A detection method of effective components in a drop pill of gastro-intestinal health care of polygonum orientale

Abstract

The application discloses a detection method of effective components in Fengliaochangkang drop pills, and is based on supercritical fluid chromatography, adopts a photodiode array detector, uses an octadecylsilane bonded silica gel filler chromatographic column for solid-liquid separation, uses supercritical carbon dioxide fluid as a mobile phase A, uses methanol as a mobile phase B, carries out isocratic elution, the flow rate is 3 mL / min, the column temperature is 35 degrees Celsius, and analysis is carried out by selecting a wavelength of 256 nm, compared with common traditional detection methods, the method relies on the unique physical and chemical properties of supercritical fluid chromatography, and each component of the Fengliaochangkang drop pills can be efficiently separated, the experimental speed is fast, the preparation period is short, and the method has extremely important significance for product quality control of the Fengliaochangkang drop pills.

Description

Technical Field

[0001] This invention belongs to the field of drug detection technology, and relates to supercritical fluid chromatography in detection and analysis technology. Specifically, it relates to a method for detecting the active ingredients in Fengliao Changweikang Dripping Pills. Background Technology

[0002] Fengliao Changweikang is a famous Li medicine product from Hainan, mainly composed of *Daphniphyllum calycinum* and *Polygonum hydropiper*, with a ratio of 2:1. It is prepared by decocting in water, concentrating the extract, adding excipients, and then processing. It has the effect of removing dampness and resolving stagnation, and is clinically used to treat acute and chronic gastroenteritis with significant effects and no adverse reactions. *Daphniphyllum calycinum* is the dried leafy stem and branches of *Daphniphyllum calycinum* Benth, a plant in the genus *Daphniphyllum* of the family Lauraceae. It has the effects of dispelling wind and relieving pain, detoxifying and reducing swelling, and is used for damp-heat diarrhea, dysentery, hematochezia, and rheumatic swelling and pain. It is the principal ingredient in Fengliao Changweikang. *Polygonum hydropiper* is the whole herb of *Polygonum hydropiper* L., a plant in the genus *Polygonum* of the family Polygonaceae. It has the effects of clearing heat and detoxifying, dispersing blood stasis and stopping bleeding. (Zhao Dan, Annie, Chen Changyu, et al. Research progress of Fengliao Changweikang [J]. Modern Drugs and Clinical, 2014, 29(12):1446-1450.) After research by many doctors and scholars, it was found that quercetin and rutin in Fengliao Changweikang have significant anti-inflammatory, antispasmodic and antidiarrheal effects, and are the main effective components of Fengliao Changweikang in the treatment of acute gastroenteritis (Tan Yinfeng, Li Hailong, Zhang Junqing, Fu Naiguang, Liu Mingsheng. Preliminary study on the pharmacodynamic material basis of Fengliao Changweikang in the treatment of acute gastroenteritis [J]. Shizhen Guoyi Guoyao, 2009, 20(12):2.DOI:10.3969 / j.issn.1008-0805.2009.12.014.).

[0003] The chemical structural formulas of quercetin and rutin are as follows:

[0004]

[0005] Currently, to ensure the quality of the finished drug during production, it is necessary to promptly test the content of its main active ingredients in the drop pills for quality control. Furthermore, to control the quality of the finished drug, ensure efficacy, and monitor potential changes in the active ingredients during storage, it is also necessary to test the active ingredients in Fengliao Changweikang drop pills. However, existing technologies primarily use high-performance liquid chromatography (HPLC) for detection. The disadvantages of this method are that the detection process is time-consuming, often requiring several tens of minutes per test, and necessitates the preparation of relatively complex mobile phase solutions, making the operation cumbersome. Excessive waiting time for testing affects the production progress of the drop pills, resulting in low production efficiency. Summary of the Invention

[0006] The purpose of this invention is to provide a method for detecting the active ingredients in Fengliao Changweikang Dripping Pills. This invention employs a novel detection method based on the unique physicochemical properties of the active ingredients in Fengliao Changweikang Dripping Pills using supercritical fluid chromatography. This method can effectively separate the active ingredients of Fengliao Changweikang Dripping Pills while significantly shortening the detection time. The preparation stage of this method is simple and convenient, and the testing process is short, efficient, and accurate.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] A method for detecting the active ingredient in Fengliao Changweikang Dripping Pills uses supercritical fluid chromatography as the detection method. The sample is separated using an octadecylsilane-bonded silica gel column, and isocratic elution is performed with mobile phase A and mobile phase B. The sample is then detected by a detector. Mobile phase A is supercritical carbon dioxide fluid, and mobile phase B is methanol.

