used to perform quantitative accurate analysis of polyflavonoid components based on the received qualified verification results, which is used to analyze the degree of qualified separation of polyflavonoid components. By performing quantitative accurate monitoring and analysis, it helps to accurately verify the accuracy of chromatographic peak identification results, effectively distinguish baseline drift signals from target peak signals, and prevent the missed detection and misjudgment of chromatographic peaks of low-content flavonoid components.

[0027] It should be added that, prior to the design of the gradient chromatography analysis system for simultaneous quantitative analysis of polyflavonoid components provided in this application, a database storing various setting data was established. This database provides dynamic resources for method development, system verification, and operation monitoring. The database includes, but is not limited to, predefined organic phase change interference characteristic values ​​and predefined baseline signal change amplitudes. These values ​​are directly set by technical personnel. Furthermore, the database supports updates and expansions under access control. Technical personnel can calibrate and supplement the parameters in the database based on the addition of new compounds or changes in instrument status to ensure that the system is always in optimal working condition.

[0028] like Figure 2 The diagram shown is an overall flowchart of a gradient chromatographic analysis system for simultaneous quantitative analysis of polyflavonoid components provided in an embodiment of this invention. Figure 2 It can be seen that: by analyzing the degree of interference of changes in the organic phase, the characteristic value of interference of organic phase changes is obtained. When the monitored characteristic value of interference of organic phase changes is greater than the predefined characteristic value of interference of organic phase changes, the mobile phase stability qualification test is triggered; otherwise, the accurate verification of chromatographic peak identification is triggered. The accurate verification of chromatographic peak identification is divided into two cases: Case 1: when the detected chromatographic peak masking value is greater than the predefined chromatographic peak masking value, the chromatographic peak masking optimization operation is performed; otherwise, the accurate quantitative analysis of polyflavonoid components is performed. Case 2: when the detected target peak overlap influence factor is not within the predefined target peak overlap qualification range, the target peak overlap interference optimization operation is initiated; otherwise, the accurate quantitative analysis of polyflavonoid components is performed.

[0029] In this embodiment, the organic phase change monitoring module, the chromatographic peak identification monitoring module, and the quantitative accuracy monitoring and analysis module are interconnected and influence each other, which helps to achieve closed-loop quality control of the entire process of gradient chromatographic detection of polyflavonoid components. This systematically solves the problem of inaccurate quantitative data caused by baseline drift, thereby helping to improve the quality control level of polyflavonoid preparations and the accuracy of effective component content determination.

[0030] Furthermore, an interference degree analysis is performed on the changes in the organic phase. The specific process is as follows: Organic phase change interference characteristic values ​​are output based on the organic phase ratios at the gradient endpoint and the gradient starting point to reflect the changes in the organic phase ratio during gradient elution. The organic phase change interference characteristic value is represented by the absolute value corresponding to the difference between the organic phase ratios at the gradient endpoint and the gradient starting point. The organic phase ratio at the gradient endpoint is represented by the volume percentage of the organic phase in the mobile phase at the end of elution, as monitored by liquid chromatography. The organic phase ratio at the gradient starting point is represented by the volume percentage of the organic phase in the mobile phase at the beginning of elution, as monitored by liquid chromatography. The interference degree is determined based on the organic phase change interference characteristic value. The specific determination method is as follows: if the organic phase change interference characteristic value is greater than a predefined organic phase change interference characteristic value used to reflect the maximum tolerable degree of organic phase change, the corresponding organic phase change interference characteristic value is marked as the organic phase change interference characteristic value to be tested, and a mobile phase stability qualification test is triggered; otherwise, a chromatographic peak identification accuracy verification is triggered. The predefined organic phase change interference characteristic value is represented by the average value of organic phase change interference characteristic values ​​over a historical time period.

[0031] like Figure 3 The diagram shown is a schematic representation of the mobile phase stability test of a gradient chromatographic analysis system for simultaneous quantitative analysis of polyflavonoid components provided in an embodiment of this invention. Figure 3 It can be seen that: first, preliminary interference optimization of the mobile phase is performed. After the preliminary interference optimization of the mobile phase is completed, the mobile phase qualification test factor is read based on the parameter combination of the organic phase change interference characteristic value and the mobile phase stability judgment value. When the monitored mobile phase qualification test factor is within the predefined mobile phase qualification range, the accurate verification of chromatographic peak identification is triggered; otherwise, the mobile phase stability optimization is triggered.

