A thin layer chromatography method for detecting pediatric security formulations

Abstract

The present application relates to a kind of pediatric security preparation thin layer chromatography detection method, including using thin layer chromatography to identify Bupleurum in the pediatric security preparation, Qianhu. The thin layer chromatography detection method of Bupleurum includes: respectively preparing Bupleurum test solution and Bupleurum control solution, test solution and control solution are on the same thin layer plate, with volume ratio (7-9) : (1-5) :1 mixture of ethyl acetate, ethanol and water as developing agent is developed, after coloration, first viewing is carried out;The thin layer chromatography detection method of Qianhu includes: respectively preparing Qianhu test solution and Qianhu control solution, test solution and control solution are on the same thin layer chromatography plate, with volume ratio (2-4) :1 mixture of petroleum ether and ethyl acetate as developing agent is developed, second viewing is carried out.The method has the advantages of rapid analysis, simple operation, high accuracy, wide adaptability and the like, and is conducive to the establishment and overall evaluation of pediatric security preparation quality standard.

Description

A thin-layer chromatography detection method for a pediatric safety preparation Technical Field

[0001] This invention relates to the field of traditional Chinese medicine detection technology, and in particular to a thin-layer chromatography detection method for pediatric safety preparations. Background Technology

[0002] Xiao'er Baoan Wan (Children's Safety Pill) has the effects of dispelling wind, calming the nerves, and eliminating phlegm. It is mainly used for vomiting and diarrhea, indigestion, early stage of cold, infantile convulsions, and cough with excessive phlegm. It is composed of 28 medicinal materials, including Pinellia ternata (processed), Gastrodia elata (processed), Atractylodes lancea (soaked), Pogostemon cablin, Notopterygium incisum, Hordeum vulgare (fried), Areca catechu peel, Aucklandia lappa, Prunus armeniaca (oil removed), Platycodon grandiflorus, Uncaria rhynchophylla, Pearl, Cinnamon, Saposhnikovia divaricata, Mentha haplocalyx, Poria cocos, Magnolia officinalis (fried with ginger juice), Bombyx mori (processed), Coptis chinensis, Citrus reticulata peel, Massa fermentata, Asarum heterotropoides, Cinnamomum cassia, Peucedanum praeruptorum, Bupleurum chinense, Amber, Borneol, and Glycyrrhiza uralensis. The chemical composition of this traditional Chinese medicine is complex and diverse, and its efficacy is not solely due to the action of a single component. However, there is currently a lack of comprehensive and accurate testing methods for its quality control. Summary of the Invention

[0003] To address the aforementioned problems, this invention provides a thin-layer chromatography detection method for pediatric safety preparations, which can rapidly and accurately detect Bupleurum chinense and Peucedanum praeruptorum, improving the accuracy and comprehensiveness of quality control of pediatric safety preparations and facilitating the establishment and overall evaluation of quality standards for pediatric safety preparations.

[0004] The technical solution is as follows:

[0005] A thin-layer chromatography detection method for a pediatric safety preparation, wherein the raw materials of the pediatric safety preparation include: Pinellia ternata, Gastrodia elata, Atractylodes lancea, Pogostemon cablin, Notopterygium incisum, Hordeum vulgare, Areca catechu, Aucklandia lappa, Prunus armeniaca, Platycodon grandiflorus, Uncaria rhynchophylla, Pearl, Cinnamon, Saposhnikovia divaricata, Mentha haplocalyx, Poria cocos, Magnolia officinalis, Bombyx mori, Coptis chinensis, Citrus reticulata, Massa fermentata, Asarum heterotropoides, Cinnamomum cassia, Peucedanum praeruptorum, Bupleurum chinense, Amber, Borneol, and Glycyrrhiza uralensis;

[0006] The thin-layer chromatography detection method for the pediatric safety preparation includes using thin-layer chromatography to identify Bupleurum chinense and Peucedanum praeruptorum in the pediatric safety preparation;

[0007] The thin-layer chromatography detection method for Bupleurum chinense includes the following steps:

[0008] Prepare Bupleurum test solution and Bupleurum reference solution separately. Spot the test solution and reference solution on the same thin-layer chromatography plate. Develop the plate with a mixture of ethyl acetate, ethanol and water in a volume ratio of (7-9):(1-5):1 as the developing solvent. After color development, perform the first inspection.

