Method for determination of dissolution of new heparinoid marine drug by ion chromatography

Abstract

The application provides a method for detecting the dissolution rate of a new type of heparinoid marine medicine by using ion chromatography, and relates to the field of medicine detection, and comprises the following steps: weighing a poly-mannuronic acid propyl ester sodium sulfate control sample to prepare a control sample solution; taking one test sample, dissolving to obtain a test sample dissolution solution; taking the dissolution solution, filtering and discarding the initial filtrate, and taking the subsequent filtrate as the test sample solution; setting ion chromatograph detection conditions: selecting a polymer matrix cation chromatographic column, the detector is an inhibition conductivity detector, setting the eluent feed flow, column temperature and detector temperature; respectively injecting the control sample solution and the test sample solution into the ion chromatograph, recording the peak area in the chromatogram, and determining the dissolution rate of the poly-mannuronic acid propyl ester sodium sulfate tablet according to the peak area of the control sample solution and the test sample solution. The application uses the ion chromatograph to detect the sodium element in the poly-mannuronic acid propyl ester sodium sulfate to calculate the dissolution rate, and overcomes the problems of poor reproducibility and unsatisfactory detection precision of the existing detection method.

Description

Technical Field

[0001] This invention relates to the field of drug detection technology, specifically a method for detecting the dissolution rate of novel heparin-like marine drugs using ion chromatography. Background Technology

[0002] Sodium polymannuronate propyl sulfate tablets are a novel heparin-like marine drug with the effects of lowering blood cholesterol and triglycerides, and increasing high-density lipoprotein (HDL). This product is an oral solid dosage form, and to ensure product quality, dissolution is a mandatory testing item and dissolution curve studies are required. However, the national drug standards for this product do not include methods for dissolution and dissolution curve testing, which is detrimental to product quality control.

[0003] The general methods for determining the content of sulfated polysaccharides such as sodium polymannuronate sulfate (direct staining method, acid degradation-color development method, spectrophotometric titration method, and liquid chromatography method) all have limitations, such as poor reproducibility and unsatisfactory detection precision. Summary of the Invention

[0004] This invention provides a method for detecting the dissolution rate of novel heparin-like marine drugs using ion chromatography, thereby solving the technical problems mentioned in the background.

[0005] To address the aforementioned technical problems, this invention discloses a method for detecting the dissolution rate of novel heparin-like marine drugs using ion chromatography, comprising: S1: Weigh out sodium polymannuronate propyl sulfate reference standard, add it to the dissolution medium to dissolve it, and obtain the reference standard solution; Take one sodium polymannuron sulfate test tablet and put it into a dissolution apparatus containing a preheated dissolution medium of a set volume. Stir to dissolve the test sample and obtain the test sample dissolution solution. Take the test sample dissolution solution, filter it, discard the initial filtrate, and take the subsequent filtrate as the test sample solution. S2: Set the detection conditions for the ion chromatograph: Select the chromatographic column for ion chromatography detection, the detector is a suppressed conductivity detector, and set the eluent feed flow rate, column temperature, and detector temperature; S3: Inject the reference solution and the test solution into the ion chromatograph separately, record the peak areas in the chromatograms, and determine the dissolution rate of the test sample based on the peak areas of the reference solution and the test solution.

[0006] Preferably, the rinsing solution is an aqueous solution of methanesulfonic acid, and the dissolution medium in S1 is a pH 6.8 medium.

[0007] Preferably, the concentration of the methanesulfonic acid aqueous solution is 20 mmol / L.

[0008] Preferably, the method for preparing the pH 6.8 medium is as follows: weigh 1.7g of anhydrous potassium dihydrogen phosphate and 1.745g of anhydrous dipotassium hydrogen phosphate, add 1000ml of degassed water, stir until completely dissolved and measure the pH, and adjust the pH to 6.8 using phosphoric acid.

[0009] Preferably, when used for testing 0.1g test samples, the concentration of sodium polymannuronate propyl sulfate in the reference solution is 0.10–0.12 mg / mL; when used for testing 50mg test samples, the concentration of sodium polymannuronate propyl sulfate in the reference solution is 0.050–0.060 mg / mL.

[0010] Preferably, the chromatographic column for ion chromatography detection includes a cation exchange analytical column and a matching cation exchange guard column; The cation exchange analysis column is a polymer matrix cation exchange analysis column bonded with carboxylic acid functional groups; The cation exchange guard column is a polymer matrix cation exchange guard column bonded with carboxylic acid functional groups.

[0011] Preferably, in S2, the eluent feed flow rate is 0.8–1.2 mL / min, the column temperature is 35–45 °C, and the detector temperature is 25–35 °C.

[0012] Preferably, in step S2, the eluent feed flow rate is 1 mL / min, the column temperature is 40°C, and the detector temperature is 30°C.

[0013] Preferably, during batch detection in the ion chromatograph, the detection and analysis process is performed periodically, and the detection and analysis process includes: S3a: The target stage before sample injection and eluent flow into the ion chromatograph involves detecting key instrument operating parameters, including: the flow rate at the outlet of the eluent feed pump, the operating current of the suppression conductivity detector, and the steady-state conductivity collected by the suppression conductivity detector when no chromatographic peak signal of the sample component is detected; one initial detection and at most one pre-conditioning re-detection are completed within the target stage. S3b: Obtain the baseline values ​​of preset key instrument operating parameters, and combine them with the key instrument operating parameters detected by S3a to determine the actual feed flow coefficient, actual operating current coefficient, and actual steady-state conductivity coefficient; if any of the actual feed flow coefficient, actual operating current coefficient, and actual steady-state conductivity coefficient does not meet the corresponding allowable operating range, a first-level alarm will be triggered. S3c: When there is no first-level alarm, the flow-baseline influence coefficient is obtained by combining the actual feed flow coefficient and the actual steady-state conductivity coefficient, and the suppression state coefficient of the suppression conductivity detector is determined by combining the actual operating current coefficient and the actual steady-state conductivity coefficient. When either the flow-baseline impact coefficient or the suppression status coefficient meets the corresponding alarm range, a second-level alarm is triggered, and the corresponding warning type prompt is output. S3d: When S3b and S3c do not generate alarms: When the flow-baseline influence coefficient meets the corresponding pre-control range, the first pre-control is executed: the output flow rate of the eluent feed pump is adjusted in the direction of reducing the coefficient; When the suppression state coefficient meets the corresponding pre-control range, the second pre-control is executed: the operating current of the suppressor is increased. If the flow-baseline impact coefficient or the suppression state coefficient meets the corresponding alarm range after the pre-control is performed and then S3a is performed, then the second-level alarm is executed directly.