[0032] It should be added that the specific process for mobile phase stability qualification inspection is as follows: First, perform preliminary mobile phase interference optimization: Input the parameter combination of organic phase change interference characteristic value, mobile phase gas pressure monitored by a pressure sensor, and initial mobile phase flow rate monitored by a flow sensor into a predefined flow rate correlation model. Read the flow rate of the mobile phase to be adjusted. If the output flow rate of the mobile phase to be adjusted is within the predefined range of the set mobile phase flow rate, then the adjustment command corresponding to the flow rate of the mobile phase to be adjusted is fed back to the flow rate execution terminal (such as an infusion pump). Based on the corresponding command, the mobile phase flow rate is adjusted to the flow rate of the mobile phase to be adjusted, and the second step is executed; otherwise, the second step is not executed and a mobile phase flow rate adjustment warning is sent. Preliminary mobile phase interference optimization is used to reduce baseline drift and pressure fluctuations caused by changes in the organic phase ratio, thereby reducing the risk of target peak superposition and masking. Second, perform flow qualification inspection: After the preliminary mobile phase interference optimization is completed, substitute the parameter combination of the newly acquired organic phase change interference characteristic value and mobile phase stability judgment value into the set mobile phase stability qualification checklist. After querying the mobile phase compliance test factors, the third step is performed based on these factors. The mobile phase stability judgment value is represented by the ratio of the predefined baseline signal change amplitude to the linear signal change amplitude, used to measure the stability of the mobile phase during gradient elution. The maximum and minimum values ​​of the baseline signal intensity within the window corresponding to the mobile phase stability compliance test are monitored using a UV-Vis detector and a diode array detector, and the difference between the two is represented as the baseline signal change amplitude. The third step is the mobile phase stability compliance assessment: within the window corresponding to the next adjacent mobile phase stability compliance test, optimization is selectively performed based on the mobile phase compliance test factors. Specifically, when the mobile phase compliance test factors are detected to be within the predefined mobile phase compliance range set by preset personnel, accurate peak identification verification is triggered; otherwise, when the mobile phase compliance test factors are detected to be outside the predefined mobile phase compliance range, mobile phase stability optimization is triggered. Mobile phase stability optimization is used to control the balance and transition of the mobile phase, thereby achieving smooth switching during gradient changes.

[0033] According to the adjustment command corresponding to the flow rate of the mobile phase to be adjusted, the flow rate of the mobile phase is adjusted to the desired flow rate, and a flow qualification test is performed. That is, based on the parameter combination of the organic phase change interference characteristic value and the mobile phase stability judgment value re-acquired after the initial mobile phase interference optimization, a mobile phase qualification test factor is obtained. This helps to achieve precise targeted adjustment of the mobile phase flow rate and real-time closed-loop verification of the optimization effect. By combining two parameters, the comprehensive effect of flow rate adjustment on the suppression of organic phase interference and the improvement of mobile phase stability is evaluated, avoiding optimization deviations caused by single parameter judgment, further reducing baseline drift interference caused by flow rate fluctuations, and ensuring the stability of elution intensity at each stage of gradient elution.

[0034] Specifically, the process involves randomly dividing the organic phase change disturbance feature values ​​acquired over historical time periods into a training set, along with the parameter combination of the mobile phase pressure and initial mobile phase velocity, and a preset mobile phase velocity to be adjusted. This training set is then input into a predefined velocity correlation model (e.g., using a decision tree model or random forest model to learn the mapping relationship between the organic phase change disturbance feature values, the parameter combination of the mobile phase pressure and initial mobile phase velocity, and the preset mobile phase velocity to be adjusted) to obtain the training velocity correlation model. By inputting the re-acquired organic phase change disturbance feature values, the parameter combination of the mobile phase pressure and initial mobile phase velocity into the training velocity correlation model, the mobile phase velocity to be adjusted can be output.

[0035] Specifically, the process of mobile phase stability optimization is as follows: Within the predefined range of column temperature, the adjustment step size is set based on the amplitude corresponding to the optimized column temperature value. The corresponding adjustment command is fed back to the column temperature execution terminal (such as a column oven). Based on the corresponding command, the column temperature is gradually increased. This helps to improve the precision and stability of column temperature adjustment, avoid fluctuations in chromatographic column performance caused by sudden changes in column temperature, and thus achieve synergistic optimization of organic phase ratio changes and column temperature in gradient elution, precise control of baseline signal stability, effective display and peak shape optimization of low-content flavonoid peaks. Simultaneously, it provides a basis for full-wavelength scanning and peak purity identification using a DAD detector. It provides a clear spectral signal basis, reducing the interference of stray light signal superposition on peak purity judgment; each time the column temperature is increased step by step, if the re-acquired mobile phase qualification test factor is within the predefined mobile phase qualification range, it triggers accurate peak identification verification; if the flow rate column temperature is increased to the predefined maximum value, and the mobile phase qualification test factor is still outside the predefined mobile phase qualification range, a mobile phase interference warning is sent; the column temperature optimization value is obtained by inputting the mobile phase stability judgment value and the mobile phase flow rate corresponding to the final state of the initial interference optimization monitored by the flow sensor into the predefined column temperature reading table.

[0036] It should be explained that the implementation scheme of this application details tables or mapping groups obtained from the database, including a mobile phase stability qualification comparison table, a predefined column temperature reading table, a predefined sampling rate adjustment range lookup table, a predefined DAD sampling rate reading table, a target peak overlap interference degree table, and a target peak overlap correlation lookup table. It is worth noting that the corresponding mapping relationships are not fixed but dynamically changing. This characteristic enables the scheme to not only achieve a precise one-to-one mapping between individual parameters but also to handle complex situations, achieving a many-to-one mapping between multiple parameters and a single parameter.