[0009] The colorimetric reagent is a mixture of p-dimethylaminobenzaldehyde and sulfuric acid ethanol solution, or a mixture of p-dimethylaminobenzaldehyde and sulfuric acid aqueous solution.

[0010] The mass-to-volume ratio of p-dimethylaminobenzaldehyde to the sulfuric acid ethanol solution is (1-5) g: 100 mL, and the volume percentage of sulfuric acid in the sulfuric acid ethanol solution is 9%-11%.

[0011] The mass-to-volume ratio of p-dimethylaminobenzaldehyde to the sulfuric acid aqueous solution is (1-5) g: 100 mL, and the volume percentage of sulfuric acid in the sulfuric acid aqueous solution is 30%-50%.

[0012] The thin-layer chromatography detection method for the aforementioned Peucedanum praeruptorum includes the following steps:

[0013] Prepare separate test solutions and reference solutions of Peucedanum praeruptorum. Spot the test solutions and reference solutions onto the same thin-layer chromatography plate and develop them with a mixture of petroleum ether and ethyl acetate in a volume ratio of (2-4):1 as the developing solvent for a second inspection.

[0014] In some embodiments, the preparation of the Bupleurum chinense test solution includes the following steps:

[0015] The pediatric safety preparation, water, and water-saturated n-butanol were mixed and extracted to obtain an organic phase solution. The organic phase was washed with an alkaline solution, and the organic phase solution was concentrated and diluted to prepare the Bupleurum chinense test solution.

[0016] The preparation of the Bupleurum reference solution includes the following steps:

[0017] Take saikosaponin a reference standard and saikosaponin d reference standard, and dissolve them in the first organic solvent to obtain saikosaponin a reference standard solution and saikosaponin d reference standard solution, respectively.

[0018] In some embodiments, the extraction method is to mix the pediatric safety preparation, water, and water-saturated n-butanol for extraction, and the number of extractions and the number of washings with the alkaline solution are each 1-5 times.

[0019] In some embodiments, the first organic solvent is one or more of methanol, ethanol, and ethyl acetate.

[0020] In some embodiments, the preparation of the Peucedanum praeruptorum test solution includes the following steps:

[0021] The pediatric safety preparation is mixed with a second organic solvent for extraction to obtain an extract, which is then concentrated, diluted, and used to prepare the Peucedanum praeruptorum test solution.

[0022] The preparation of the Peucedanum praeruptorum reference solution includes the following steps:

[0023] Dissolve the white peucedanin reference standard in a third organic solvent to obtain the white peucedanin reference standard solution.

[0024] In some embodiments, the pediatric safety preparation is extracted by mixing with a second organic solvent using ultrasonic extraction for 10-40 minutes.

[0025] In some embodiments, the second organic solvent is one or more of ethyl acetate, chloroform, diethyl ether, and dichloromethane; and the third organic solvent is one or more of methanol, ethanol, and ethyl acetate.

[0026] In some embodiments, the temperature of the color development treatment is 100°C-120°C.

[0027] In some embodiments, the first light source for inspection is a fluorescent lamp or natural light, and the second light source for inspection is a 365nm ultraviolet lamp.

[0028] In some embodiments, the first organic solvent is one or more of methanol, ethanol, or ethyl acetate; and the second organic solvent is one or more of ethyl acetate, chloroform, diethyl ether, or dichloromethane.

[0029] In some embodiments, the dosage form of the pediatric safety preparation is selected from granules, powders, tablets, capsules, or pills.

[0030] The present invention has at least the following beneficial effects:

[0031] Bupleurum and Peucedanum are the main components of pediatric safety preparations and are closely related to their indications and functions. The thin-layer chromatography method for detecting Bupleurum and Peucedanum in pediatric safety preparations provided by this invention can rapidly detect these components, producing clear thin-layer chromatographic spots without interference from negative controls. This improves the accuracy and comprehensiveness of quality control for the pediatric safety preparations and facilitates the establishment and overall evaluation of their quality standards. Furthermore, this method has the advantages of simple operation and wide applicability. Attached Figure Description

[0032] Figure 1 is a thin-layer chromatogram of Bupleurum chinense, where 1 is a mixed reference standard of saikosaponins a and d, 2 is a negative control solution of Bupleurum chinense, 3 is pediatric safety preparation D00001M, 4 is pediatric safety preparation D00002M, and 5 is pediatric safety preparation D00003M.

