Characteristic spectrum of shuangge antidiarrheal preparation and construction method thereof

Abstract

The application relates to the technical field of traditional Chinese medicine, and particularly provides a characteristic spectrum of a Shuangge anti-diarrhea preparation and a construction method thereof. The application adopts a multi-wavelength switching technology by screening a sample processing method and optimizing chromatographic conditions, and simultaneously characterizes main efficacy components of six medicines in a prescription. The method can evaluate the overall curative effect of the Shuangge anti-diarrhea preparation from a qualitative point of view, and significantly improves the quality control level of the Shuangge anti-diarrhea preparation.

Description

Technical Field

[0001] This application relates to the field of traditional Chinese medicine technology, specifically providing a characteristic spectrum of a Shuangge antidiarrheal preparation and its construction method. Background Technology

[0002] Shuangge Antidiarrheal Oral Liquid is an oral liquid preparation made from six traditional Chinese medicines: honeysuckle, kudzu root, scutellaria baicalensis, coptis chinensis, white peony root, and prepared licorice root, using modern extraction technology. Clinically, it has the effects of clearing heat and drying dampness, detoxifying and stopping diarrhea, and can be used for damp-heat diarrhea in piglets. Shuangge Antidiarrheal Granules are an improved dosage form product based on Shuangge Antidiarrheal Oral Liquid.

[0003] The material basis for the disease prevention and treatment and pharmacological effects of traditional Chinese medicine lies in its active ingredients. Chlorogenic acid in Shuangge Zhixie Oral Liquid exhibits strong antiviral activity and is a characteristic component of honeysuckle. Kudzu root has the effects of relieving muscle tension and fever, promoting body fluid production and quenching thirst; puerarin is the most important monitoring component for evaluating the quality of kudzu root products. Scutellaria baicalensis mainly contains flavonoids (primarily baicalin and baicalein), and has the effects of clearing heat and drying dampness, purging fire and detoxifying. Coptis chinensis mainly contains alkaloids, flavonoids, lignans, polysaccharides, and other active ingredients, all of which have certain medicinal effects. Alkaloids are considered to have the highest efficacy, with berberine hydrochloride as the main active ingredient, having the effects of clearing heat and drying dampness, purging fire and detoxifying. White peony root has the effects of nourishing blood and regulating menstruation, astringing yin and stopping sweating; monoterpenoid glycosides, represented by paeoniflorin, are the main active ingredients of white peony root. Licorice has the functions of tonifying the spleen and replenishing qi, clearing heat and detoxifying, resolving phlegm and relieving cough, relieving spasms and pain, and harmonizing various medicines. Among them, glycyrrhizin and glycyrrhizic acid ammonium are the main quality control components.

[0004] Due to the complexity of the chemical composition of traditional Chinese medicine (TCM), no single active ingredient in a TCM compound preparation can fully represent its overall therapeutic effect. Characteristic chromatograms of TCM preparations, as a recognized and effective means of identifying the quality of TCM, take a macroscopic perspective, better reflecting the complexity and holistic nature of TCM, and are widely used in the quality control of multiple TCM varieties in Part I of the 2020 edition of the Chinese Pharmacopoeia. A characteristic chromatogram of TCM preparations refers to a chromatogram established by appropriately processing TCM preparation samples and using suitable analytical methods, reflecting information about multiple components and demonstrating their quality characteristics. Characteristic chromatograms of TCM preparations are of great significance for identifying key quality attributes of TCM preparations, studying quantity-quality transfer, evaluating the uniformity and stability of TCM preparation quality, and improving the overall quality control level of TCM preparations.

[0005] Currently, the quality standards for Shuangge Zhixie Oral Liquid and Shuangge Zhixie Granules include thin-layer chromatography (TLC) identification for many herbs, but lack TLC identification for licorice. Furthermore, the current quality standards identify each herb individually, making the methods cumbersome, time-consuming, and inefficient. There is a lack of a comprehensive evaluation method for all components in this drug preparation, and no literature reports on fingerprint spectroscopy for this series of preparations or quality control methods for licorice in the prescription. This is detrimental to product quality stability and evaluation, and may lead to significant differences in product quality and clinical efficacy.

[0006] In view of the above, this application is hereby submitted. Summary of the Invention

[0007] One of the purposes of this application is to provide a characteristic spectrum of Shuangge antidiarrheal preparations and its construction method, which can analyze and evaluate the complex component system of Shuangge antidiarrheal preparations, and can more comprehensively and effectively characterize the quality of the product, providing an effective solution for simple and stable quality control of the product.

[0008] To achieve the above objectives, this application adopts the following technical solution:

[0009] A method for constructing a characteristic spectrum of a Shuangge antidiarrheal preparation includes the following steps:

[0010] (1) Preparation of reference solutions: Weigh chlorogenic acid, puerarin, paeoniflorin, baicalin, berberine hydrochloride and ammonium glycyrrhizate as reference standards, and dissolve them in methanol to prepare reference solutions.

[0011] Preparation of the test solution: Weigh the Shuangge antidiarrheal preparation into a solvent, mix well, and filter to obtain the test solution; the solvent is 0 to 100 v / v% methanol or 0 to 50 v / v% ethanol;

[0012] (2) High-performance liquid chromatography (HPLC) detection conditions: A column packed with octadecylsilane-bonded silica gel; mobile phase A was acetonitrile, mobile phase B was 0.05%–0.4% aqueous phosphoric acid solution, gradient elution; detection wavelength was 280 nm from 0 min to 22 min, 345 nm from 22 min to 41 min, and 250 nm from 41 min to 60 min; column temperature was 28℃–32℃; flow rate was 0.9 ml / min–1.1 ml / min; injection volume was 1 μl–50 μl.