[0037] Specifically, various relevant information collected by pre-defined personnel over a historical time period, such as parameter combinations of organic phase change interference characteristic values ​​and mobile phase stability judgment values, combinations of mobile phase flow rates corresponding to the final state of mobile phase initial interference optimization, chromatographic peak masking values, parameter combinations of initial sampling rate and gradient elution time, combination of chromatographic peak masking values ​​and gradient elution time, target peak overlap values ​​greater than predefined target peak overlap values, target peak overlap values, and parameter combinations of mobile phase flow rates and column temperatures at the start of the target peak overlap interference optimization operation, are input into a machine learning model (e.g., a decision tree model, a major advantage of which is its ability to reveal the criticality of each feature). This information is then processed using the model's feature analysis... The mechanism obtains relevant weight values ​​or other data information, including but not limited to mobile phase compliance inspection factors, column temperature optimization values, combinations of predefined maximum and minimum values ​​corresponding to the sampling rate of the DAD detector, predefined sampling rate adjustment step size, target peak overlap influence factors, combinations of predefined detection wavelength optimization values ​​and the number of times the detection wavelength is progressively increased for the DAD detector, etc.; the data obtained based on the historical time period model are associated and matched with the corresponding weights or data to generate a mobile phase stability compliance comparison table, a predefined column temperature reading table, a predefined sampling rate adjustment range lookup table, a predefined DAD sampling rate reading table, and a target peak overlap interference table. The table of degree, the target peak overlap correlation lookup table, etc., these tables or mapping groups will provide important reference standards for subsequent real-time data analysis and judgment; the parameter combination of the real-time collected organic phase change interference characteristic value and mobile phase stability judgment value, the combination of mobile phase flow rate corresponding to the mobile phase stability judgment value and the final state of the mobile phase initial interference optimization, the parameter combination of chromatographic peak masking value, the parameter combination of initial sampling rate and gradient elution time, the parameter combination of chromatographic peak masking value and gradient elution time, the target peak overlap value greater than the predefined target peak overlap value, the target peak overlap value, the parameter combination of mobile phase flow rate and column temperature at the beginning of the target peak overlap interference optimization operation, etc., are input into the pre-constructed mobile phase stability qualified reference table and the predefined The column temperature reading table, the predefined sampling rate adjustment range lookup table, the predefined DAD sampling rate reading table, the target peak overlap interference degree table, and the target peak overlap correlation lookup table are used to read relevant results such as the mobile phase qualification inspection factor, the column temperature optimization value, the combination of the predefined maximum value and the predefined minimum value corresponding to the DAD detector sampling rate, the predefined sampling rate adjustment step size, the target peak overlap influence factor, the combination of the predefined detection wavelength optimization value of the DAD detector and the number of times the detection wavelength is incremented stepwise (these results are obtained based on predefined mapping relationships). In order to ensure the standardization and consistency of the results, the value range of these results is strictly limited to the range of 0-1.

[0038] In this embodiment, interference characteristic values ​​of organic phase change are obtained by analyzing the degree of interference of organic phase changes. When the interference characteristic value of organic phase change is greater than the predefined interference characteristic value of organic phase change, the mobile phase stability qualification test is triggered; otherwise, the accurate verification of chromatographic peak identification is triggered. This helps to achieve a deep integration of quantitative assessment of the degree of interference of organic phase change, pre-screening of mobile phase stability and targeted verification of the accuracy of chromatographic peak identification. Through the graded triggering mechanism, the control requirements of different interference scenarios are accurately matched, thereby realizing the prediction, graded handling and source avoidance of baseline drift risk in multiflavonoid gradient chromatographic analysis. This effectively avoids the problem of background signal masking low-content flavonoid peaks caused by organic phase fluctuations, thereby improving the completeness and reliability of the preliminary identification of chromatographic peaks of multiflavonoid components (such as hyperoside, rutin and quercetin).

[0039] By conducting mobile phase stability compliance tests to obtain mobile phase compliance test factors, and then optimizing mobile phase stability when these factors fall outside the predefined mobile phase compliance range, the interaction between mobile phase stability compliance tests and mobile phase stability optimization is strengthened. This leads to a dynamic balance between the interaction between the mobile phase composition and the stationary phase of the chromatographic column, significantly reducing baseline fluctuations caused by changes in the organic phase ratio during gradient elution, and laying a stable separation foundation for subsequent peak identification and quantitative analysis.

[0040] As an embodiment of the first aspect, the specific process for accurate verification of chromatographic peak identification is as follows: Based on baseline noise, a chromatographic peak masking value is output to quantify the masking status of polyflavonoid chromatographic peaks; the degree of chromatographic peak masking is assessed based on the chromatographic peak masking value; baseline noise within a predefined time period is monitored using a UV-Vis detector and a diode array detector, and its average value is represented as the chromatographic peak masking value; the chromatographic peak masking degree assessment indicates that when the detected chromatographic peak masking value is not greater than the predefined chromatographic peak masking value, the corresponding polyflavonoid gradient chromatographic data (such as sampling amount, extraction solvent, ultrasonic time, dilution factor, etc.) is marked as qualified polyflavonoid gradient chromatographic data, and accurate quantitative analysis of polyflavonoid components is initiated; otherwise, the polyflavonoid gradient chromatographic data within the window period corresponding to the current accurate verification of chromatographic peak identification is fed back to the predefined personnel, and a chromatographic peak masking optimization operation is initiated in the next adjacent window period, wherein the predefined chromatographic peak masking value is represented by the average value of chromatographic peak masking values ​​over historical time periods; the chromatographic peak masking optimization operation is used to collect more data points for a chromatographic peak, reduce the degree of masking of the flavonoid target peak by baseline noise, thereby providing reliable chromatographic data for subsequent quantitative analysis.