[0014] Preferably, the detection and analysis process also includes: S3e: When the first-level alarm and the second-level alarm are not triggered, the sample is injected sequentially when the sample is separated from the peak. The detector collects the chromatographic signal of the sample and obtains at least one of the relative retention time, peak asymmetry factor and peak area based on the chromatographic signal. S3f: Compare the relative retention time and peak asymmetry factor obtained from S3e with the corresponding preset reference range. If either the relative retention time or peak asymmetry factor is outside the corresponding allowable reference range, a level three alarm will be triggered. If no Level 3 alarm is triggered and the relative retention time and peak asymmetry factor meet the corresponding correction feedback range, the flow adjustment range and current adjustment range of the next S3d will be corrected based on the relative retention time and peak asymmetry factor determined by S3e.

[0015] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention employs ion chromatography to calculate dissolution rate by detecting sodium in sodium polymannuronate propyl sulfate (and dissolution curves can be constructed using conventional methods). This fills the gap in the national drug standards for this product, which lack methods for detecting dissolution rate and dissolution curves, and also solves the problems of low response and poor specificity of HPLC methods in the literature. Furthermore, the instruments used in this method are common in pharmaceutical R&D and production laboratories, eliminating the need for cumbersome sample solution pretreatment; the dissolution solution can be directly analyzed after extraction, making it suitable for product quality control in industrial production processes. Attached Figure Description

[0017] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 This is a chromatogram of the test solution of the present invention; Figure 2 This is a chromatogram of the reference solution of the present invention; Figure 3 The graph shows the concentration-peak area relationship of sodium polymannuronate propyl sulfate in the pH 6.8 dissolution medium, as determined by ion chromatography in the linearity test. Detailed Implementation

[0018] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0019] Furthermore, in this invention, the use of terms such as "first" and "second" is for descriptive purposes only and does not specifically refer to any order or sequence, nor is it intended to limit the invention. They are merely used to distinguish components or operations described using the same technical terms and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Additionally, the technical solutions and features of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. If a combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by this invention.

[0020] The present invention provides the following embodiments: Example 1: This embodiment of the invention provides a method for detecting the dissolution rate of sodium polymannuronate sulfate tablets using ion chromatography, comprising: S1: Weigh out sodium polymannuronate propyl sulfate reference standard, add it to the dissolution medium to dissolve it, and obtain the reference standard solution; Take one sodium polymannuron sulfate test tablet and put it into a dissolution apparatus containing a set volume V (500ml, 900ml, or 1000ml) of dissolution medium preheated to 37℃±0.5℃. Stir to dissolve the test sample and obtain the test sample dissolution solution. Take the test sample dissolution solution, filter it, discard the initial filtrate, and take the subsequent filtrate as the test sample solution. S2: Set the ion chromatography detection conditions: Select the chromatographic column for ion chromatography detection, the detector is a suppressed conductivity detector, and set the eluent feed flow rate, column temperature, and detector temperature; injection volume: 10 μl; S3: Inject the reference solution and the test solution into the ion chromatograph separately, record the peak areas in the chromatograms, and determine the dissolution rate of the test sample based on the peak areas of the reference solution and the test solution.

[0021] ; in, The peak area of ​​the reference solution; The peak area of ​​the test solution; This refers to the mass concentration of the reference solution. The mass concentration of the test solution; Dissolution rate of sodium polymannuron sulfate tablets ; The indicated mass of sodium polymannuronate sulfate contained in each sample shall be specified. The set volume in S1; The rinsing solution is an aqueous solution of methanesulfonic acid, and the dissolution medium in S1 is a pH 6.8 medium (1.7g of anhydrous potassium dihydrogen phosphate and 1.745g of anhydrous dipotassium hydrogen phosphate are weighed, 1000ml of degassed water is added, stirred until completely dissolved and the pH is measured, and the pH is adjusted to 6.8 with phosphoric acid).

[0022] The concentration of the methanesulfonic acid aqueous solution is 20 mmol / L.

[0023] When used for testing 0.1g samples, the concentration of sodium polymannuronate propyl sulfate in the reference solution is 0.10–0.12 mg / mL; when used for testing 50mg samples, the concentration of sodium polymannuronate propyl sulfate in the reference solution is 0.050–0.060 mg / mL.

[0024] The chromatographic column for ion chromatography detection includes a cation exchange analytical column and a matching cation exchange guard column; Cation exchange analytical columns are polymer-based cation exchange analytical columns with carboxylic acid functional groups bonded together, such as the Dionex IonPac CS12A 4.0mm. 250mm; The cation exchange guard column is a polymer-based cation exchange guard column with carboxylic acid functional groups bonded together, such as the Dionex IonPac CG 12A 4.0mm. 50mm.

[0025] In S2, the eluent feed flow rate is 0.8–1.2 mL / min, the column temperature is 35–45 °C, and the detector temperature is 25–35 °C.