[0009] Furthermore, the isocratic elution process is as follows: the proportion of mobile phase A in the system is 85%-75%, and the proportion of mobile phase B is 15%-25%, lasting for 7 minutes.

[0010] Furthermore, the detection wavelength for supercritical fluid chromatography is 256 nm.

[0011] Furthermore, the chromatographic column model and specifications are XSelect HSS C18 SB Column. 5μm, 4.6mm×250mm.

[0012] Furthermore, the flow rate of the mobile phase is 3 mL / min.

[0013] Furthermore, the detector is a photodiode array detector, model ACQUITY UPC2 PDA.

[0014] Furthermore, the samples to be tested include rutin reference solution, quercetin reference solution, mixed reference solution containing rutin and quercetin, and Fengliao Changweikang Dripping Pills test solution.

[0015] Furthermore, the active ingredients detected in Fengliao Changweikang Dripping Pills include rutin and quercetin.

[0016] Furthermore, the automatic back pressure regulator for supercritical fluid chromatography is set at 1800 psi.

[0017] Furthermore, the temperature of the chromatographic column was 35 degrees Celsius.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This invention relates to a method for detecting the active ingredients in Fengliao Changweikang Dripping Pills. The active ingredients to be detected are rutin and quercetin. The detection method employs supercritical fluid chromatography, which rapidly detects the active ingredients in Fengliao Changweikang Dripping Pills by limiting the interaction of detection conditions, including the chromatographic column, mobile phase A, and mobile phase B. This invention provides a detection method that is both time-efficient and highly accurate.

[0020] Specifically, compared to the common high-performance liquid chromatography (HPLC) method for determining the active ingredients in Fengliao Changweikang Dripping Pills, the method of this invention relies on the unique physicochemical properties of supercritical fluid chromatography (SLC) for the active ingredients in Fengliao Changweikang Dripping Pills. Supercritical fluids have diffusion coefficients and viscosities close to those of gases, resulting in low mass transfer resistance of the solute. Using it as a mobile phase allows for rapid and efficient separation of the active ingredients in Fengliao Changweikang Dripping Pills. Furthermore, supercritical fluids have densities similar to liquids, possessing high solubility. The high operating pressure and flow rate of the entire supercritical fluid chromatograph can shorten the detection time from tens of minutes to less than ten minutes. Moreover, this invention is relatively safe and environmentally friendly, specifically because the mobile phase A used is a supercritical carbon dioxide fluid, which is non-toxic and harmless; and the mobile phase B is a methanol solution, which is simple to prepare. Compared to existing technologies, there is no need to prepare complex mobile phase solutions, thus the mobile phase preparation process is convenient and quick, the experimental preparation time is short, and the testing efficiency is high. Shortening the detection time reduces the waiting time for drug quality control, thereby accelerating the drug production process and improving the overall drug production efficiency. Detailed Implementation

[0021] The present invention will be further described below through specific embodiments. Experimental methods in preferred embodiments that do not specify specific conditions are generally performed under conventional conditions. The embodiments provided are for better illustration of the invention, but are not intended to limit the scope of the invention to these embodiments. Therefore, non-essential improvements and adjustments made to the implementation schemes by those skilled in the art based on the invention's content still fall within the protection scope of the present invention.

[0022] Furthermore, it should be understood that the one or more method steps mentioned in this invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, unless otherwise stated. Moreover, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not intended to limit the order of the method steps or to limit the scope of the invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the invention.