[0041] It should be added that the specific process of chromatographic peak masking optimization is as follows: Determine the sampling rate adjustment range of the DAD detector: Input the combination of the chromatographic peak masking value, the initial sampling rate monitored by the DAQ (Data Acquisition System), and the gradient elution time monitored by the timer into the predefined sampling rate adjustment range lookup table of the DAD detector. Look up the predefined maximum and minimum values ​​corresponding to the sampling rate of the DAD detector, and use the range formed by these two values ​​as the acceptable sampling rate adjustment range of the DAD detector. Determine the sampling rate adjustment step size of the DAD detector: Input the chromatographic peak masking value and the gradient elution time monitored by the timer into the predefined DAD sampling rate readout table, and read the predefined sampling rate adjustment step size of the DAD detector. Adjust the DAD sampling rate: Within the acceptable sampling rate adjustment range of the DAD detector, set the adjustment step size based on the predefined sampling rate adjustment step size, and implement the sampling rate increment operation based on the initial sampling rate of the DAD detector. Determine the pass / fail status of chromatographic peak optimization: Each time the sampling rate is adjusted... The process involves incremental operations to acquire optimized peak masking values. It then determines whether these values ​​meet the acceptable criteria for peak masking optimization. If they do, a peak masking qualification assessment is triggered; otherwise, a warning is issued. The optimized peak masking value is represented by the difference between the initial peak masking value and the newly acquired value after each incremental sampling rate operation. The acceptable criteria for peak masking optimization are indicated by the optimized peak masking value being greater than a predefined value. The qualification assessment is as follows: after each incremental sampling rate operation, if the newly acquired peak masking value is not greater than the predefined value, the corresponding polyflavonoid gradient chromatographic data is marked as acceptable, and quantitative analysis of polyflavonoids is initiated. If, after a predefined number of incremental sampling rate operations, the peak masking value is still greater than the predefined value, a peak masking warning is issued.

[0042] By defining the range between the predefined maximum and minimum sampling rates of the DAD detector as the acceptable sampling rate adjustment range, it is helpful to standardize the control of the DAD detector's sampling rate, avoiding signal noise superposition caused by excessively fast sampling or peak distortion caused by excessively slow sampling. This establishes stable parameter boundaries for the acquisition of chromatographic peak signals of multiflavonoid components and full-wavelength spectral analysis, ensuring that the sampling process adapts to the detection requirements of dynamic changes in the organic phase during gradient elution. By setting the adjustment step size based on a predefined sampling rate adjustment step size within the acceptable sampling rate adjustment range of the DAD detector, and implementing incremental sampling rate operations based on the initial sampling rate of the DAD detector, it is helpful to improve the precision and targeting of sampling rate adjustment, avoid the decrease in detection efficiency caused by blind adjustment, and thus achieve accurate capture of weak flavonoid peak signals and effective differentiation between baseline drift signals and target peak signals, providing clear spectral information support for peak purity identification.

[0043] In this embodiment, the peak masking value is obtained by accurately verifying the chromatographic peak identification. When the peak masking value is not greater than the predefined peak masking value, the quantitative analysis of polyflavonoid components is performed. Otherwise, the peak masking is optimized. This helps to realize the interrelation between the accurate verification of chromatographic peak identification and the optimization of chromatographic peak masking. This interrelation leads to the effective unlocking of chromatographic peaks of low-content flavonoid components, the improvement of the integrity and accuracy of the identification of target flavonoid peaks (such as hyperoside, rutin, and quercetin), the optimization of the clarity of peak boundary determination, and the reliability of peak purity identification by the DAD detector. This avoids the peak masking and misjudgment problems caused by baseline drift from the source.

[0044] As an embodiment of the second aspect, the specific process for accurate verification of chromatographic peak identification is as follows: Based on the peak area of ​​the overlapping portion of adjacent target peaks, a target peak overlap value is output to reflect the overlapping status of the target peaks of polyflavonoid components; the peak area of ​​the overlapping portion of adjacent target peaks is monitored by high-performance liquid chromatography (HPLC), and the ratio of its average value to the total detected area of ​​the target peaks is expressed as the target peak overlap value; a judgment is made based on the target peak overlap value and a predefined target peak overlap value. Specifically, if the target peak overlap value is greater than the predefined target peak overlap value, the target peak overlap degree analysis is initiated; otherwise, the corresponding polyflavonoid gradient chromatographic data is marked as qualified polyflavonoid gradient chromatographic data, and accurate quantitative analysis of polyflavonoid components is initiated. In this process, the predefined target peak overlap value is represented by the average value of target peak overlap values ​​over a historical period. The specific process for analyzing the degree of target peak overlap is as follows: Input the target peak overlap value that is greater than the predefined target peak overlap value into the set target peak overlap interference degree table, and query the target peak overlap influence factor; determine whether the target peak overlap influence factor is within the predefined target peak overlap qualification range. If so, mark the corresponding polyflavonoid gradient chromatographic data as qualified polyflavonoid gradient chromatographic data and start the accurate quantitative analysis of polyflavonoids. Otherwise, start the target peak overlap interference optimization operation to reduce peak overlap interference from the detection response level, and provide an accurate data basis for the identification and synchronous quantification of polyflavonoids.