[0013] (3) Determination: The reference solution and the test solution were determined by high performance liquid chromatography to obtain the chromatogram of the reference solution and the characteristic chromatogram of the test solution. The index components in the characteristic chromatogram of the test solution were assigned and located by the retention time of the index components in the reference solution.

[0014] The characteristic spectrum of the test solution should include 12 characteristic peaks. With peak 6 as the reference peak S, the characteristic peaks in the characteristic spectrum of the test solution are located by relative retention time, thereby obtaining the characteristic spectrum of the Shuangge antidiarrheal preparation.

[0015] Using peak 6 as the reference peak S with a retention time of 1.0000, the relative retention times of the 12 common characteristic peaks are as follows: peak 1 (0.2680), peak 2 (0.3191), peak 3 (0.4115), peak 4 (0.4522), peak 5 (0.5374), peak 6 (1.0000), peak 7 (1.4616), peak 8 (1.5944), peak 9 (1.7580), peak 10 (1.8952), peak 11 (2.0249), and peak 12 (2.4253). Their relative retention times should be within ±10% of the specified value. Among them, peak 3 is chlorogenic acid, peak 5 is puerarin, peak 6 is paeoniflorin, peak 9 is baicalin, peak 10 is berberine hydrochloride, and peak 12 is ammonium glycyrrhizate.

[0016] Furthermore, in step (1), the Shuangge antidiarrheal preparation is mixed by ultrasound, and the ultrasound time can be from 0 min to 60 min, preferably 30 min.

[0017] Furthermore, the gradient elution procedure in step (2) is as follows: .

[0018] Furthermore, in step (2), the chromatographic column is a Kromasil 100-5 C18 column with a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5.0 μm.

[0019] Furthermore, the Shuangge antidiarrheal preparation includes Shuangge antidiarrheal oral liquid or Shuangge antidiarrheal granules.

[0020] Furthermore, the solvent of the reference solution is 70 v / v % methanol.

[0021] Furthermore, the Shuangge antidiarrheal preparation is prepared by dissolving 2g in 50ml of solvent.

[0022] Further, the high performance liquid chromatography detection conditions in step (2) are as follows: the chromatographic column is a Kromasil 100-5 C18 column, the mobile phase A is acetonitrile, the mobile phase B is 0.1% phosphoric acid aqueous solution, the column temperature is 30℃, the flow rate is 1.0 ml / min, and the injection volume is 10 μl.

[0023] The characteristic spectrum of the Shuangge antidiarrheal preparation constructed by the above method.

[0024] The above-mentioned construction method is used in the quality testing of Shuangge antidiarrheal preparations.

[0025] Compared with the prior art, the technical effects of this application are as follows:

[0026] 1. This application employs high-performance liquid chromatography (HPLC) characteristic chromatographic method, which is simple to operate, has a short detection time, high sensitivity, is unaffected by temperature and humidity, and has high detection efficiency. For the first time, a characteristic chromatogram of Shuangge antidiarrheal preparation has been constructed for the quality control and qualitative evaluation of this product.

[0027] 2. This pharmaceutical preparation contains six traditional Chinese medicines and has a high concentration of active ingredients. If the pretreatment or chromatographic detection conditions are not adjusted satisfactorily, the resulting chromatogram will contain numerous impurity peaks, with severe overlap or tailing between peaks, making analysis impossible. This application addresses these issues by screening sample processing methods and optimizing chromatographic conditions, employing high-performance liquid chromatography gradient elution technology. This solves the problems of incomplete extraction of characteristic peaks, difficulty in separating multiple characteristic peaks, and interference from impurity peaks, providing a new analytical method for the internal quality control of Shuangge antidiarrheal preparations.

[0028] 3. This pharmaceutical preparation contains numerous active ingredients, each with different maximum UV absorption wavelengths. This can lead to significant differences in the response values ​​of the chromatographic peaks within the same chromatogram, preventing the effective detection of all active ingredients. This application employs multi-wavelength switching technology to ensure good separation and signal response for the six main active ingredients—chlorogenic acid, puerarin, paeoniflorin, baicalin, berberine hydrochloride, and ammonium glycyrrhizate—within the same chromatogram.

[0029] 4. The characteristic chromatograms provided by this invention are the first to perform quality control on ammonium glycyrrhizate, the main active ingredient in licorice.

[0030] 5. The characteristic spectrum constructed in this application contains 12 common peaks, filling the research gap in the characteristic spectrum of Shuangge antidiarrheal preparations. It can identify chlorogenic acid, puerarin, paeoniflorin, baicalin, berberine hydrochloride, and ammonium glycyrrhizate in Shuangge antidiarrheal preparations, and characterize the main active ingredients of each of the six herbs in the prescription. This method can qualitatively evaluate the overall efficacy of Shuangge antidiarrheal preparations and significantly improve the quality control level of Shuangge antidiarrheal preparations.

[0031] 6. The characteristic spectral method constructed in this application is simple to operate, and exhibits high stability, precision, and repeatability. It can provide a technical foundation for internal quality control in manufacturing enterprises, monitor drug quality, and ensure batch-to-batch stability; it can also provide an effective analytical means for aquaculture enterprises to identify the quality and authenticity of Shuangge antidiarrheal preparations on the market.