[0026] In S2, the eluent feed flow rate is 1 mL / min, the column temperature is 40℃, and the detector temperature is 30℃.

[0027] like Figures 1-2 The figures shown are the chromatograms of the test solution and the reference solution of the present invention, respectively. (1) Among them, the test solution: according to the pharmacopoeia dissolution test method, the test sample (sodium polymannuronate sulfate test sample tablet) is put into the dissolution medium and stirred in the dissolution apparatus at the specified speed (50-75 r / min for paddle method and 50-100 r / min for basket method) and temperature (e.g., 37℃). The tablet disintegrates and the test sample is released and dissolved in the medium. The resulting liquid is the test sample dissolution solution. Take the dissolution solution and filter it (you can choose a 0.45μm polyethersulfone (PES) aqueous needle filter). The first part of the filtrate that flows out at the beginning of filtration is called the initial filtrate. After discarding the initial filtrate, the filtrate collected later is called the subsequent filtrate.

[0028] (2) The suppression-type conductivity detector used in this method is essentially a type of conductivity detector. Its core detection principle is to quantify substances by measuring changes in the conductivity of the solution. This method uses methanesulfonic acid aqueous solution as the eluent for ion chromatography. This solution itself has a high ion content and strong conductivity. If a common conductivity detector is used for direct detection, the high-intensity background conductivity signal generated by the eluent will completely mask the sample response signal of the sodium polymannuronate propyl sulfate to be tested, making effective quantification impossible.

[0029] The suppression-type conductivity detector achieves two key optimizations through a built-in suppressor to ensure that detection can proceed normally: Reduce background interference: Convert methanesulfonic acid in the eluent into water with extremely weak conductivity, significantly reduce the background conductivity of the mobile phase, and eliminate the masking of sample signal by high background. Enhanced conductivity response of analytes: By replacing the counterions of the sodium polymannuronate sulfate component with ion exchange, the analyte is transformed into an ion pair with a stronger conductivity response, thereby improving the conductivity output of the analyte itself. Combined with the reduction of background conductivity of the mobile phase, the signal-to-noise ratio and sensitivity of the detection are improved.

[0030] Finally, the conductivity change of the chromatographic effluent is measured by the conductivity detection unit of the detector to obtain the peak area of ​​the corresponding chromatographic peak; the peak area is proportional to the content of sodium polymannuronate propyl sulfate, thereby completing the quantitative calculation of sodium polymannuronate propyl sulfate.

[0031] The beneficial effects of the above technical solution are as follows: This invention uses an ion chromatograph to calculate the dissolution rate by detecting the sodium element in sodium polymannuronate propyl sulfate (and the dissolution curve can be constructed according to the commonly used dissolution curve construction method). This fills the gap in the national drug standard for this product, which lacks methods for detecting dissolution rate and dissolution curve, and also solves the problems of low response and poor specificity of HPLC methods in the literature. Furthermore, the instruments used in this method are common in pharmaceutical R&D and production laboratories, eliminating the need for cumbersome pretreatment of the test solution; the dissolution solution can be directly analyzed after extraction, making it suitable for product quality control in industrial production processes.

[0032] To verify the reliability of the above-mentioned ion chromatography method for determining the dissolution rate of sodium polymannuronate sulfate tablets, methodological validation experiments were conducted on specificity, linearity, accuracy, and solution stability. The specific results are as follows: To investigate the specificity of this method, determinations were performed on blank solvent, reference solution, pH 6.8 medium, and test solution with pH 6.8 medium as the dissolution medium. The results are shown in Table 1. Table 1 Results of specificity test

[0033] The results showed that: (1) the blank solvent and blank excipient did not interfere with the detection; (2) the theoretical plate number of the main peak of the reference solution was 8475 (>2000), and the tailing factor was 1.06 (between 0.8 and 1.8); the method has good specificity.

[0034] Blank excipients refer to excipient matrices that do not contain active pharmaceutical ingredients.

[0035] A series of sodium polymannuron sulfate standard solutions of various concentrations were prepared, and the linear relationship between chromatographic peak area and concentration was investigated. The results are shown in Table 2. Table 2 Results of linear experiments

[0036] The linear relationship diagram corresponding to the linear experiment is shown in the attached figure of the instruction manual. Figure 3 ; For the 0.1g and 50mg formulations, sodium polymannuronate propyl sulfate solutions of different concentrations were prepared, and the recovery rates were determined. The results are shown in Table 3. Table 3 Accuracy Test Results

[0037] The peak area changes of the test solution after being placed at room temperature for different times were investigated to evaluate the stability of the solution. The results are shown in Table 4. Table 4 Results of solution stability test

[0038] The results showed that the ratio of dissolution at each time point to 0 h for the test solution (50 mg specification) was between 98.0% and 102.0%, and the solution was stable after 60 hours at room temperature; the ratio of dissolution at each time point to 0 h for the test solution (0.1 g specification) was between 98.0% and 102.0%, and the solution was stable after 60 hours at room temperature.

[0039] The 100% concentration level mentioned above refers to the concentration level of the completely dissolved test sample solution.