[0045] It should be added that the specific process for target peak overlap interference optimization is as follows: The target peak overlap value, the mobile phase velocity monitored by the flow velocity sensor at the start of the optimization operation, and the column temperature monitored by the column temperature chamber are combined and input into a pre-set target peak overlap correlation lookup table. The table then queries the predefined detection wavelength optimization value and the number of increments of the detection wavelength. Within the predefined range of the detection wavelength, the amplitude of the predefined detection wavelength optimization value is used as the adjustment step size, and the number of increments of the detection wavelength is used as the corresponding number of operations. Based on the initial detection wavelength, performing a step-by-step increase in the detection wavelength helps improve the detection wavelength optimization... The precision and adaptability of the section avoid the ambiguity of peak identification caused by blind adjustment, thereby optimizing and improving the separation of adjacent flavonoid peaks and effectively distinguishing overlapping peak signals. This provides suitable spectral detection conditions for the accurate identification and peak purity determination of target peaks (such as hyperoside, rutin, and quercetin), alleviating the separation problem caused by the decline in column efficiency. After the operation of gradually increasing detection wavelength is completed, if the target peak overlap influence factor is within the predefined target peak overlap qualification range, the corresponding polyflavonoid gradient chromatographic data will be marked as qualified polyflavonoid gradient chromatographic data, and the quantitative and accurate analysis of polyflavonoid components will be initiated. Otherwise, a target peak overlap warning will be sent.

[0046] In this embodiment, after repeated use of the chromatographic column, the decreased column efficiency leads to reduced resolution and increased overlap of adjacent flavonoid peaks. This verification calculates the target peak overlap value to determine whether the chromatographic column can still meet the separation requirements for polyflavonoids. Specifically, it obtains the target peak overlap value through accurate peak identification verification. When the target peak overlap value is greater than a predefined target peak overlap value, target peak overlap degree analysis is initiated to obtain the target peak overlap influence factor; otherwise, accurate quantitative analysis of polyflavonoid components is initiated. When the target peak overlap influence factor is within the predefined acceptable range for target peak overlap, accurate quantitative analysis of polyflavonoid components is initiated; otherwise, target peak overlap interference optimization is initiated. This helps to avoid quantitative errors caused by the overlap of adjacent flavonoid peaks due to decreased column efficiency, ensuring the stability and accuracy of polyflavonoid component separation and quantitative analysis, thereby specifically addressing the technical pain point of reduced resolution after repeated use of the chromatographic column.

[0047] Furthermore, the specific process for accurate quantitative analysis of polyflavonoid components is as follows: The component separation performance value is obtained based on the average resolution of the target peak, reflecting the high efficiency of polyflavonoid component separation; the target peak resolution is monitored using high-performance liquid chromatography (HPLC), and its average value is used as the component separation performance value; a quantitative deviation determination is performed to reflect the quantitative deviation of polyflavonoid components. The specific process is as follows: It is determined whether the component separation performance value is greater than a predefined component separation performance value, where the predefined component separation performance value is represented by the average value of component separation performance values ​​over a historical time period; if so, a polyflavonoid component quantitative qualification notification is sent, and the corresponding reference solution and test solution are marked as qualified reference solution and qualified test solution, respectively; otherwise, a polyflavonoid component quantitative failure notification is sent.

[0048] The preparation of the reference solution provided in this application embodiment is as follows: Take appropriate amounts of rutin reference standard, hyperoside reference standard, and quercetin reference standard, accurately weigh them, and dilute them with methanol to prepare a mixed reference solution containing 100 μg of rutin, 5 μg of hyperoside, and 5 μg of quercetin per 1 ml.

[0049] The test solution is prepared as follows: Take an appropriate amount of this product, remove the film coating, grind it into a fine powder, take about 2g, weigh it accurately, place it in a stoppered conical flask, accurately add 20 ml of 70% methanol, weigh it, sonicate it (power 350W, 40kHz) for 1 hour, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0050] Furthermore, in the embodiments of this application, octadecylsilane-bonded silica gel is used as the filler; acetonitrile is used as mobile phase A, and 0.2% phosphoric acid solution is used as mobile phase B, and gradient elution is performed according to the provisions of Table 1.

[0051] Table 1

[0052] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～25 10 90 25～27 10→16 90→84 27～50 16 84 50～52 16→27 84→73 52～65 27 73 65～67 27→95 73→5 67～72 95 5 72～75 95→10 5→90

[0053] The test method provided in this application showed a good linear relationship between rutin concentration and peak area in the range of 1.06310 to 88.59160 μg / ml: y = 40.558x + 8.4356 (R 2 =0.9994); hyperoside showed a good linear relationship with peak area in the concentration range of 0.50563–5.05627 μg / ml: y = 35.673x + 0.2458 (R = 0.9994). 2 =0.9991); Quercetin showed a good linear relationship with peak area in the concentration range of 0.44000–4.40004 μg / ml: y = 89.248x - 1.1367 (R = 0.9991). 2 =0.9991), where R 2 This represents the linear correlation coefficient.