[0023] A method for detecting the active ingredients in Fengliao Changweikang Dripping Pills uses supercritical fluid chromatography (SFCLC) as the detection method. The sample is separated using an octadecylsilane-bonded silica gel column, and isocratic elution is performed with mobile phases A and B before detection by a detector. The active ingredients in Fengliao Changweikang Dripping Pills include rutin and quercetin. Specific detection conditions are as follows:

[0024] Chromatographic column: Octadecylsilane-bonded silica gel column;

[0025] Column model and specifications: XSelect HSS C18 SB Column 5μm, 4.6mm × 250mm;

[0026] Mobile phase A: Supercritical carbon dioxide fluid;

[0027] Mobile phase B: Methanol;

[0028] Detector: Photodiode array detector;

[0029] Detector model: ACQUITY UPC2 PDA;

[0030] Automatic back pressure regulator pressure: 1800 psi;

[0031] Detection wavelength: 256nm;

[0032] Flow rate: 3 mL / min;

[0033] Temperature: 35 degrees Celsius;

[0034] Isocratic elution process:

[0035] Mobile phase A accounts for 85%-75%, and mobile phase B accounts for 15%-25%, for 7 minutes;

[0036] (1) Investigation of the mobile phase: The mobile phase was investigated using the method of this invention, i.e., different solutions were prepared as the mobile phase for testing, and the test results were observed. Specific operation: Acetonitrile-methanol (70:30), acetonitrile-methanol (50:50), acetonitrile-methanol (20:80), and methanol were prepared as mobile phase B. Rutin and quercetin reference solutions were taken for testing, respectively. The results showed that in the acetonitrile-methanol (70:30), acetonitrile-methanol (50:50), acetonitrile-methanol (20:80), and methanol systems, methanol as mobile phase B provided the best separation effect.

[0037] (2) Selection of wavelength for determination: Ultraviolet spectroscopy was performed on rutin and quercetin reference standards respectively. According to the scanning results, since both have strong absorption around 256nm produced by benzoyl chromophore, 256nm was selected as the detection wavelength.

[0038] (3) Optimization of isocratic elution protocol: Different ratios of mobile phase were tested to investigate isocratic elution protocols, including mobile phase A: mobile phase B (80:20), mobile phase A: mobile phase B (85:15), and mobile phase A: mobile phase B (75:25), all of which were effective for detection. Among them, it was found that when the mobile phase A was 75% and the mobile phase B was 25%, the separation of the detectable components could be completed in a shorter time of 7 minutes. Therefore, mobile phase A: mobile phase B (75:25) was selected as the optimal isocratic elution protocol.

[0039] Example 1

[0040] 1. Instruments and chromatographic conditions

[0041] 1.1 Instruments

[0042] The supercritical fluid chromatograph required for this method is the Waters ACQUITY UPC2 system, which includes the ACQUITY UPC2 Binary Solvent Manager (BSM); ACQUITY UPC2 Sample Manager; ACQUITY UPC2 Phase Manager; ACQUITY 30cm Single-Zone Column Manager (CM-30S); ACQUITY UPC2 PDA Photodiode Array Detector; and the Empower 3 software system, which plays a control and data collection role.

[0043] 1.2 Chromatographic conditions

[0044] Selected chromatographic column: XSelect HSS C18 SB Column 5μm, 4.6mm × 250m;

[0045] Photodiode array detector detection wavelength: 256nm;

[0046] Automatic back pressure regulator pressure: 1800 psi;

[0047] Column temperature: 35 degrees Celsius;

[0048] Autosampler temperature: 10 degrees Celsius;

[0049] Injection volume: 10 μL;

[0050] Flow rate: 3 mL / min;

[0051] Mobile phase A: Carbon dioxide (high purity);

[0052] Mobile phase B: Methanol;

[0053] Isocratic elution process: mobile phase A accounts for 75%, mobile phase B accounts for 25%, and the process lasts for 7 minutes;

[0054] 2. Solution preparation

[0055] 2.1 Preparation of rutin reference solution: Weigh 3 mg of rutin reference standard, place it in a 1 mL volumetric flask, dissolve it in methanol and dilute to the mark, shake well to obtain a rutin reference solution containing 3 mg per 1 mL.

[0056] 2.2 Preparation of quercetin reference solution: Weigh 2 mg of quercetin reference standard, place it in a 1 mL volumetric flask, dissolve it in methanol and dilute to the mark, shake well to obtain a quercetin reference solution containing 2 mg per 1 mL.