[0040] Example 2, based on Example 1, involves periodically executing a detection and analysis process during batch detection in an ion chromatograph. This batch detection includes sequential continuous injection and detection of multiple samples from the same batch, relay continuous detection of multiple batches of samples, and long-term continuous detection by the instrument. The periodic execution of the detection and analysis process means that the detection and analysis are triggered according to a set cycle during the batch detection process. The set cycle can be configured by a time interval or by the number of samples to be tested: detection and analysis can be performed before each sample injection, or at certain intervals, or after several samples. The detection and analysis process includes: S3a: The target stage before sample injection and eluent flow to the ion chromatograph involves detecting key instrument operating parameters, including: the flow rate at the eluent feed pump outlet, the operating current of the suppression conductivity detector, and the steady-state conductivity collected by the suppression conductivity detector when no chromatographic peak signal of the sample component is detected; one initial detection and at most one pre-adjustment are completed within the target stage, followed by a re-detection; iterative parameter adjustments are not allowed within this target stage. S3b: Obtain the baseline values ​​of preset key instrument operating parameters, and combine them with the key instrument operating parameters detected by S3a to determine the actual feed flow coefficient, actual operating current coefficient, and actual steady-state conductivity coefficient; if any of the actual feed flow coefficient, actual operating current coefficient, and actual steady-state conductivity coefficient does not meet the corresponding allowable operating range, a first-level alarm will be triggered. S3c: When there is no first-level alarm, the flow-baseline influence coefficient is obtained by combining the actual feed flow coefficient and the actual steady-state conductivity coefficient, and the suppression state coefficient of the suppression conductivity detector is determined by combining the actual operating current coefficient and the actual steady-state conductivity coefficient. When either the flow-baseline impact coefficient or the suppression status coefficient meets the corresponding alarm range, a second-level alarm is triggered, and the corresponding warning type prompt is output. S3d: When S3b and S3c do not generate alarms: When the flow-baseline influence coefficient meets the corresponding pre-control range, the first pre-control is executed: the output flow rate of the eluent feed pump is adjusted in the direction of reducing the coefficient; When the suppression state coefficient meets the corresponding pre-control range, the second pre-control is executed: the operating current of the suppressor is increased. If, after the pre-positioned control is implemented and S3a is executed, either the flow-baseline influence coefficient or the suppression state coefficient meets the corresponding alarm range, then the second-level alarm is executed directly.

[0041] 1) The entire process of eluent flow into the ion chromatograph without sample injection is defined as T; therefore, the range of 0.2T-0.7T is selected as the target stage; the first 20% of the eluent flow period is discarded to avoid pump start-up disturbances; the target stage ends when there are 30%T remaining before sample injection. Within this target stage, one initial detection and at most one retest after pre-adjustment are completed. Both the initial detection and the retest after pre-adjustment are limited to completion within this target stage; after this target stage, parameter acquisition, alarm judgment, and instrument parameter adjustment are no longer performed, and the process awaits sample injection. Initial detection: Preferably, the steady-state conductivity corresponding to the first time the conductivity steady-state condition is met in the target stage (i.e., the difference between the maximum and minimum conductivity within the sampling time (10s-30s) is less than the preset conductivity (e.g., 0.05μS / cm)), the average flow rate of the outlet of the eluent feed pump obtained within the sampling time corresponding to the first time the conductivity steady-state condition is met, and the average working current of the suppressed conductivity detector are used to calculate the coefficients corresponding to the first initial detection; The flow rate at the outlet of the eluent feed pump: the actual output flow rate of the eluent pump.

[0042] The suppressor's operating current is the electrolytic current of the detector. The suppressor achieves suppression by electrolyzing water, and the magnitude of the current determines the suppression effect.

[0043] Suppressed steady-state conductivity acquired by the conductivity detector when no sample component chromatographic peak signal is detected: when there is no sample peak, the detector acquires a stable baseline signal, in μS / cm, which is the background conductivity of pure eluent passing through the detector.

[0044] Steady-state conductivity: The difference between the maximum and minimum conductivity within the sampling period (10s-30s) is less than a preset conductivity (e.g., 0.05μS / cm); the steady-state conductivity is the average of the conductivity values ​​detected during the above sampling period. Eluent pump set flow rate: The selectable pump flow rate range is 0.8-1.2 mL / min as described above. The target set flow rate is selected from this range. The target set flow rate (which can be 1 mL / min) is selected based on the following criteria: Under the selected eluent (e.g., 20 mmol / L methanesulfonic acid eluent) system, the sodium alginate diester component can achieve peak elution with normal peak shape, meeting the requirements for external standard method peak area quantification. Therefore, 1 mL / min is set as the eluent pump set flow rate (target input parameter).

[0045] Suppressor setting operating current: Under the selected eluent (such as 20 mmol / L methanesulfonic acid eluent), select the setting current in combination with the eluent system and the pump set flow rate; Selection criteria: The setting current can effectively suppress the eluent, reduce the interference of the eluent background on the dissolution detection of sodium alginate diester tablets, and ensure the accuracy of the dissolution detection results. The target setting operating current of the suppressor (target input parameter) is determined accordingly.

[0046] Set steady-state conductivity: The column is newly installed or replaced, and the instrument is in normal working condition; under the selected eluent (e.g., 20 mmol / L methanesulfonic acid eluent), the pump flow rate is set to 1 mL / min, and the suppressor current is set as determined above; the eluent is introduced, and a baseline window with no sample interference is captured for 10-30 s; the difference between the maximum and minimum conductivity values ​​within the window is <0.05 μS / cm, and the average conductivity of this window is taken to obtain the set steady-state conductivity (target input parameter) corresponding to the instrument-eluent system.

[0047] The above three target input parameters are fixed and saved as the baseline values ​​of the preset key instrument operating parameters. During batch testing, the instrument operating parameters obtained by actual measurement are allowed to fluctuate to a certain extent relative to the baseline values. Batch testing directly calls this set of baseline values ​​and compares them with the real-time measured parameters obtained in the 10-30s sampling window of step S3a to calculate various coefficients. The instrument hardware uses the set flow rate and set current in the baseline values ​​as the target values ​​for hardware closed-loop control for output control. The measured instrument operating parameters will fluctuate relative to the baseline values ​​due to the influence of hardware performance and system status. This invention defines the tolerable boundary of this fluctuation as the allowable operating range corresponding to each actual coefficient. Baseline value revision trigger conditions: The baseline value is determined and updated only when the chromatographic conditions change: the eluent concentration is changed, the pump flow rate is adjusted, or the suppressor current is adjusted. If the chromatographic conditions remain unchanged, the baseline value remains fixed and is not repeated with batches or single injections.