[0033] Figure 2 is a thin-layer chromatogram of Peucedanum praeruptorum, where 1 is the reference standard of Peucedanum praeruptorum A, 2 is the negative control solution of Peucedanum praeruptorum, 3 is the pediatric safety preparation D00001M, 4 is the pediatric safety preparation D00002M, and 5 is the pediatric safety preparation D00003M.

[0034] Figure 3 shows the thin-layer chromatograms of Bupleurum chinense after different extraction times with water-saturated n-butanol and washing times with ammonia solution. In the figure, 1 is the mixed reference standard of saikosaponins a and d, 2 is the reference medicinal material of Bupleurum chinense, 3 is the test solution 1, 4 is the test solution 2, and 5 is the test solution 3.

[0035] Figure 4 shows the thin-layer chromatograms of Bupleurum chinense washed with different amounts of ammonia solution, where 1 is the mixed reference standard of saikosaponins a and d, 2 is test solution 1, 3 is test solution 2, and 4 is test solution 3.

[0036] Figure 5 shows the thin-layer chromatograms of Bupleurum chinense with different developing solvent ratios, where A is 8:2:1, B is 8:3:1, C is 8:4:1, D is 7:2:1, 1 is a mixed reference standard of saikosaponins a and d, 2 is pediatric safety preparation D00001M, 3 is pediatric safety preparation D00002M, and 4 is pediatric safety preparation D00003M.

[0037] Figure 6 shows the thin-layer chromatograms of Bupleurum chinense under different colorimetric reagents, where A is a 2% p-dimethylaminobenzaldehyde solution in 10% sulfuric acid ethanol, B is a 2% p-dimethylaminobenzaldehyde solution in 40% sulfuric acid, 1 is a mixed reference standard of saikosaponins a and d, 2 is a pediatric safety preparation D00001M, 3 is a pediatric safety preparation D00002M, and 4 is a pediatric safety preparation D00003M.

[0038] Figure 7 shows the thin-layer chromatogram of Bupleurum obtained from the Bupleurum specificity experiment. Among them, 1 is the mixed reference standard of Bupleurum saponins a and d, 2 is the Bupleurum negative control solution, 3 is the pediatric safety preparation D00001M, 4 is the pediatric safety preparation D00002M, and 5 is the pediatric safety preparation D00003M.

[0039] Figure 8 shows the thin-layer chromatograms of Bupleurum chinense on different thin-layer pre-prepared plates, where A is the German Merck plate, B is the Silver Dragon plate, 1 is the mixed reference standard of saikosaponins a and d, 2 is the negative control solution of Bupleurum chinense, 3 is the pediatric safety preparation D00001M, 4 is the pediatric safety preparation D00002M, and 5 is the pediatric safety preparation D00003M.

[0040] Figure 9 shows the thin-layer chromatograms of Bupleurum chinense at different relative humidity levels, where A represents 40% relative humidity, B represents 70% relative humidity, 1 is a mixed reference standard of saikosaponins a and d, 2 is pediatric safety preparation D00001M, 3 is pediatric safety preparation D00002M, and 4 is pediatric safety preparation D00003M.

[0041] Figure 10 shows the thin-layer chromatograms of Bupleurum chinense at different temperatures, where A is 4℃, B is 32℃, 1 is a mixed reference standard of saikosaponins a and d, 2 is pediatric safety preparation D00001M, 3 is pediatric safety preparation D00002M, and 4 is pediatric safety preparation D00003M.

[0042] Figure 11 shows the thin-layer chromatograms of Peucedanum praeruptorum extracted by different methods, where 1 is Peucedanum praeruptorum A, 2 is test solution 1, 3 is test solution 2, 4 is test solution 3, and 5 is test solution 4.

[0043] Figure 12 shows the thin-layer chromatograms of Peucedanum praeruptorum extracted with different solvents, where 1 is Peucedanum praeruptorum A, 2 is test solution 1, 3 is test solution 2, 4 is test solution 3, and 5 is test solution 4.

[0044] Figure 13 shows the thin-layer chromatograms of Peucedanum praeruptorum obtained by different pretreatment methods, where 1 is Peucedanum praeruptorum A, 2 is the unground sample, 3 is the sample after grinding and 10 μL is taken for detection, and 4 is the sample after grinding and 15 μL is taken for detection.

[0045] Figure 14 is a thin-layer chromatogram of Peucedanum praeruptorum obtained from the Peucedanum praeruptorum specificity experiment. 1 is Peucedanum praeruptorum A, 2 is Peucedanum praeruptorum negative control, 3 is pediatric safety preparation D00001M, 4 is pediatric safety preparation D00002M, and 5 is pediatric safety preparation D00003M.