[0054] like Figure 4 The figure shows the linear relationship between rutin concentration and peak area in a gradient chromatographic analysis method for simultaneous quantitative analysis of polyflavonoid components provided in this application embodiment. The concentration of rutin in the figure is in the range of 1.06310 to 88.59160 μg / ml. The rutin concentration and peak area are fitted with a linear equation y=40.558x+8.4356. The peak area itself does not have a fixed physical unit, but is defined according to the detection method of the instrument, and can be considered as a response unit or a detection signal unit.

[0055] The test methods provided in this application yielded the following limits of detection (LOD) for rutin reference standard: 7.1 ng, LOD for quantification: 21.3 ng; for hyperoside reference standard: 10.6 ng, LOD for quantification: 30.3 ng; and for quercetin reference standard: 5.3 ng, LOD for quantification: 8.8 ng.

[0056] like Figure 5 The diagram shows a flowchart of a gradient chromatographic analysis method for simultaneous quantitative determination of polyflavonoid components provided in this application embodiment. This method, applied to a gradient chromatographic analysis system for simultaneous quantitative determination of polyflavonoid components, includes the following steps:

[0057] S1, Organic Phase Change Monitoring: During the gradient chromatographic analysis of polyflavonoid components, the degree of interference of organic phase changes is analyzed to obtain analytical results that reflect the changes in the proportion of organic phase during gradient elution. Based on the analytical results, it is determined whether to perform accurate peak identification verification to assess the accuracy of peak identification. If so, the corresponding judgment result is transmitted to S2; otherwise, the mobile phase stability qualification test is performed to assess the stability of the mobile phase during gradient elution.

[0058] S2, Chromatographic Peak Identification Monitoring: Based on the received judgment results, perform chromatographic peak identification accuracy verification to assess the accuracy of chromatographic peak identification. After the verification is completed, transmit the qualified verification results to S3.

[0059] S3, Quantitative and accurate monitoring and analysis: Based on the received qualified verification results, perform quantitative and accurate analysis of polyflavonoid components to analyze the degree of qualified separation of polyflavonoid components.

[0060] In this embodiment, the separation performance value of polyflavonoid components is obtained by accurate quantitative analysis. When the separation performance value is greater than the predefined separation performance value, the corresponding qualified reference solution and qualified test solution are obtained. Otherwise, the quantitative analysis of polyflavonoid components is not qualified. This helps to achieve quantitative evaluation and qualification screening of polyflavonoid component separation effect, timely interception and early warning of unqualified separation data, and precise control of the quality of reference and test solutions. This ensures the accuracy of polyflavonoid component content calculation, the strictness of the implementation of quality standards for polyflavonoid-containing preparations, and the reliability of product quality control results.

[0061] In summary, the embodiments of this invention analyze the degree of interference in the changes of the organic phase to obtain analytical results reflecting the changes in the proportion of the organic phase during gradient elution. Based on the analytical results, it determines whether to perform accurate verification of chromatographic peak identification. This helps to quantitatively predict and classify the baseline drift risk caused by fluctuations in the organic phase proportion, identify interference sources that may mask low-content peaks in advance, and avoid ineffective peak identification verification processes. By performing accurate verification of chromatographic peak identification and, after verification, performing accurate quantitative analysis of polyflavonoid components to evaluate the separation of polyflavonoid components and obtaining qualified test solutions, it helps to accurately control the identification and separation effect of target peaks. This ensures that target peaks (such as hyperoside and rutin) are not masked by baseline drift and verifies the reliability of the basic data for quantitative calculation through indicators such as resolution. In this way, it improves the accuracy of simultaneous quantitative data of polyflavonoid components based on gradient chromatography and solves the problem of low accuracy of simultaneous quantitative data of polyflavonoid components based on gradient chromatography in the prior art.