[0057] 2.3 Preparation of mixed reference solution containing rutin and quercetin: Accurately transfer 0.5 mL of rutin reference solution and 0.5 mL of quercetin reference solution into a 5 mL volumetric flask, and dilute to the mark with methanol solution to obtain a mixed reference solution of the two components (containing rutin at a concentration of 300 μg / mL and quercetin at a concentration of 200 μg / mL). Store at 5℃ for later use.

[0058] 2.4 Preparation of the test solution for Fengliao Changweikang Dripping Pills: Take approximately 3.0 g of Fengliao Changweikang Dripping Pills, grind them using a mortar and pestle, pass them through a No. 3 sieve, weigh them, then place them in an Erlenmeyer flask, add 70 mL of methanol, and weigh them again. Extract by sonication for 40 min, cool, weigh again, replenish the lost weight with methanol, shake well, and filter. Take the filtrate and filter it through a 0.45 μm microporous membrane to obtain the test solution.

[0059] 3. Instrument preparation and debugging

[0060] The specific operation steps are as follows:

[0061] 3.1Place XSelect HSS C18 SB Column, A 5μm, 4.6mm×250mm chromatographic column was placed inside an ACQUITY 30cm single-zone column manager (CM-30S) and connected to tubing.

[0062] 3.2 Prepare mobile phase A: Check the purity and current pressure of carbon dioxide gas in the cylinder, then connect the prepared carbon dioxide cylinder to the Waters ACQUITY UPC2 system supercritical fluid generator pump, flush the pipeline and standby.

[0063] 3.3 Prepare mobile phase B: 250 mL of methanol solution, sonicate for 20 minutes to remove gas from the solution, then place it on the mobile phase tray and connect it to the solvent management system.

[0064] 3.4 Open Empower 3 software and perform routine checks on the equipment before sample testing, checking the operating status of each component, whether the pipeline pressure is stable, and whether the detector baseline is stable.

[0065] 3.5 After filtering the prepared rutin-containing standard solution through an ultrafiltration membrane, inject it into sample vial 1 and place sample vial 1 into the autosampler.

[0066] 3.6 After filtering the prepared reference solution containing quercetin through an ultrafiltration membrane, inject it into sample vial 2 and place sample vial 2 into the autosampler.

[0067] 3.7 After filtering the prepared mixed reference solution containing rutin and quercetin through an ultrafiltration membrane, inject it into sample vial 3 and place sample vial 3 into the autosampler.

[0068] 3.8 After filtering the prepared Fengliao Changweikang Dripping Pills test solution through an ultrafiltration membrane, inject it into sample bottle 4 and place sample bottle 4 into the autosampler.

[0069] 3.9 Create a new detection method in Empower 3 software. Set the automatic back pressure regulator to 1800 psi, the detector wavelength to 256 nm, the column temperature to 35 degrees Celsius, the autosampler temperature to 10 degrees Celsius, the injection volume to 10 μL, the flow rate to 3 mL / min, and the isocratic elution protocol to 75% mobile phase A and 25% mobile phase B for 7 minutes. After detection, rinse with mobile phase B.

[0070] 4. Determination method

[0071] Accurately measure 10 μL each of the rutin reference solution, quercetin reference solution, mixed reference solution containing rutin and quercetin, and test solution, and inject them into a supercritical fluid chromatograph. Determine the content of quercetin and rutin in the test solution according to the supercritical fluid chromatography method (Chinese Pharmacopoeia 2020 Edition 0531). Record the peak area and calculate the content of quercetin and rutin in the test solution using the external standard one-point method.

[0072] 5. Methodological Validation

[0073] 5.1 Specificity and System Suitability Test

[0074] Preparation of negative samples of effective components of Polygonum aviculare: Except for the absence of Acer buergerianum and Polygonum hydropiper, the other raw materials and preparation methods are the same as those of Polygonum aviculare gastrointestinal health drops.

[0075] Take a negative sample of the active ingredient from *Polygonum hydropiper* and prepare a negative control solution of the active ingredient from *Polygonum hydropiper* gastrointestinal health drops according to the preparation method of the test solution. Inject rutin, quercetin reference solutions, the test solution, and the above negative control solution into the sample for determination.