[0032] 7. The feature map method constructed in this application is applicable to the quality control of both Shuangge Zhixie oral liquid and Shuangge Zhixie granules. Attached Figure Description

[0033] The various technical features of this application and their relationships will be further explained below with reference to the accompanying drawings. The drawings are exemplary; some technical features are not shown to scale, and some drawings may omit technical features commonly used in the art to which this application pertains that are not essential for understanding and implementing this application, or additionally show technical features that are not essential for understanding and implementing this application. In other words, the combination of various technical features shown in the drawings is not intended to limit this application. Furthermore, throughout this application, the same reference numerals refer to the same things. Specific descriptions of the drawings are as follows:

[0034] Figure 1 This is a characteristic chromatogram of the Shuangge antidiarrheal preparation of this application;

[0035] Figure 2 This is a chromatogram of different extraction solvents used in Example 2 of this application;

[0036] Figure 3 This is a chromatogram showing the selection of different detection wavelengths in Example 2 of this application;

[0037] Figure 4 This is a chromatogram of different ultrasonic times in Example 2 of this application;

[0038] Figure 5 This is a chromatogram of different injection volumes for Example 2 of this application;

[0039] Figure 6 The chromatograms are of 10 batches of Shuangge Antidiarrheal Oral Solution in Example 3 of this application;

[0040] Figure 7 Examples of different chromatographic columns used in Example 4 of this application are chromatograms.

[0041] Figure 8 This is a chromatogram of different flow rates and column temperatures for Example 4 of this application;

[0042] Figure 9 This is a chromatogram of different phosphoric acid concentrations used in Example 4 of this application;

[0043] Figure 10 This is the chromatogram for the injection precision assessment in Example 4 of this application;

[0044] Figure 11 This is the chromatogram for the repeatability test of Example 4 of this application;

[0045] Figure 12 This is the chromatogram for the stability study of the solution in Example 4 of this application;

[0046] Figure 13 The chromatograms are for three batches of Shuangge Zhixie Granules as described in Example 5 of this application. Detailed Implementation

[0047] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions described in this application will be further described in detail below with reference to specific embodiments.

[0048] In this application, terms such as "further," "even more," and "particularly" are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of this invention.

[0049] The Shuangge antidiarrheal preparations in this application include Shuangge antidiarrheal oral liquid or Shuangge antidiarrheal granules, or other dosage forms prepared from six traditional Chinese medicines: honeysuckle, kudzu root, scutellaria, coptis, white peony root, and prepared licorice root.

[0050] This application provides a method for constructing the characteristic spectrum of a Shuangge antidiarrheal preparation, comprising the following steps:

[0051] (1) Preparation of reference solutions: Weigh chlorogenic acid, puerarin, paeoniflorin, baicalin, berberine hydrochloride and ammonium glycyrrhizate as reference standards, and dissolve them in methanol to prepare reference solutions.

[0052] In some embodiments, the concentrations of chlorogenic acid in the reference solution are 0.059 mg / ml, puerarin in the reference solution are 0.27 mg / ml, paeoniflorin in the reference solution are 0.79 mg / ml, baicalin in the reference solution are 0.15 mg / ml, berberine hydrochloride in the reference solution is 0.0069 mg / ml, and glycyrrhizic acid ammonium in the reference solution are 0.033 mg / ml.

[0053] In some embodiments, the solvent for the reference solution is 70 v / v % methanol.

[0054] Preparation of the test solution: Weigh the Shuangge antidiarrheal preparation into a solvent, mix well, and filter to obtain the test solution; the solvent is 0 to 100 v / v% methanol or 0 to 50 v / v% ethanol.

[0055] In some embodiments, the Shuangge antidiarrheal preparation is 2g dissolved in 50ml of solvent.

[0056] In some embodiments, the solvent may be, but is not limited to, water, 10 v / v% methanol, 50 v / v% methanol, 100 v / v% methanol, 10 v / v% ethanol or 50 v / v% ethanol, preferably 50 v / v% methanol or 50 v / v% ethanol.

[0057] In some embodiments, the Shuangge antidiarrheal preparation is mixed by ultrasound in step (1), and the ultrasound time can be 0 min to 60 min, preferably 30 min.

[0058] (2) High performance liquid chromatography detection conditions: chromatographic column with octadecylsilane-bonded silica gel as packing material; mobile phase A is acetonitrile, mobile phase B is 0.05% to 0.4% aqueous phosphoric acid solution, gradient elution; detection wavelength is 280 nm from 0 min to 22 min, detection wavelength is 345 nm from 22 min to 41 min, and detection wavelength is 250 nm from 41 min to 60 min; column temperature is 28℃ to 32℃; flow rate is 0.9 ml / min to 1.1 ml / min; injection volume is 1 μl to 50 μl.

[0059] In some implementations, the gradient elution procedure is as follows: .

[0060] In some embodiments, the chromatographic column is a Kromasil 100-5 C18 column with a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5.0 μm.

[0061] In some implementations, the high-performance liquid chromatography (HPLC) detection conditions are as follows: the chromatographic column is a Kromasil 100-5 C18 column, mobile phase A is acetonitrile, mobile phase B is 0.1% aqueous phosphoric acid solution, the column temperature is 30°C, the flow rate is 1.0 ml / min, and the injection volume is 10 μl.

[0062] (3) Determination: The reference solution and the test solution were determined by high performance liquid chromatography to obtain the chromatogram of the reference solution and the characteristic chromatogram of the test solution. The index components in the characteristic chromatogram of the test solution were assigned and located by the retention time of the index components in the reference solution.

[0063] The characteristic spectrum of the test solution should include 12 characteristic peaks. With peak 6 as the reference peak S, the characteristic peaks in the characteristic spectrum of the test solution are located by relative retention time, thereby obtaining the characteristic spectrum of the Shuangge antidiarrheal preparation.