[0062] The above are merely specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A gradient chromatographic analysis system for simultaneous quantitative analysis of polyflavonoid components, characterized in that, include: Organic phase change monitoring module, chromatographic peak identification monitoring module, and quantitative accurate monitoring and analysis module; The organic phase change monitoring module is used to analyze the degree of interference of organic phase changes during gradient chromatographic analysis of polyflavonoid components, and obtain analytical results that reflect the changes in the proportion of organic phase during gradient elution. This allows the module to determine whether to perform accurate peak identification verification to assess the accuracy of peak identification. If so, the corresponding judgment result is transmitted to the peak identification monitoring module; otherwise, a mobile phase stability qualification test is performed to assess the stability of the mobile phase during gradient elution. The chromatographic peak identification and monitoring module is used to verify the accuracy of chromatographic peak identification in order to evaluate the accuracy of chromatographic peak identification. After the verification is completed, the qualified verification results are transmitted to the quantitative accuracy monitoring and analysis module. The quantitative and accurate monitoring and analysis module is used to perform quantitative and accurate analysis of polyflavonoid components based on the received qualified verification results, which is used to analyze the degree of qualified separation of polyflavonoid components. The specific process for analyzing the degree of interference with changes in the organic phase is as follows: The organic phase change interference characteristic value is output based on the organic phase ratio at the gradient endpoint and the organic phase ratio at the gradient starting point to reflect the change in the organic phase ratio during gradient elution. The organic phase ratio at the gradient endpoint is expressed as the volume percentage of the organic phase in the mobile phase at the end of elution. The gradient starting point organic phase ratio is expressed as the volume percentage of the organic phase in the mobile phase at the initial elution time. The degree of interference is determined based on the characteristic value of organic phase change interference. The specific determination method is as follows: if the characteristic value of organic phase change interference is greater than the predefined characteristic value of organic phase change interference, the corresponding characteristic value of organic phase change interference is marked as the characteristic value of organic phase change interference to be tested, and the mobile phase stability qualification test is triggered; otherwise, the accurate verification of chromatographic peak identification is triggered. The specific procedure for the mobile phase stability compliance test is as follows: The first step is to perform preliminary interference optimization of the mobile phase: The combination of the characteristic value of the organic phase change interference and the parameters of the mobile phase pressure and the initial mobile phase velocity is input into the predefined velocity correlation model. If the output mobile phase velocity to be adjusted is within the predefined range of the set mobile phase velocity, the adjustment command corresponding to the mobile phase velocity to be adjusted is fed back to the velocity execution terminal. Based on the corresponding command, the mobile phase velocity is adjusted to the mobile phase velocity to be adjusted, and the second step is executed. Otherwise, the second step is not executed and a mobile phase velocity adjustment warning prompt is sent. The initial disturbance optimization of the mobile phase is used to reduce baseline drift and pressure fluctuations caused by changes in the organic phase ratio, thereby reducing the risk of target peak overlap and masking. The second step is to conduct a flow qualification test: After the initial interference optimization of the mobile phase is completed, the parameter combination of the newly acquired organic phase change interference characteristic value and the mobile phase stability judgment value is substituted into the set mobile phase stability qualification comparison table, and the mobile phase qualification test factor is queried before proceeding to the third step. The mobile phase stability judgment value is used to measure the degree of stability of the mobile phase during gradient elution. The third step is to evaluate the stability of the mobile phase: within the window period corresponding to the next adjacent mobile phase stability test, optimization is performed based on the selectivity of the mobile phase stability test factor. Specifically, when the mobile phase stability test factor is detected to be within the predefined mobile phase test range, accurate verification of chromatographic peak identification is triggered; otherwise, mobile phase stability optimization is triggered. The mobile phase stability optimization is used to control the balance and transition of the mobile phase, thereby achieving smooth switching during gradient changes. The specific process for optimizing the stability of the mobile phase is as follows: Within the predefined range of column temperature, the adjustment step size is set based on the amplitude corresponding to the optimized column temperature value, and the corresponding adjustment command is fed back to the column temperature execution terminal. Based on the corresponding command, the column temperature is gradually increased. Each time the column temperature is increased step by step, if the mobile phase qualification test factor is within the predefined mobile phase qualification range, the accurate peak identification verification is triggered. If the mobile phase flow rate and column temperature are increased to the predefined maximum value of the column temperature, and the mobile phase qualification test factor is still not within the predefined mobile phase qualification range, a mobile phase interference warning is sent. The optimized column temperature value is obtained by inputting the mobile phase stability judgment value and the mobile phase flow rate corresponding to the final state of the initial disturbance optimization of the mobile phase into a predefined column temperature reading table.

2. The gradient chromatographic analysis system for simultaneous quantitative determination of polyflavonoid components according to claim 1, characterized in that, The specific procedure for verifying the accuracy of chromatographic peak identification is as follows: Based on the baseline noise output, the chromatographic peak masking value is used to quantify the masking of chromatographic peaks of polyflavonoid components, and the degree of chromatographic peak masking is evaluated based on the chromatographic peak masking value. The chromatographic peak masking assessment means that when the detected chromatographic peak masking value is not greater than the predefined chromatographic peak masking value, the corresponding polyflavonoid gradient chromatographic data is marked as qualified polyflavonoid gradient chromatographic data, and the quantitative and accurate analysis of polyflavonoid components is initiated. Otherwise, the polyflavonoid gradient chromatographic data within the window period corresponding to the current accurate verification of chromatographic peak identification is fed back to the predefined personnel, and the chromatographic peak masking optimization operation is initiated in the next adjacent window period.

3. The gradient chromatographic analysis system for simultaneous quantitative analysis of polyflavonoid components according to claim 2, characterized in that, The specific process of the chromatographic peak masking and optimization operation is as follows: Determine the sampling rate adjustment range of the DAD detector: Input the parameter combination of chromatographic peak masking value, initial sampling rate and gradient elution time into the predefined sampling rate adjustment range lookup table of the DAD detector, look up the predefined maximum value and predefined minimum value corresponding to the sampling rate of the DAD detector, and then take the range formed by the two as the qualified sampling rate adjustment range of the DAD detector. Determine the sampling rate adjustment step size of the DAD detector: Input the chromatographic peak masking value and gradient elution time into the predefined DAD sampling rate reading table, and read the predefined sampling rate adjustment step size of the DAD detector; Adjusting the DAD sampling rate: Within the acceptable sampling rate adjustment range of the DAD detector, set the adjustment step size based on the predefined sampling rate adjustment step size, and implement the sampling rate increment operation based on the initial sampling rate of the DAD detector. Determining the pass / fail status of chromatographic peak optimization: After each sampling rate increment operation, obtain the chromatographic peak masking optimization value, and determine whether the chromatographic peak masking optimization value meets the pass / fail conditions. If it does, trigger the chromatographic peak masking pass / fail determination; otherwise, send a chromatographic peak optimization level warning. The optimized chromatographic peak masking value is represented by the difference between the chromatographic peak masking value at the beginning of the chromatographic peak masking tuning operation and the chromatographic peak masking value re-acquired after each sampling rate increment operation. The qualified condition for chromatographic peak masking optimization means that the chromatographic peak masking optimization value is greater than the predefined chromatographic peak masking optimization value; Determining the pass / fail status of chromatographic peak masking: If the newly acquired chromatographic peak masking value is not greater than the predefined chromatographic peak masking value after each sampling rate increment operation, the corresponding polyflavonoid gradient chromatographic data is marked as qualified polyflavonoid gradient chromatographic data, and quantitative accurate analysis of polyflavonoid components is initiated. If the chromatographic peak masking value is still greater than the predefined chromatographic peak masking value after a predefined number of sampling rate increment operations, a chromatographic peak masking warning is sent.