[0076] The results showed that the recorded DAD spectra and chromatograms were located at the same positions as the reference chromatographic peaks, with no interference from the negative control. The test sample chromatogram exhibited chromatographic peaks with the same retention times as the reference chromatogram. Calculated based on the quercetin and rutin chromatographic peaks, the theoretical plate number was greater than 20,000, the resolution was greater than 2.0, and the peak purity was greater than 99.5. The results indicate that the method has good specificity and system suitability, meeting the requirements for content determination.

[0077] 5.2 Sample solution stability

[0078] The same sample solution of Fengliao Changweikang Dripping Pills was taken and injected for determination at 0, 4, 8, 12 and 24 h after preparation. The RSD values ​​of the peak areas of quercetin and rutin were calculated. The values ​​of quercetin and rutin were 0.5% and 0.8%, respectively, both not greater than 2%, indicating that the sample solution was stable within 24 h.

[0079] 5.3 Examination of Linear Relationships

[0080] Precisely measured 2 μL, 5 μL, 10 μL, 20 μL, and 40 μL of the mixed reference solution (containing rutin at a concentration of 300 μg / mL and quercetin at a concentration of 200 μg / mL) were injected for analysis. The linear regression equation for quercetin was y = 3.2752x + 1.0531, r = 0.99997; the linear regression equation for rutin was y = 1.7536x - 34.025, r = 0.99996, indicating good linearity within the determination range.

[0081] 5.4 Instrument Precision Assessment

[0082] Accurately measure 10 μL of the mixed reference solution and inject it into the liquid chromatograph. Repeat the determination 6 times and calculate the RSD value of the peak area of ​​each pair of index components. The peak area RSD value of quercetin is 0.21% and the peak area RSD value of rutin is 0.85%, indicating that the instrument precision of this method is good.

[0083] 5.5 Repeatability Experiment

[0084] Six test solutions were prepared in parallel using the same batch of Fengliao Changweikang Dripping Pills according to the test solution preparation method. The solutions were then measured according to the method, and the RSD values ​​were calculated based on the measured content of each indicator component. The RSD value of quercetin content was 0.54%, and the RSD value of rutin content was 0.66%, indicating that the method has good repeatability.

[0085] 5.6 Intermediate Precision Experiment

[0086] The intermediate precision of the same batch of Fengliao Changweikang Dripping Pills was investigated under three varying factors: time, personnel, and instrument. The results showed that the RSD values ​​of the intermediate precision under different time, personnel, and instruments were all less than 2.0%, indicating that the method has good intermediate precision.

[0087] Comparative Example 1

[0088] 1. Instruments and chromatographic conditions

[0089] 1.1 The high-performance liquid chromatograph used in Comparative Example 1 was a Waters Arc HPLC, and the detector was a Waters 2998PDA photodiode array detector.

[0090] 1.2 Selected chromatographic column: XSelect HSS C18 SB Column 5μm, 4.6mm×250m; photodiode array detector detection wavelength: 256nm; column temperature: 35 degrees Celsius; autosampler temperature: 10 degrees Celsius; injection volume: 10μL; flow rate: 1mL / min; mobile phase A: 0.5% phosphoric acid aqueous solution, mobile phase B: methanol.

[0091] 1.3 Isocratic elution protocol: The system consists of 75% mobile phase A and 25% mobile phase B, for 40 minutes.

[0092] 2. The solution is prepared in the same manner as in Example 1.

[0093] 3. Instrument preparation and debugging

[0094] The specific operation steps are as follows:

[0095] 3.1Place XSelect HSS C18 SB Column, A 5μm, 4.6mm×250mm chromatographic column was placed in the column oven and the tubing was connected.

[0096] 3.2 Preparation of mobile phase A: Prepare a 0.5% phosphoric acid aqueous solution. Transfer 5.88 mL of 85% phosphoric acid solution to a 1000 mL volumetric flask, add distilled water to the mark, make up to volume, mix well, place in a mobile phase bottle, sonicate for 20 minutes to degas, place on a mobile phase tray, and connect to the solvent management system.

[0097] 3.3 Prepare mobile phase B: 250 mL of methanol solution, sonicate for 20 minutes to remove gas from the solution, then place it on the mobile phase tray and connect it to the solvent management system.

[0098] 3.4 Open Empower 3 software and perform routine checks on the equipment before sample testing, checking the operating status of each component, whether the pipeline pressure is stable, and whether the detector baseline is stable.