[0046] Figure 15 shows the thin-layer chromatograms of Peucedanum praeruptorum on different silica gel G thin-layer plates, where A is the German Merck plate, B is the Yantai plate, 1 is Peucedanum praeruptorum A, 2 is Peucedanum praeruptorum negative control, 3 is pediatric safety preparation D00001M, 4 is pediatric safety preparation D00002M, and 5 is pediatric safety preparation D00003M.

[0047] Figure 16 shows the thin-layer chromatograms of Peucedanum praeruptorum at different relative humidity levels. A is 40% relative humidity, B is 76% relative humidity, 1 is Peucedanum praeruptorum A, 2 is Peucedanum praeruptorum negative control solution, 3 is pediatric safety preparation D00001M, 4 is pediatric safety preparation D00002M, and 5 is pediatric safety preparation D00003M.

[0048] Figure 17 shows the thin-layer chromatograms of Peucedanum praeruptorum at different temperatures. A is 4℃, B is 32℃, 1 is Peucedanum praeruptorum A, 2 is Peucedanum praeruptorum negative control, 3 is pediatric safety preparation D00001M, 4 is pediatric safety preparation D00002M, and 5 is pediatric safety preparation D00003M. Detailed Implementation

[0049] The present invention will be further described in detail below with reference to specific embodiments. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of the present invention.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0051] In this invention, the terms "preferredly," "more preferably," "better," and "even better" refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of the invention. That is, in this invention, "preferredly," "more preferably," "better," and "even better" are merely descriptions of more effective implementations or examples, but do not constitute a limitation on the scope of protection of the invention.

[0052] In this invention, terms such as "further," "even more," and "particularly" are used for descriptive purposes and to indicate differences in content, but should not be construed as limiting the scope of protection of this invention.

[0053] In this invention, "at least one" means one or more, such as one, two, or more. "Multiple" or "several" means at least two, such as two, three, etc., and "multi-layered" means at least two layers, such as two layers, three layers, etc., unless otherwise explicitly specified. In the description of this invention, "several" means at least one, such as one, two, etc., unless otherwise explicitly specified.

[0054] When a numerical range is disclosed in this invention, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Further, when the range refers to an integer, it includes every integer between the minimum and maximum values ​​of the range. Moreover, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0055] Unless otherwise specified, all steps of this invention may be performed sequentially or randomly. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the method may also include step (c), indicating that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0056] Unless otherwise stated, a singular term may include a plural term and should not be understood as having a quantity of one.

[0057] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined with "first" or "second" may explicitly or implicitly include at least one of that feature.

[0058] In this invention, the terms "comprising," "including," "containing," "having," or other variations are intended to cover non-closed inclusion, and no distinction is made between these terms. The term "comprising" means that other steps and components may be added without affecting the final result. The compositions and methods / processes of this invention comprise, consist of, and substantially consist of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, steps, or limitations described herein.

[0059] In this invention, the terms "efficacy", "performance", "effect" and "potential" are not distinguished.

[0060] The weights of the relevant components mentioned in the embodiments of this invention can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this invention is within the scope disclosed in the embodiments of this invention. Specifically, the weights mentioned in the embodiments of this invention can be well-known units of mass in the chemical industry, such as μg, mg, g, and kg.

[0061] The pediatric health supplement is made from a variety of ingredients including processed Pinellia ternata, processed Gastrodia elata, soaked Atractylodes lancea, Pogostemon cablin, Notopterygium incisum, stir-fried Hordeum vulgare malt, Areca catechu peel, Aucklandia lappa, dried bitter almond, Platycodon grandiflorus, Uncaria rhynchophylla, pearl, cinnamon, Saposhnikovia divaricata, Mentha haplocalyx, Poria cocos, Magnolia officinalis (stir-fried with ginger juice), processed Bombyx mori, Coptis chinensis, Citrus reticulata peel, Massa fermentata, Asarum heterotropoides, Cinnamomum cassia, Peucedanum praeruptorum, Bupleurum chinense, amber, borneol, and Glycyrrhiza uralensis. It consists of a total of 28 medicinal materials. Among them, Bupleurum chinense and Peucedanum praeruptorum are the main ingredients in the pediatric health supplement and are closely related to the functions and indications of this preparation.