[0064] Using peak 6 as the reference peak S with a retention time of 1.0000, the relative retention times of the 12 common characteristic peaks are as follows: peak 1 (0.2680), peak 2 (0.3191), peak 3 (0.4115), peak 4 (0.4522), peak 5 (0.5374), peak 6 (1.0000), peak 7 (1.4616), peak 8 (1.5944), peak 9 (1.7580), peak 10 (1.8952), peak 11 (2.0249), and peak 12 (2.4253). Their relative retention times should be within ±10% of the specified value. Among them, peak 3 is chlorogenic acid, peak 5 is puerarin, peak 6 is paeoniflorin, peak 9 is baicalin, peak 10 is berberine hydrochloride, and peak 12 is ammonium glycyrrhizate.

[0065] In one implementation, the construction method steps are as follows:

[0066] Preparation of reference solutions: Weigh chlorogenic acid, puerarin, paeoniflorin, baicalin, berberine hydrochloride, and ammonium glycyrrhizate as reference standards, and dissolve them in 70% v / v methanol to prepare reference solutions containing 0.059 mg / ml chlorogenic acid, 0.27 mg / ml puerarin, 0.79 mg / ml paeoniflorin, 0.15 mg / ml baicalin, 0.0069 mg / ml berberine hydrochloride, and 0.033 mg / ml ammonium glycyrrhizate.

[0067] Accurately weigh approximately 2g of Shuangge Antidiarrheal Oral Liquid, place it in a 50ml volumetric flask, add 50% v / v methanol to near the mark, sonicate for 30 minutes, cool, make up to volume, shake well, and filter to obtain the final product.

[0068] A Kromasil 100-5C18 column (4.6 mm × 250 mm, 5 μm) was used as the packing material, with acetonitrile as mobile phase A and 0.1% aqueous phosphoric acid solution as mobile phase B. Gradient elution was performed as shown in the table below. The detection wavelength was 280 nm from 0 min to 22 min, 345 nm from 22 min to 41 min, and 250 nm from 41 min to 60 min. The column temperature was 30 °C, the flow rate was 1.0 mL / min, and the injection volume was 10 μL.

[0069] Accurately measure 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and obtain the chromatograms of the reference solution and the characteristic chromatograms of the test solution.

[0070] The characteristic spectrum of the test solution should include 12 characteristic peaks. Peak 6 is used as the reference peak S. The characteristic peaks in the characteristic spectrum of the test solution are located by relative retention time, so as to obtain the characteristic spectrum of Shuangge antidiarrheal preparation.

[0071] Using peak 6 as the reference peak (S peak) with a retention time of 1.0000, the relative retention times of the 12 common characteristic peaks are as follows: peak 1 (0.2680), peak 2 (0.3191), peak 3 (0.4115), peak 4 (0.4522), peak 5 (0.5374), peak 6 (1.0000), peak 7 (1.4616), peak 8 (1.5944), peak 9 (1.7580), peak 10 (1.8952), peak 11 (2.0249), and peak 12 (2.4253). Their relative retention times should be within ±10% of the specified value. Peak 3 is chlorogenic acid, peak 5 is puerarin, peak 6 is paeoniflorin, peak 9 is baicalin, peak 10 is berberine hydrochloride, and peak 12 is ammonium glycyrrhizate. The results are as follows: Figure 1As shown.

[0072] This application provides the feature spectrum obtained by the above construction method, and the construction method and feature spectrum can be used for quality testing of Shuangge antidiarrheal preparations.

[0073] This application provides a quality testing method for a Shuangge antidiarrheal preparation, which includes the step of comparing the spectrum of the product to be tested with the characteristic spectrum obtained by the construction method of this application.

[0074] Example 1: Test materials used in all examples

[0075] 1. Main Instruments

[0076] An Agilent 1260 high-performance liquid chromatograph (equipped with a G7117C DAD detector, Agilent Technologies, USA), an electronic analytical balance (XSR205DU, Mettler Toledo Technologies), an ultrasonic cleaner (KQ-500DB, Kunshan Ultrasonic Instrument Co., Ltd.), and chromatographic columns (Kromasil 100-5 C18 4.6mm×250mm, 5μm, Akzonobel, Sweden; Thermo Hypersil GOLD C18 4.6mm×250mm, 5μm, Thermo Technologies)

[0077] 2. Main reagents and standard substances

[0078] Phosphoric acid (chromatographic grade, Tianjin Kemeo Chemical Reagent Co., Ltd., batch number: 20231023); Acetonitrile (chromatographic grade, Shanghai Anpu Testing Technology Co., Ltd., batch number: O8371440); Chlorogenic acid (China National Institutes for Food and Drug Control, batch number: 110753-202119, content: 96.3%); Puerarin (China National Institutes for Food and Drug Control, batch number: 110752-202217, content: 96.8%); Paeoniflorin (China National Institutes for Food and Drug Control, batch number: 110752-202217, content: 96.8%); The following ingredients were tested: Baicalin (China National Institutes for Food and Drug Control, batch number: 110736-202447, content: 98.1%); Berberine hydrochloride (China National Institutes for Food and Drug Control, batch number: 110715-202223, content: 97.2%); berberine hydrochloride (China National Institutes for Food and Drug Control, batch number: 110713-202316, content: 91.0%); ammonium glycyrrhizate (China National Institutes for Food and Drug Control, batch number: 110731-202122, 94.4%).

[0079] 3 samples

[0080] Shuangge Antidiarrheal Oral Solution (Luoyang Huizhong Veterinary Medicine Co., Ltd., batch numbers: 20230102, 20230302, 20230401, 20230503, 20230504, 20230702, 20230801, 20240302, 20240603, 20240801); Shuangge Antidiarrheal Granules (Luoyang Huizhong Veterinary Medicine Co., Ltd., batch numbers: 20240301, 20240602, 20240801).