[0048] 2) ① Actual feed flow rate coefficient = |Flow rate at the outlet of the rinsing liquid feed pump detected by S3a - Set flow rate of the rinsing liquid pump corresponding to S3a detection| ÷ Set flow rate of the rinsing liquid pump corresponding to S3a detection. Physical meaning: The relative absolute deviation of the eluent feed flow rate characterizes the degree to which the actual flow rate deviates from the set flow rate. The larger the value, the more severe the deviation from the set value. Function: Quantifies the flow drift, pulsation, and pressure disturbance in the pump flow path; serves as the basic input for flow path disturbances, providing flow-side disturbance information for the subsequent flow-baseline influence coefficient.

[0049] ② Actual operating current coefficient = Operating current of the suppression conductivity detector during S3a detection ÷ Suppressor setting operating current corresponding to S3a detection; Physical meaning: The ratio of the actual operating current of the suppressor to the set current. Greater than 1: The actual operating current is higher than the set current; less than 1: The actual operating current is lower than the set current; The actual operating current coefficient is affected by the power supply and the suppressor load.

[0050] ③ Actual steady-state conductivity coefficient = steady-state conductivity detected by S3a ÷ set steady-state conductivity corresponding to S3a detection; Physical meaning: The ratio of the measured baseline conductivity to the reference baseline conductivity; greater than 1: steady-state conductivity increases relative to the baseline; less than 1: steady-state conductivity decreases relative to the baseline; the actual steady-state conductivity coefficient is affected by flow paths, suppressors, etc.

[0051] ④ Flow rate-baseline influence coefficient = actual feed flow rate coefficient × |actual steady-state conductivity coefficient - 1|; Only when the flow rate deviates and the actual steady-state conductivity coefficient deviates synchronously will the coefficient be significantly greater than 0. If the flow rate is almost constant, even if the actual steady-state conductivity coefficient deviates significantly, the coefficient will approach 0; if the actual steady-state conductivity coefficient does not deviate, even if the flow rate fluctuates greatly, the coefficient will also approach 0.

[0052] Function: Specifically identifies hidden faults caused by flow disturbances that result in abnormal actual steady-state conductivity; filters out actual steady-state conductivity drift unrelated to flow (such as suppressor aging and rinsing fluid contamination), avoiding misjudgments caused by irrelevant drift.

[0053] ⑤ The suppression state coefficient of the suppression conductivity detector = actual steady-state conductivity coefficient ÷ actual operating current coefficient; A coefficient much greater than 1 indicates that the actual steady-state conductivity coefficient increases significantly under the same current conditions, suggesting a worsening suppression effect under the same current, which points to the suppression of membrane performance degradation, membrane fouling, and membrane aging. A coefficient significantly less than 1 indicates that the actual steady-state conductivity coefficient is abnormally low under the same current coefficient conditions, which can reflect other abnormal operating conditions.

[0054] Function: Actual steady-state conductivity is affected by fluctuations in the actual operating current. By dividing by the actual operating current coefficient, the actual steady-state conductivity coefficient is normalized to the reference level of the current coefficient, eliminating the contribution of the current's own deviation at the sampling moment. This distinguishes two types of effects: one is the change in actual steady-state conductivity caused by current fluctuations, and the other is the increase in actual steady-state conductivity caused by the degradation of the suppression membrane's own performance. It eliminates the interference of power supply fluctuations, evaluates the operating status of the suppression membrane, and enables early identification of suppressor faults.

[0055] Preferably, the allowable operating ranges for the actual feed flow rate coefficient, actual operating current coefficient, and actual steady-state conductivity coefficient are [0.97, 1.03], [0.95, 1.05], and [0.92, 1.08], respectively. When the first-level alarm is triggered, the analysis process for this batch test is terminated, and subsequent correlation analysis, second-level alarm, and pre-control steps are not executed. An instrument malfunction warning is then output.

[0056] 3) ① Flow-baseline influence coefficient: Alarm range: Flow-baseline influence coefficient ≥ 0.05, pre-control range [0.03, 0.05); Suppression state coefficient of the suppression conductivity detector: Alarm range: The suppression state coefficient of the suppression conductivity detector is greater than 1.1 or less than 0.9, and the pre-adjustment range is [1.05, 1.1]. When either the flow-baseline influence coefficient or the suppression state coefficient meets the corresponding level 2 alarm range, a level 2 alarm is triggered. When a level 2 alarm is triggered, the pre-control hardware automatic control action is not executed, an alarm prompt is output, and the instrument parameters at the alarm time are saved. The currently started sample detection sequence can be completed, but the next batch of sample injection is prohibited. After the current sequence detection is completed, troubleshooting and maintenance are performed, the steady-state timing verification is completed again, and all coefficients return to the normal operating range before batch sample detection can be resumed.

[0057] ② If the flow-baseline influence coefficient triggers a Level 2 alarm: The system will display the message "Hidden disturbance in the flow path, abnormal flow-baseline coupling" (Class 1 warning type). Sample testing of the currently injected sequence is allowed, but starting the next batch of new samples is prohibited. After the current sequence test is completed, the pump, piping, and flow path connectors must be checked and maintained. The S3a-S3c steady-state sequence must be re-executed, and the baseline and coefficients must return to their normal ranges before batch testing can resume.

[0058] ③ If the suppression state coefficient triggers the second-level alarm: Inhibition state coefficient > 1.10: This indicates "the inhibitor's inhibition efficiency has decreased significantly and the inhibition membrane performance has deteriorated" (Class II warning type). You are allowed to complete the detection of the remaining samples in the current sequence, but you are prohibited from starting the next batch of samples. After the test, the inhibitor should be activated and maintained or the inhibition membrane should be replaced. The baseline reference parameters should be recalibrated. Batch detection can only be resumed after all coefficients have returned to normal.