4. The gradient chromatographic analysis system for simultaneous quantitative determination of polyflavonoid components according to claim 1, characterized in that, The specific procedure for verifying the accuracy of chromatographic peak identification is as follows: The peak area of ​​the overlapping portion of adjacent target peaks is used to output the target peak overlap value to reflect the target peak superposition status of polyflavonoid components; The target peak overlap value is represented by the ratio of the average peak area of ​​the overlapping portion of adjacent target peaks to the total detected area of ​​the target peaks; The judgment is based on the target peak overlap value and the predefined target peak overlap value. The specific judgment criteria are as follows: if the target peak overlap value is greater than the predefined target peak overlap value, the target peak overlap degree analysis is initiated; otherwise, the corresponding polyflavonoid gradient chromatographic data is marked as qualified polyflavonoid gradient chromatographic data, and the quantitative and accurate analysis of polyflavonoid components is initiated. The specific process for analyzing the overlap of the target peaks is as follows: Input the target peak overlap value that is greater than the predefined target peak overlap value into the set target peak overlap interference degree table, and query the target peak overlap influence factor. Determine whether the target peak overlap influence factor is within the predefined target peak overlap qualification range. If so, mark the corresponding polyflavonoid gradient chromatographic data as qualified polyflavonoid gradient chromatographic data and start accurate quantitative analysis of polyflavonoids. Otherwise, start the target peak overlap interference optimization operation to reduce peak overlap interference from the detection response level, and provide an accurate data basis for the identification and synchronous quantification of polyflavonoids.

5. The gradient chromatographic analysis system for simultaneous quantitative determination of polyflavonoid components according to claim 4, characterized in that, The specific process for optimizing the target peak overlap interference is as follows: Input the target peak overlap value, the combination of mobile phase flow rate and column temperature at the start of the target peak overlap interference optimization operation into the pre-set target peak overlap correlation lookup table, and query the predefined detection wavelength optimization value of the DAD detector and the number of times the detection wavelength is incremented step by step; Within the predefined range of the detection wavelength, the amplitude of the predefined detection wavelength tuning value is used as the adjustment step size, and the number of times the detection wavelength is increased step by step is used as the corresponding number of operations. Based on the initial detection wavelength, the detection wavelength is increased step by step. After the detection wavelength is increased step by step, if the target peak overlap influence factor is within the predefined target peak overlap qualification range, the corresponding polyflavonoid gradient chromatographic data will be marked as qualified polyflavonoid gradient chromatographic data, and the quantitative and accurate analysis of polyflavonoid components will be initiated; otherwise, a target peak overlap warning will be sent.

6. A gradient chromatographic analysis system for simultaneous quantitative determination of polyflavonoid components according to claim 2 or 4, characterized in that, The specific process for accurate quantitative analysis of the polyflavonoid components is as follows: The component separation performance value is obtained based on the average value of the target peak resolution, which is used to reflect the degree of efficient separation of polyflavonoid components; The degree of quantitative deviation is determined to reflect the quantitative deviation of polyflavonoid components. The specific process is as follows: Determine whether the component separation performance value is greater than the predefined component separation performance value; If yes, send a notification that the quantification of polyflavonoids is qualified, and mark the corresponding reference solution and test solution as qualified reference solution and qualified test solution, respectively; otherwise, send a notification that the quantification of polyflavonoids is unqualified.

7. A gradient chromatographic analysis method for simultaneous quantitative determination of polyflavonoid components, applied to the gradient chromatographic analysis system for simultaneous quantitative determination of polyflavonoid components according to any one of claims 1-5, characterized in that, Includes the following steps: S1, during the gradient chromatographic analysis of polyflavonoid components, analyze the degree of interference of changes in the organic phase, obtain analytical results to reflect the changes in the proportion of organic phase in gradient elution, and determine whether to perform accurate peak identification verification to assess the accuracy of peak identification. If so, the corresponding judgment result is transmitted to S2; otherwise, perform mobile phase stability qualification test to assess the stability of the mobile phase in gradient elution. S2, perform accurate verification of chromatographic peak identification to evaluate the accuracy of chromatographic peak identification, and after the verification is completed, transmit the qualified verification results to S3; S3. Based on the received qualified verification results, perform accurate quantitative analysis of polyflavonoid components to analyze the degree of qualified separation of polyflavonoid components.

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