[0081] Example 2: Establishment of a characteristic chromatographic method for Shuangge antidiarrheal preparations

[0082] 1. Investigation of extraction solvent

[0083] 1.1 Preparation of reference solution: Accurately weigh appropriate amounts of chlorogenic acid, puerarin, paeoniflorin, baicalin, berberine hydrochloride, and ammonium glycyrrhizate, and add 70% v / v methanol to prepare a reference solution containing 0.059 mg / ml chlorogenic acid, 0.27 mg / ml puerarin, 0.79 mg / ml paeoniflorin, 0.15 mg / ml baicalin, 0.0069 mg / ml berberine hydrochloride, and 0.033 mg / ml ammonium glycyrrhizate.

[0084] 1.2 Preparation of the test solution: Accurately weigh about 2g of Shuangge Antidiarrheal Oral Liquid, place it in a 50ml volumetric flask, add water, 10% v / v methanol, 50% v / v methanol, 100% v / v methanol, 10% v / v ethanol, 50% v / v ethanol, and 100% v / v ethanol to near the mark, sonicate for 30min, cool, make up to volume, shake well, and filter to obtain the test solution.

[0085] 1.3 Chromatographic conditions: A Kromasil 100-5 C184.6 mm × 250 mm, 5 μm column packed with octadecylsilane-bonded silica gel was used; mobile phase A was acetonitrile, and mobile phase B was 0.1% aqueous phosphoric acid solution, with gradient elution; detection wavelength was 280 nm from 0 min to 22 min, 345 nm from 22 min to 41 min, and 250 nm from 41 min to 60 min; column temperature was 30 ℃; flow rate was 1.0 ml / min; and injection volume was 10 μl.

[0086] The gradient elution procedure is shown in the table below;

[0087] 1.4 Determination: Accurately measure 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, obtain the chromatogram of the reference solution and the characteristic chromatogram of the test solution, and compare the peak shape and resolution of different solvents used as extraction solvents.

[0088] 1.5 Results

[0089] In the chromatogram using 100% v / v ethanol as the solvent, no ammonium glycyrrhizate peak was observed. While peak sizes varied with other solvents, 12 characteristic peaks were obtained in all cases, with 50% v / v methanol and 50% v / v ethanol showing the best results. Therefore, suitable solvents include water to 100% v / v methanol and water to 50% v / v ethanol, with 50% v / v methanol and 50% v / v ethanol being preferred. See Figure 2 .

[0090] 2 Selection of detection wavelength

[0091] 2.1 Preparation of reference solution: Same as in Example 2 "1.1".

[0092] 2.2 Preparation of test solution: Accurately weigh about 2g of Shuangge antidiarrheal oral liquid, place it in a 50ml volumetric flask, add 50% v / v methanol to near the mark, sonicate for 30min, cool, make up to volume, shake well, filter, and the test solution is obtained.

[0093] 2.3 Chromatographic conditions:

[0094] A Kromasil 100-5C18 column (4.6 mm × 250 mm, 5 μm) was used as the packing material, with acetonitrile as mobile phase A and 0.1% aqueous phosphoric acid solution as mobile phase B. Gradient elution was performed, and the gradient elution program is shown in the table below. The column temperature was 30 °C, the flow rate was 1.0 mL / min, and the injection volume was 10 μL.

[0095] Wavelength condition 1: 250nm

[0096] Wavelength condition two: 327nm

[0097] Wavelength condition three: 345nm

[0098] Wavelength condition four: 280nm

[0099] Wavelength condition 5: The detection wavelength is 280nm from 0min to 22min, 345nm from 22min to 41min, and 250nm from 41min to 60min.

[0100] 2.4 Results:

[0101] Shuangge Antidiarrheal Oral Liquid contains six traditional Chinese medicines with complex components. The maximum absorption of different components varies, and the absorption rates were investigated at 250 nm, 327 nm, 345 nm, and 280 nm. Results showed that glycyrrhizic acid did not show peaks at 327 nm, 345 nm, and 280 nm, while chlorogenic acid peaks were indistinguishable at 250 nm. To ensure the separation and response of the six known components, a multi-wavelength switching method was used: detection wavelength of 280 nm from 0 min to 22 min, 345 nm from 22 min to 41 min, and 250 nm from 41 min to 60 min. The results showed good response and separation for each component. (See...) Figure 3 .

[0102] 3. Examination of extraction time

[0103] 3.1 Preparation of reference solution: Same as in Example 2, "1.1".

[0104] 3.2 Preparation of test solution: Accurately weigh about 2g of Shuangge antidiarrheal oral liquid, place it in a 50ml volumetric flask, add 50% v / v methanol to near the mark, shake well without sonication, sonicate for 30 minutes, and sonicate for 60 minutes respectively, cool, make up to volume, shake well, and filter to obtain the test solution.

[0105] 3.3 Chromatographic conditions: Same as in Example 2, "1.3".

[0106] 3.4 Determination: Accurately measure 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and obtain the characteristic chromatograms of the reference solution and the test solution.

[0107] 3.5 Results: After shaking without sonication, sonication for 30 min, and sonication for 60 min all resulted in good elution of characteristic peaks. Considering both extraction completeness and efficiency, 30 min was the optimal extraction time. See [link / details]. Figure 4 .

[0108] 4. Investigation of injection volume

[0109] 4.1 Preparation of reference solution: Same as in Example 2, "1.1".

[0110] 4.2 Preparation of test solution: Accurately weigh about 2g of Shuangge antidiarrheal oral liquid, place it in a 50ml volumetric flask, add 50% v / v methanol to near the mark, sonicate for 30 minutes, cool, make up to volume, shake well, filter, and the test solution is obtained.