[0059] Inhibition state coefficient < 0.90: This indicates "Abnormally low inhibition state, non-membrane degradation type fault" (Category 3 warning type). The remaining sample in the current sequence can be tested, but the next batch of samples is prohibited. The next step is to investigate issues such as the preparation of the eluent, the power supply of the inhibitor, and the status of the detection cell. Testing can continue after the fault is eliminated.

[0060] 4) ① When the flow-baseline influence coefficient meets the corresponding pre-control range, execute the first pre-control: adjust the output flow rate of the eluent feed pump in the direction of reducing the coefficient; If the outlet flow rate of the rinsing fluid feed pump during S3a testing is greater than the set flow rate of the rinsing fluid pump corresponding to S3a testing, then the adjusted output flow rate of the rinsing fluid feed pump = the adjusted output flow rate of the rinsing fluid feed pump before adjustment × (1 - flow rate adjustment ratio); the flow rate adjustment ratio is 1% to 3%. If the outlet flow rate of the rinsing fluid feed pump detected by S3a is less than the set flow rate of the rinsing fluid pump corresponding to the S3a detection, then the output flow rate of the rinsing fluid feed pump after adjustment = the output flow rate of the rinsing fluid feed pump before adjustment × (1 + flow rate adjustment ratio). Furthermore, the actual feed flow coefficient calculated by substituting the output flow rate of the rinsing fluid feed pump into the adjusted output flow rate meets the corresponding allowable range; After stabilizing for 10-30 seconds following adjustment, repeat the S3a test. ② When the suppression state coefficient meets the corresponding pre-adjustment range, execute the second pre-adjustment: increase the operating current of the suppressor, and the operating current of the suppressedor after adjustment = the operating current of the suppressedor before adjustment × (1 + current adjustment ratio); the current adjustment ratio is 2% to 3%; The beneficial effects of the above technical solution are as follows: This solution addresses the challenges of continuous batch detection in ion chromatography. Before sample injection, it strictly limits the target time window and mandates a maximum of one pre-treatment retest. Focusing on three key parameters—eluent pump outlet flow rate, suppressor operating current, and steady-state conductivity—it constructs a complete workflow encompassing parameter acquisition, characteristic coefficient calculation, two-stage alarms, pre-treatment, and retest verification. This approach accurately identifies early potential defects in the flow path and suppressor, performing risk assessment and parameter compensation while ensuring uninterrupted detection sequence. This effectively prevents the scrapping of entire batches of samples and improves the reliability of long-term batch operation. Details are as follows: 1. Innovatively setting a target stage with strict time boundaries, limiting a maximum of one pre-control retest, and resolving the conflict between self-testing and sample injection from the root of the time sequence: This scheme selects 0.2T-0.7T of the total eluent infusion time T as the target stage, discarding the first 20% of the time period when the eluent is just started, avoiding the shock and disturbance caused by the instrument startup; the target stage ends when there are 30%T left before sample injection, to avoid affecting the sample injection detection effect.

[0061] Within this target phase, only one initial test and a maximum of one retest after pre-adjustment are allowed. Once the target phase is exceeded, no further parameter acquisition, alarm judgment, or instrument parameter adjustment will be performed; the system will directly wait for sample injection. This constraint avoids unlimited repeated adjustments that occupy sample injection time and prevents the self-test process from disrupting the overall test sequence.

[0062] Baseline parameters are fixed and saved. They are only recalibrated and updated when chromatographic conditions such as eluent concentration, pump flow rate setting, and suppressor current setting change. The reference can be reused directly under the same conditions, eliminating the error and operational burden caused by repeated calibration between batches. The instrument status can be quantitatively compared, and potential equipment problems can be identified before the sample is injected. It is suitable for single batches of large numbers of samples, multiple batches in succession, and long-term uninterrupted instrument detection.

[0063] 2. Compare the actual operating status of the equipment with the baseline. The baseline coefficients (actual feed flow coefficient, actual operating current coefficient, and actual steady-state conductivity coefficient) reflect the degree of deviation of the flow rate, suppressor current, and steady-state conductivity from the baseline, respectively. If the baseline coefficient exceeds the allowable operating range, a first-level alarm will be triggered directly, and the batch test will be terminated.

[0064] The flow-baseline impact coefficient is specifically designed to identify baseline anomalies caused by flow fluctuations, filtering out baseline drift unrelated to flow and avoiding false alarms. The suppression state coefficient eliminates the impact of current fluctuations on baseline readings, distinguishing whether baseline anomalies originate from current fluctuations or from suppression membrane aging and fouling, and capturing early signals of suppressor performance degradation.

[0065] 3. Set up a two-level alarm mechanism and implement differentiated handling based on the severity of the fault. The first-level alarm is for hardware parameters that deviate significantly from the baseline. It will directly terminate the batch testing for this batch, without proceeding with subsequent coefficient calculations and adjustment processes, and output a hardware anomaly message.

[0066] When the basic parameters are within the allowable range, but the flow-baseline influence coefficient and suppression state coefficient reach the alarm threshold, a level 2 alarm is triggered. At this time, the currently initiated sample sequence is allowed to continue testing, but the initiation of the next batch of new samples is prohibited. This avoids continuing to test unknown samples under risky conditions while preserving samples already on the machine and preventing unnecessary sample scrapping. Furthermore, corresponding prompts are output for different fault types to guide subsequent equipment troubleshooting and maintenance.