[0099] 3.5 After filtering the prepared rutin-containing standard solution through an ultrafiltration membrane, inject it into sample vial 1 and place sample vial 1 into the autosampler.

[0100] 3.6 After filtering the prepared reference solution containing quercetin through an ultrafiltration membrane, inject it into sample vial 2 and place sample vial 2 into the autosampler.

[0101] 3.7 After filtering the prepared mixed reference solution containing rutin and quercetin through an ultrafiltration membrane, inject it into sample vial 3 and place sample vial 3 into the autosampler.

[0102] 3.8 After filtering the prepared Fengliao Changweikang Dripping Pills test solution through an ultrafiltration membrane, inject it into sample bottle 4 and place sample bottle 4 into the autosampler.

[0103] 3.9 Create a new detection method in Empower 3 software, set the detector wavelength to 256 nm, the column temperature to 35 degrees Celsius, the autosampler temperature to 10 degrees Celsius, the injection volume to 10 μL, the flow rate to 1 mL / min, and the isocratic elution protocol to 75% mobile phase A and 25% mobile phase B for 40 minutes. After detection, rinse with mobile phase B.

[0104] 4. The determination method is the same as in Example 1.

[0105] Conclusion: Comparative Example 1 is a comparative experiment with Example 1, the difference being that Comparative Example 1 used high-performance liquid chromatography (HPLC) for detection, while Example 1 of this application used supercritical fluid chromatography (SFC). Furthermore, the detection conditions in Comparative Example 1 were adjusted according to the principles of HPLC, and are slightly different from the SFC detection conditions in Example 1. The detection results show that the detection method and corresponding conditions in Comparative Example 1 can also determine the content of quercetin and rutin, but the method in Comparative Example 1 is time-consuming, reducing production efficiency. A comparison between Comparative Example 1 and Example 1 shows that the detection method and corresponding conditions in Example 1 of this application can rapidly determine the content of quercetin and rutin, with a shorter detection time, thereby improving production efficiency.

[0106] Comparative Example 2

[0107] Accurately measure 10 μL of the mixed reference solution containing rutin and quercetin from Example 1, and perform detection using supercritical fluid chromatography. The detection conditions are as follows:

[0108] Column: XSelect HSS C18 SB Column 5μm, 4.6mm×250m; Automatic back pressure regulator pressure: 1800psi; Photodiode array detector detection wavelength: 256nm; Column temperature: 35 degrees Celsius; Autosampler temperature: 10 degrees Celsius; Injection volume: 10μL; Flow rate: 3mL / min; Mobile phase A: Carbon dioxide (high purity grade), Mobile phase B: Methanol.

[0109] Isocratic elution protocol: Mobile phase A is 90% and mobile phase B is 10%, for 7 minutes.

[0110] The detection procedure is the same as in Example 1.

[0111] Conclusion: Comparative Example 2 is a comparative experiment with Example 1. Both Comparative Example 2 and Example 1 used supercritical fluid chromatography for detection, the difference being the isocratic elution scheme. The isocratic elution scheme used in Comparative Example 2 failed to accurately determine the content of quercetin and rutin within a short time. Therefore, the detection effect of Comparative Example 2 was poor, and the effective substances were not detected. A comparison between Comparative Example 2 and Example 1 shows that the isocratic elution scheme of Example 1 of this application can accurately determine the content of quercetin and rutin within a short time of 7 minutes, demonstrating excellent detection performance.

[0112] Comparative Example 3

[0113] Accurately measure 10 μL of the mixed reference solution containing rutin and quercetin from Example 1, and perform detection using supercritical fluid chromatography. The detection conditions are as follows:

[0114] Column: XSelect HSS C18 SB Column 5μm, 4.6mm×250m; Automatic back pressure regulator pressure: 1800psi; Photodiode array detector detection wavelength: 256nm; Column temperature: 35 degrees Celsius; Autosampler temperature: 10 degrees Celsius; Injection volume: 10μL; Flow rate: 3mL / min; Mobile phase A: Carbon dioxide (high purity grade), Mobile phase B: Methanol.

[0115] Isoclinity elution protocol: The system consists of 60% mobile phase A and 40% mobile phase B, for 7 minutes.