[0111] 4.3 Chromatographic conditions: The injection volumes were set to 1 μl, 5 μl, 10 μl, 20 μl, and 50 μl, respectively, and the remaining chromatographic conditions were the same as in Example 2, "1.3".

[0112] 4.4 Determination: Accurately measure 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and obtain the characteristic chromatograms of the reference solution and the test solution.

[0113] 4.5 Results:

[0114] Using injection volumes of 1 μl to 50 μl, the obtained characteristic chromatograms showed good peak shape and resolution, with 10 μl being the preferred volume. See the figure for 10 μl injection volume; 1 μl, 5 μl, 20 μl, and 50 μl injection volumes are shown in the figure. Figure 5 .

[0115] Example 3: Confirmation of common characteristic peaks in the characteristic spectrum

[0116] 1. Preparation of reference solution

[0117] Accurately weigh appropriate amounts of chlorogenic acid, puerarin, paeoniflorin, baicalin, berberine hydrochloride, and ammonium glycyrrhizate, and add 70% v / v methanol to prepare a reference solution containing 0.059 mg / ml chlorogenic acid, 0.27 mg / ml puerarin, 0.79 mg / ml paeoniflorin, 0.15 mg / ml baicalin, 0.0069 mg / ml berberine hydrochloride, and 0.033 mg / ml ammonium glycyrrhizate.

[0118] 2. Preparation of the test solution

[0119] Accurately weigh approximately 2g of 10 batches of Shuangge Antidiarrheal Oral Solution (batch numbers: 20230102, 20230302, 20230401, 20230503, 20230504, 20230702, 20230801, 20240302, 20240603, 20240801), place in a 50ml volumetric flask, add 50% v / v methanol to near the mark, sonicate for 30min, cool, make up to volume, shake well, and filter to obtain the final product.

[0120] 3 Chromatographic conditions

[0121] A Kromasil 100-5 C18 column (4.6 mm × 250 mm, 5 μm) was used as the packing material, with acetonitrile as mobile phase A and 0.1% aqueous phosphoric acid solution as mobile phase B. Gradient elution was used, and the gradient elution program is shown in the table below. The detection wavelength was 280 nm from 0 min to 22 min, 345 nm from 22 min to 41 min, and 250 nm from 41 min to 60 min. The column temperature was 30 °C, the flow rate was 1.0 mL / min, and the injection volume was 10 μL.

[0122] 4. Measurement

[0123] Accurately measure 10 μl each of the mixed reference solution and the test solution, and inject them into the liquid chromatograph to obtain the chromatograms of the reference solution and the characteristic chromatograms of the test solution.

[0124] 5 Results

[0125] Test solutions were prepared using 10 batches of Shuangge Antidiarrheal Oral Solution, and chromatograms were obtained for 10 batches. Twelve stable chromatographic peaks were identified as common characteristic peaks of the product. Reference solutions were used to identify the main characteristic peaks: peak 3 was chlorogenic acid, peak 5 was puerarin, peak 6 was paeoniflorin, peak 9 was baicalin, peak 10 was berberine hydrochloride, and peak 12 was ammonium glycyrrhizate. The paeoniflorin peak, with a moderate and stable retention time, was selected as the reference peak (S peak). The relative retention times of the 12 common characteristic peaks were as follows: peak 1 (0.2680), peak 2 (0.3191), peak 3 (0.4115), peak 4 (0.4522), peak 5 (0.5374), peak 6 (1.0000), peak 7 (1.4616), peak 8 (1.5944), peak 9 (1.7580), peak 10 (1.8952), peak 11 (2.0249), and peak 12 (2.4253). Their relative retention times should be within ±10% of the specified value. See [link / reference]. Figure 6 .

[0126] Example 4: Verification of the Feature Mapping Method

[0127] 1. Durability assessment

[0128] 1.1 Investigation of different chromatographic columns

[0129] (1) Preparation of reference solution: Accurately weigh appropriate amounts of chlorogenic acid, puerarin, paeoniflorin, baicalin, berberine hydrochloride and ammonium glycyrrhizate, and add 70% v / v methanol to prepare a reference solution containing 0.059 mg / ml chlorogenic acid, 0.27 mg / ml puerarin, 0.79 mg / ml paeoniflorin, 0.15 mg / ml baicalin, 0.0069 mg / ml berberine hydrochloride and 0.033 mg / ml ammonium glycyrrhizate.

[0130] (2) Preparation of test solution: Accurately weigh about 2g of Shuangge antidiarrheal oral liquid, place it in a 50ml volumetric flask, add 50% v / v methanol to near the mark, sonicate for 30min, cool, make up to volume, shake well, filter, and the test solution is obtained.

[0131] (3) Chromatographic conditions: Column 1: Kromasil 100-5 C18 column (4.6mm×250mm, 5μm, SN: P455935); Column 2: Kromasil 100-5 C18 column (4.6mm×250mm, 5μm, SN: P455942); Column 3: ThermoHypersil GOLD C18 column (4.6mm×250mm, 5μm, SN: USWZ001555). All other conditions were the same as in Example 3, "3 Chromatographic Conditions".

[0132] (4) Determination: Accurately measure 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and obtain the characteristic chromatograms of the reference solution and the test solution.

[0133] (5) Results: Using columns from different manufacturers, columns from different batches and manufacturers showed good peak shapes and resolutions for all characteristic peaks, with relative retention times within the specified range. The Kromasil 100-5 C18 column (4.6mm × 250mm, 5μm) performed best; therefore, the Kromasil 100-5 C18 column (4.6mm × 250mm, 5μm) is the preferred choice. See the chromatogram below. Figure 7 .