[0067] 4. A pre-adjustment range is set before the alarm threshold. When the indicator falls within the adjustment range but has not yet triggered an alarm, the corresponding parameter is adjusted: if the flow-baseline influence coefficient exceeds the standard, the output flow rate of the eluent pump is finely adjusted; if the suppression state coefficient exceeds the standard, the suppressor operating current is increased. The instrument autonomously compensates for minor drifts during operation, reducing the frequency of manual maintenance shutdowns. All adjustments and retests are completed within the target phase, without modifying the experimental conditions of the sample being injected or interfering with the test results of already injected samples.

[0068] 5. A 10-30s sampling window is used for steady-state judgment. Only when the difference between the maximum and minimum conductivity within the window is less than the preset threshold is it considered to have reached a steady state. The mean value within the window is used to participate in the calculation of various coefficients to avoid misjudgment caused by instantaneous signal spikes and noise.

[0069] Example 3, based on Example 2, further includes the following detection and analysis process: S3e: When the first-level alarm and the second-level alarm are not triggered, the sample is injected in the order of peak separation, the detector collects the chromatographic signal of the sample, and the relative retention time, at least one of the peak asymmetry factor and the peak area are obtained based on the chromatographic signal. S3f: Compare the relative retention time and peak asymmetry factor obtained from S3e with the corresponding preset reference range. If either the relative retention time or peak asymmetry factor is outside the corresponding allowable reference range, a level three alarm will be triggered. If no Level 3 alarm is triggered and the relative retention time and peak asymmetry factor meet the corresponding correction feedback range, the flow adjustment amplitude and current adjustment amplitude of the next S3d are corrected based on the relative retention time and peak asymmetry factor determined by S3e. The allowable reference range, correction feedback range, and normal operating range corresponding to the relative retention time and peak asymmetry factor are shown in Table 5 below; among them, the correction feedback range is divided into: mild correction feedback range and moderate correction feedback range. The relative retention time is the ratio of the adjusted retention time of the target analyte to the adjusted retention time of the reference analyte; the adjusted retention time equals the measured retention time of the analyte minus the dead time. The reference analyte peak is taken from the chromatogram of the sample, preferably using the ion peak inherent in the dissolution medium, or a stable impurity peak coexisting in the sample, without the need for additional injection. The relative retention time is a ratio parameter that can eliminate the overall retention shift caused by minor fluctuations in the eluent flow rate; this ratio will only shift significantly when the selectivity of the column itself changes. If the reference analyte peak cannot be reliably identified due to matrix interference, the relative retention time is not used.

[0070] The peak asymmetry factor, also known as the tailing factor, is measured and calculated at 10% of the peak height of the target analyte. It is equal to the ratio of the width of the trailing edge to the width of the leading edge. An ideal chromatographic peak has a peak asymmetry factor close to 1. When the column is contaminated with impurities, tailing often occurs, and the peak asymmetry factor is greater than 1. Damaged packing material can result in a leading edge peak. This parameter primarily characterizes the degree of non-specific adsorption within the chromatographic column.

[0071] Table 5. Permissible reference range, correction feedback range, and normal operating range for relative retention time and peak asymmetry factor.

[0072] Then, based on the relative retention time and peak asymmetry factor determined in S3e, the flow rate adjustment range and current adjustment range of the next S3d are corrected. Specifically, the maximum correction feedback degree corresponding to the relative retention time and peak asymmetry factor is first determined, and the flow rate adjustment ratio is corrected based on the maximum correction feedback degree. Specifically, when there is no amplitude correction from the previous detection and analysis process, the current adjustment ratio is 2.5%, and the flow rate adjustment ratio is 2%. When the maximum level of correction feedback is within the range of mild correction feedback: the current adjustment ratio after correction = the current adjustment ratio before correction × 0.95, and the flow adjustment ratio after correction = the flow adjustment ratio before correction × 0.92; When the maximum level of correction feedback is within the moderate range of correction feedback: the current adjustment ratio after correction = the current adjustment ratio before correction × 0.9, and the flow adjustment ratio after correction = the flow adjustment ratio before correction × 0.85; The corrected current adjustment ratio still meets the requirement of 2% to 3%, and the corrected flow rate ratio still meets the requirement of 1% to 3%. The beneficial effects of the above technical solution are as follows: Peak asymmetry factor quantitatively characterizes chromatographic peak tailing and leading edge distortion, directly reflecting the non-specific adsorption within the column caused by column contamination and packing material damage; relative retention time can truly reflect changes in column selectivity. The two constitute a dual evaluation dimension, comprehensively judging the working status of the chromatographic system from the aspects of column selectivity changes and non-specific adsorption within the column.

[0073] When column performance deteriorates, the system deviates from its ideal operating point. If the original high gain is still used for adjustment, the adjustment output will further interfere with retention time and peak shape, forming a positive feedback loop of "deteriorating column performance - significant adjustment - increased disturbance - further deterioration of column performance". This solution actively reduces the adjustment gain when the detection parameter drifts, reducing the disturbance caused by the adjustment, cutting off the above positive feedback loop, and delaying the deterioration of the chromatographic system from the perspective of system dynamics, reducing unnecessary alarms and shutdowns.

[0074] In ion chromatography, pump flow rate is a fast kinetic component, where adjustment effects are quickly reflected in retention time; high gain adjustment can easily cause closed-loop oscillation. Suppressor current, on the other hand, is a slow kinetic component, with longer system thermal and membrane equilibrium establishment periods, resulting in a lower risk of oscillation under the same gain conditions. This scheme sets independent gain attenuation coefficients to address the differences in the kinetic responses of the two controlled components. Greater gain compression is applied to the fast-response flow rate, while a relatively higher adjustment gain is retained for the slow-response suppressor current.