[0116] The detection procedure is the same as in Example 1.

[0117] Conclusion: Comparative Example 3 is a comparative experiment with Example 1. Both Comparative Example 3 and Example 1 used supercritical fluid chromatography for detection, the difference being the isocratic elution scheme. The isocratic elution scheme used in Comparative Example 3 failed to effectively separate quercetin and rutin within a short time, resulting in significant overlap of chromatographic peaks. In contrast, the isocratic elution scheme of Example 1 of this application can accurately determine the content of quercetin and rutin within a short time of 7 minutes, demonstrating excellent detection results.

[0118] In summary, this invention provides a method for detecting the active ingredients in Fengliao Changweikang Dripping Pills. The active ingredients to be detected are rutin and quercetin. The detection method employs supercritical fluid chromatography. By defining the interaction of detection conditions, including the chromatographic column, mobile phase A, and mobile phase B, the active ingredients in Fengliao Changweikang Dripping Pills can be detected rapidly. This invention's detection method is time-efficient and highly accurate.

[0119] The beneficial effects of this embodiment are as follows: Compared with the common high-performance liquid chromatography (HPLC) method for determining the active ingredients in Fengliao Changweikang Dripping Pills, the method of this invention relies on the unique physicochemical properties of supercritical fluid chromatography (SLC) for the active ingredients in Fengliao Changweikang Dripping Pills. The diffusion coefficient and viscosity of supercritical fluids are close to those of gases, resulting in low mass transfer resistance of the solute. Using supercritical fluids as the mobile phase allows for rapid and efficient separation of the active ingredients in Fengliao Changweikang Dripping Pills. Furthermore, the density of supercritical fluids is similar to that of liquids, giving them high solubility. The high operating pressure and high flow rate of the entire supercritical fluid chromatograph can shorten the detection time from tens of minutes to less than ten minutes. Moreover, this invention is relatively safe and environmentally friendly, specifically because the mobile phase A used is a supercritical carbon dioxide fluid, which is non-toxic and harmless; and the mobile phase B is a methanol solution, which is simple to prepare. Compared with existing technologies, there is no need to prepare complex mobile phase solutions, thus making mobile phase preparation convenient and quick, reducing experimental preparation time, and increasing testing efficiency. Shortening the detection time reduces the waiting time for drug quality control, thereby accelerating drug production and improving overall drug production efficiency. This addresses the problem of low production efficiency caused by the long testing time in existing technologies.

[0120] Although the present invention has been described using the above preferred embodiments, it is not intended to limit the scope of protection of the present invention. Any changes and modifications made by those skilled in the art to the above embodiments without departing from the spirit and scope of the present invention shall still fall within the scope of protection of the present invention.

Claims

1. A method for detecting the active ingredient in Fengliao Changweikang Dripping Pills, characterized in that, Supercritical fluid chromatography was used as the detection method. The sample to be tested was separated using an octadecylsilane-bonded silica gel column. The sample was eluted isocratically with mobile phase A and mobile phase B and then detected by a detector. Mobile phase A was supercritical carbon dioxide fluid and mobile phase B was methanol. The ratio of mobile phase A to mobile phase B is 75:25, and the mixture lasts for 7 minutes. The detection wavelength used in the supercritical fluid chromatography method is 256 nm; The flow rate of the mobile phase is 3 mL / min; The detector is a photodiode array detector, model ACQUITY UPC2 PDA; The automatic back pressure regulator of the supercritical fluid chromatograph is set at 1800 psi. The temperature of the chromatographic column is 35 degrees Celsius; The active ingredients detected in the Fengliao Changweikang Dripping Pills include rutin and quercetin; Grind the Fengliao Changweikang pills into a fine powder, add methanol, and extract by ultrasonication to obtain the test solution.

2. The method for detecting the effective components in Fengliao Changweikang Dripping Pills according to claim 1, characterized in that, The chromatographic column is an XSelect HSS C18 SB Column, 100Å, 5µm, 4.6mm × 250mm.

3. The method for detecting the effective components in Fengliao Changweikang Dripping Pills according to claim 1, characterized in that, The test samples include rutin reference solution, quercetin reference solution, mixed reference solution containing rutin and quercetin, and Fengliao Changweikang Dripping Pill test solution.

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