[0134] Table 1 Results of the durability study of different chromatographic columns

[0135] 1.2 Column Temperature Investigation

[0136] (1) Preparation of reference solution: Same as in Example 4 "1.1".

[0137] (2) Preparation of test solution: Same as in Example 4 "1.1".

[0138] (3) Chromatographic conditions:

[0139] Column temperature 1: 28℃; Column temperature 2: 30℃; Column temperature 3: 32℃. The remaining chromatographic conditions are the same as those in Example 3, "3 Chromatographic Conditions".

[0140] (4) Determination: Accurately measure 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and obtain the characteristic chromatograms of the reference solution and the test solution.

[0141] (5) Results: Using column temperatures of 28℃, 30℃, and 32℃, the peak shapes and separation of each characteristic peak in the characteristic chromatogram of Shuangge Antidiarrheal Oral Liquid were good, and the relative retention times were all within the specified range, meeting the requirements. 30℃ was the preferred temperature. See [link to relevant documentation]. Figure 8 .

[0142] Table 2 Results of durability tests at different column temperatures

[0143] 1.3 Investigation of Phosphoric Acid Ratio

[0144] (1) Preparation of reference solution: Same as in Example 4 "1.1".

[0145] (2) Preparation of test solution: Same as in Example 4 "1.1".

[0146] (3) Chromatographic conditions:

[0147] Phosphoric acid solution ratio 1: 0.05%; phosphoric acid solution ratio 2: 0.1%; phosphoric acid solution ratio 3: 0.4%. The remaining chromatographic conditions are the same as in Example 3, "3 Chromatographic Conditions".

[0148] (4) Determination: Accurately measure 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and obtain the characteristic chromatograms of the reference solution and the test solution.

[0149] (5) Results: Using phosphoric acid solutions at ratios of 0.05%, 0.1%, and 0.4%, the peak shapes and resolutions of each characteristic peak in the chromatogram of Shuangge Antidiarrheal Oral Solution were all good, and the relative retention times were all within the specified range, meeting the requirements. 0.1% was the preferred ratio. See Figure 9 .

[0150] Table 3 Results of durability studies with different phosphoric acid ratios

[0151] 1.4 Flow velocity investigation

[0152] (1) Preparation of reference solution: Same as in Example 4 "1.1".

[0153] (2) Preparation of test solution: Same as in Example 4 "1.1".

[0154] (3) Chromatographic conditions:

[0155] Flow rate 1: 0.9 ml / min; Flow rate 2: 1.0 ml / min; Flow rate 3: 1.1 ml / min. The remaining chromatographic conditions are the same as in Example 3, "3 Chromatographic Conditions".

[0156] (4) Determination: Accurately measure 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and obtain the characteristic chromatograms of the reference solution and the test solution.

[0157] (5) Results: At flow rates of 0.9 ml / min, 1.0 ml / min, and 1.1 ml / min, the peak shapes and resolutions of all characteristic peaks in the chromatogram of Shuangge Antidiarrheal Oral Solution were good, and the relative retention times were all within the specified range, meeting the requirements. See [link to relevant documentation]. Figure 8 .

[0158] Table 4 Results of durability tests at different flow velocities

[0159] 2. Sample injection precision test

[0160] (1) Preparation of test solution: Same as in Example 4 "1.1".

[0161] (2) Chromatographic conditions: Same as “3 Chromatographic conditions” in Example 3.

[0162] (3) Determination: Take a sample of Shuangge Antidiarrheal Oral Liquid and prepare one test solution. Inject the sample six times consecutively to obtain the characteristic chromatograms of the six test solutions. Calculate the RSD of the relative retention time of each characteristic peak.

[0163] (4) Results: The relative retention times (RSDs) of each characteristic peak were 0.2%–0.3%, all meeting the requirements. See Figure 10 .

[0164] Table 5 Results of injection precision study (relative retention time)

[0165] 3. Repeatability test

[0166] (1) Preparation of reference solution: Same as in Example 4 "1.1".

[0167] (2) Preparation of test solution: Same as in Example 4 "1.1".

[0168] (3) Chromatographic conditions: Same as in Example 3, “3 Chromatographic conditions”.

[0169] (4) Determination: Take the Shuangge Antidiarrheal Oral Liquid sample and prepare 6 test solutions. Inject the reference solution and the test solution into the liquid chromatograph to obtain the characteristic chromatograms of the 6 test solutions. Calculate the RSD of the relative retention time of each characteristic peak.

[0170] (5) Results: The relative retention times (RSDs) of the characteristic peaks in the six test solutions ranged from 0.2% to 0.3%, all meeting the requirements. See Figure 11 .

[0171] Table 6. Results of Repeatability Testing (Relative Retention Time)

[0172] 4. Solution stability study

[0173] (1) Preparation of test solution: Same as in Example 4 "1.1".

[0174] (2) Chromatographic conditions: Same as “3 Chromatographic conditions” in Example 3.

[0175] (3) Determination: Take a sample of Shuangge antidiarrheal oral liquid and prepare one test solution. Inject the sample at 0h, 3h, 6h, 9h and 12h respectively to obtain the characteristic chromatograms of the six test solutions. Calculate the RSD of the relative retention time of each characteristic peak.

[0176] (4) Results: The relative retention times (RSDs) of each characteristic peak were 0.2%–0.5%, all meeting the requirements. See Figure 12 .

[0177] Table 7 Results of solution stability study (relative retention time)

[0178] Example 5: Application of the Feature Mapping Method

[0179] (1) Preparation of reference solution: Same as in Example 4 "1.1".