[0075] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for detecting the dissolution rate of novel heparin-like marine drugs using ion chromatography, characterized in that: include: S1: Weigh out sodium polymannuronate propyl sulfate reference standard, add it to the dissolution medium to dissolve it, and obtain the reference standard solution; Take one sodium polymannuron sulfate test tablet and put it into a dissolution apparatus containing a preheated dissolution medium of a set volume. Stir to dissolve the test sample and obtain the test sample dissolution solution. Take the test sample dissolution solution, filter it, discard the initial filtrate, and take the subsequent filtrate as the test sample solution. S2: Set the detection conditions for the ion chromatograph: Select the chromatographic column for ion chromatography detection, the detector is a suppressed conductivity detector, and set the eluent feed flow rate, column temperature, and detector temperature; S3: Inject the reference solution and the test solution into the ion chromatograph separately, record the peak areas in the chromatograms, and determine the dissolution rate of the test sample based on the peak areas of the reference solution and the test solution.

2. The method for detecting the dissolution rate of novel heparin-like marine drugs using ion chromatography according to claim 1, characterized in that: The rinsing solution is an aqueous solution of methanesulfonic acid, and the dissolution medium in S1 is a pH 6.8 medium.

3. The method for detecting the dissolution rate of novel heparin-like marine drugs using ion chromatography according to claim 2, characterized in that: The concentration of the methanesulfonic acid aqueous solution is 20 mmol / L.

4. The method for detecting the dissolution rate of novel heparin-like marine drugs using ion chromatography according to claim 2, characterized in that: The method for preparing the pH 6.8 medium is as follows: Weigh 1.7g of anhydrous potassium dihydrogen phosphate and 1.745g of anhydrous dipotassium hydrogen phosphate, add 1000ml of degassed water, stir until completely dissolved and measure the pH, and adjust the pH to 6.8 using phosphoric acid.

5. The method for detecting the dissolution rate of novel heparin-like marine drugs using ion chromatography according to claim 1, characterized in that: When used for testing 0.1g of the test sample, the concentration of sodium polymannuronate propyl sulfate in the reference solution is 0.10–0.12 mg / mL; when used for testing 50mg of the test sample, the concentration of sodium polymannuronate propyl sulfate in the reference solution is 0.050–0.060 mg / mL.

6. The method for detecting the dissolution rate of novel heparin-like marine drugs using ion chromatography according to claim 1, characterized in that: The chromatographic column for ion chromatography detection includes a cation exchange analytical column and a matching cation exchange guard column. The cation exchange analysis column is a polymer matrix cation exchange analysis column bonded with carboxylic acid functional groups; The cation exchange guard column is a polymer matrix cation exchange guard column bonded with carboxylic acid functional groups.

7. The method for detecting the dissolution rate of novel heparin-like marine drugs using ion chromatography according to claim 1, characterized in that: The eluent feed flow rate in S2 is 0.8–1.2 mL / min, the column temperature is 35–45 °C, and the detector temperature is 25–35 °C.

8. The method for detecting the dissolution rate of novel heparin-like marine drugs using ion chromatography according to claim 7, characterized in that: The eluent feed flow rate in S2 is 1 mL / min, the column temperature is 40℃, and the detector temperature is 30℃.

9. The method for detecting the dissolution rate of novel heparin-like marine drugs using ion chromatography according to claim 1, characterized in that: During batch detection in an ion chromatograph, the detection and analysis process is performed periodically. The detection and analysis process includes: S3a: The target stage before sample injection and eluent flow into the ion chromatograph involves detecting key instrument operating parameters, including: the flow rate at the outlet of the eluent feed pump, the operating current of the suppression conductivity detector, and the steady-state conductivity collected by the suppression conductivity detector when no chromatographic peak signal of the sample component is detected; one initial detection and at most one pre-conditioning re-detection are completed within the target stage. S3b: Obtain the baseline values ​​of preset key instrument operating parameters, and combine them with the key instrument operating parameters detected by S3a to determine the actual feed flow coefficient, actual operating current coefficient, and actual steady-state conductivity coefficient; if any of the actual feed flow coefficient, actual operating current coefficient, and actual steady-state conductivity coefficient does not meet the corresponding allowable operating range, a first-level alarm will be triggered. S3c: When there is no first-level alarm, the flow-baseline influence coefficient is obtained by combining the actual feed flow coefficient and the actual steady-state conductivity coefficient, and the suppression state coefficient of the suppression conductivity detector is determined by combining the actual operating current coefficient and the actual steady-state conductivity coefficient. When either the flow-baseline impact coefficient or the suppression status coefficient meets the corresponding alarm range, a second-level alarm is triggered, and the corresponding warning type prompt is output. S3d: When S3b and S3c do not generate alarms: When the flow-baseline influence coefficient meets the corresponding pre-control range, the first pre-control is executed: the output flow rate of the eluent feed pump is adjusted in the direction of reducing the coefficient; When the suppression state coefficient meets the corresponding pre-control range, the second pre-control is executed: the operating current of the suppressor is increased. If the flow-baseline impact coefficient or the suppression state coefficient meets the corresponding alarm range after the pre-control is performed and then S3a is performed, then the second-level alarm is executed directly.

10. The method for detecting the dissolution rate of novel heparin-like marine drugs using ion chromatography according to claim 9, characterized in that: The detection and analysis process also includes: S3e: When the first-level alarm and the second-level alarm are not triggered, the sample is injected sequentially when the sample is separated from the peak. The detector collects the chromatographic signal of the sample and obtains at least one of the relative retention time, peak asymmetry factor and peak area based on the chromatographic signal. S3f: Compare the relative retention time and peak asymmetry factor obtained from S3e with the corresponding preset reference range. If either the relative retention time or peak asymmetry factor is outside the corresponding allowable reference range, a level three alarm will be triggered. If no Level 3 alarm is triggered and the relative retention time and peak asymmetry factor meet the corresponding correction feedback range, the flow adjustment range and current adjustment range of the next S3d will be corrected based on the relative retention time and peak asymmetry factor determined by S3e.

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