[0180] (2) Preparation of test solution: Accurately weigh about 2g of 10 batches of Shuangge Zhixie oral liquid (batch numbers: 20230102, 20230302, 20230401, 20230503, 20230504, 20230702, 20230801, 20240302, 20240603, 20240801) and Shuangge Zhixie granules (batch numbers: 20240301, 20240602, 20240801), place them in a 50ml volumetric flask, add 50% v / v methanol to near the mark, sonicate for 30min, cool, make up to volume, shake well, filter, and the test solution is obtained.

[0181] (3) Chromatographic conditions: Same as in Example 3, “3 Chromatographic conditions”.

[0182] (4) Determination: Accurately measure 10 μl of the mixed reference solution and the test solution respectively, inject them into the liquid chromatograph, and obtain the chromatogram of the reference solution and the characteristic chromatogram of the test solution.

[0183] (5) Results: Using the established characteristic chromatogram detection method, 10 batches of Shuangge Zhixie oral liquid and 3 batches of Shuangge Zhixie granules were tested, and all showed 12 characteristic peaks with good separation. The results indicate that this method is applicable to both Shuangge Zhixie oral liquid and Shuangge Zhixie granules, improving the quality control level of the Shuangge Zhixie series of preparations. Data for 10 batches of Shuangge Zhixie oral liquid are shown in Example 3, and results for 3 batches of Shuangge Zhixie granules are shown in the table below. The chromatograms are shown in... Figure 13 .

[0184] Table 8. Relative retention times of three batches of Shuangge Antidiarrheal Granules

[0185] Note that the above are merely preferred embodiments and the technical principles employed in this application. Those skilled in the art will understand that this application is not limited to the specific embodiments described herein, and various obvious changes, readjustments, and substitutions can be made without departing from the scope of protection of this application. Therefore, although this application has been described in detail through the above embodiments, this application is not limited to the above embodiments. Many other equivalent embodiments may be included without departing from the technical concept of this application, all of which fall within the scope of protection of this application.

Claims

1. A method for constructing a characteristic spectrum of a Shuangge antidiarrheal preparation, characterized in that, Includes the following steps: (1) Preparation of reference solutions: Weigh chlorogenic acid, puerarin, paeoniflorin, baicalin, berberine hydrochloride and ammonium glycyrrhizate as reference standards, and dissolve them in methanol to prepare reference solutions. Preparation of the test solution: Weigh the Shuangge antidiarrheal preparation into a solvent, mix well, and filter to obtain the test solution; the solvent is 0 to 100 v / v% methanol or 0 to 50 v / v% ethanol; (2) High-performance liquid chromatography (HPLC) detection conditions: A column packed with octadecylsilane-bonded silica gel; mobile phase A was acetonitrile, mobile phase B was 0.05%–0.4% aqueous phosphoric acid solution, gradient elution; detection wavelength was 280 nm from 0 min to 22 min, 345 nm from 22 min to 41 min, and 250 nm from 41 min to 60 min; column temperature was 28℃–32℃; flow rate was 0.9 ml / min–1.1 ml / min; injection volume was 1 μl–50 μl. (3) Determination: The reference solution and the test solution were determined by high performance liquid chromatography to obtain the chromatogram of the reference solution and the characteristic chromatogram of the test solution. The index components in the characteristic chromatogram of the test solution were assigned and located by the retention time of the index components in the reference solution. The characteristic spectrum of the test solution should include 12 characteristic peaks. With peak 6 as the reference peak S, the characteristic peaks in the characteristic spectrum of the test solution are located by relative retention time, thereby obtaining the characteristic spectrum of the Shuangge antidiarrheal preparation. Using peak 6 as the reference peak S with a retention time of 1.0000, the relative retention times of the 12 common characteristic peaks are as follows: peak 1 (0.2680), peak 2 (0.3191), peak 3 (0.4115), peak 4 (0.4522), peak 5 (0.5374), and peak 6. Peak 7 (1.0000), Peak 8 (1.4616), Peak 9 (1.5944), Peak 9 Peaks 10 (1.7580), 11 (1.8952), 12 (2.0249), and 13 (2.4253) should have relative retention times within ±10% of the specified values. Peak 3 is chlorogenic acid, peak 5 is puerarin, peak 6 is paeoniflorin, peak 9 is baicalin, peak 10 is berberine hydrochloride, and peak 12 is ammonium glycyrrhizate.

2. The construction method according to claim 1, characterized in that, In step (1), the Shuangge antidiarrheal preparation is mixed by ultrasound, and the ultrasound time can be from 0 min to 60 min, preferably 30 min.

3. The construction method according to claim 1, characterized in that, The gradient elution procedure in step (2) is as follows:

4. The construction method according to claim 1, characterized in that, In step (2), the chromatographic column is a Kromasil 100-5C18 column with a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5.0 μm.

5. The construction method according to claim 1, characterized in that, The Shuangge antidiarrheal preparation includes Shuangge antidiarrheal oral liquid or Shuangge antidiarrheal granules.

6. The construction method according to claim 1, characterized in that, The solvent for the reference solution is 70 v / v % methanol.

7. The construction method according to claim 1, characterized in that, The Shuangge antidiarrheal preparation is prepared by dissolving 2g in 50ml of solvent.

8. The construction method according to claim 1, characterized in that, The high performance liquid chromatography (HPLC) detection conditions in step (2) are as follows: the chromatographic column is a Kromasil 100-5C18 column, the mobile phase A is acetonitrile, the mobile phase B is 0.1% phosphoric acid aqueous solution, the column temperature is 30℃, the flow rate is 1.0 ml / min, and the injection volume is 10 μl.

9. The characteristic spectrum of the Shuangge antidiarrheal preparation constructed by the construction method according to any one of claims 1 to 8.

10. Use of the construction method according to any one of claims 1 to 8 in the quality testing of the Shuangge antidiarrheal preparation.

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