[0062] Thin-layer chromatography (TLC) involves coating a suitable stationary phase onto a glass plate, plastic, or aluminum substrate to form a uniform thin layer. After spotting and development, the ratio shift (Rf) of the chromatogram is compared with the ratio shift (Rf) of a suitable reference substance obtained using the same method. This method is used for drug identification, impurity detection, or content determination. As a crucial experimental technique for the rapid separation and qualitative analysis of small amounts of substances, TLC features short development time, strong separation capability, and high sensitivity, making it a vital analytical tool for the quality control of traditional Chinese medicine.

[0063] However, currently only thin-layer chromatography is available for identifying Magnolia officinalis in pediatric safety preparations, making it difficult to comprehensively and accurately evaluate the quality of these preparations.

[0064] Accordingly, in order to improve the accuracy and comprehensiveness of quality control of pediatric safety preparations, this invention provides a thin-layer chromatography method for detecting Bupleurum chinense and Peucedanum praeruptorum in pediatric safety preparations.

[0065] The technical solution is as follows:

[0066] The thin-layer chromatography detection method for Bupleurum chinense includes the following steps:

[0067] Prepare Bupleurum test solution and Bupleurum reference solution separately. Spot the test solution and reference solution on the same thin-layer chromatography plate. Develop the plate with a mixture of ethyl acetate, ethanol and water in a volume ratio of (7-9):(1-5):1 as the developing solvent. After color development, perform the first inspection.

[0068] The colorimetric reagent is a mixture of p-dimethylaminobenzaldehyde and sulfuric acid ethanol solution, or a mixture of p-dimethylaminobenzaldehyde and sulfuric acid aqueous solution.

[0069] The mass-to-volume ratio of p-dimethylaminobenzaldehyde to the sulfuric acid ethanol solution is (1-5) g: 100 mL, and the volume percentage of sulfuric acid in the sulfuric acid ethanol solution is 9%-11%.

[0070] The mass-to-volume ratio of p-dimethylaminobenzaldehyde to the sulfuric acid aqueous solution is (1-5) g: 100 mL, and the volume percentage of sulfuric acid in the sulfuric acid aqueous solution is 30%-50%.

[0071] The thin-layer chromatography detection method for the aforementioned Peucedanum praeruptorum includes the following steps:

[0072] Prepare separate test solutions and reference solutions of Peucedanum praeruptorum. Spot the test solutions and reference solutions onto the same thin-layer chromatography plate and develop them with a mixture of petroleum ether and ethyl acetate in a volume ratio of (2-4):1 as the developing solvent for a second inspection.

[0073] Understandably, the volume ratio of the mixture of ethyl acetate, ethanol and water may be, but is not limited to, 8:2:1, 8:3:1, 8:4:1, or 7:2:1; preferably, the volume ratio of the mixture of ethyl acetate, ethanol and water is 7:2:1.

[0074] Understandably, the mass-to-volume ratio of p-dimethylaminobenzaldehyde to the sulfuric acid ethanol solution includes, but is not limited to, 1g:100mL, 2g:100mL, 3g:100mL, 4g:100mL, and 5g:100mL, and the volume percentage of sulfuric acid in the sulfuric acid ethanol solution includes, but is not limited to, 9%, 10%, and 11%; preferably, the mass-to-volume ratio of p-dimethylaminobenzaldehyde to the sulfuric acid ethanol solution is 2g:100mL, and the volume percentage of sulfuric acid in the sulfuric acid ethanol solution is 10%.

[0075] Understandably, the mass-to-volume ratio of p-dimethylaminobenzaldehyde to the sulfuric acid aqueous solution includes, but is not limited to, 1g:100mL, 2g:100mL, 3g:100mL, 4g:100mL, and 5g:100mL, and the volume percentage of sulfuric acid in the sulfuric acid aqueous solution includes, but is not limited to, 30%, 40%, and 50%; preferably, the mass-to-volume ratio of p-dimethylaminobenzaldehyde to the sulfuric acid aqueous solution is 2g:100mL, and the volume percentage of sulfuric acid in the sulfuric acid aqueous solution is 40%.

[0076] Understandably, the volume ratio of the mixture of petroleum ether and ethyl acetate includes, but is not limited to, 2:1, 3:1, and 4:1; preferably, the volume ratio of the mixture of petroleum ether and ethyl acetate is 3:1.

[0077] Understandably, the petroleum ether can be a petroleum ether with a boiling point of 30°C to 60°C, or a petroleum ether with a boiling point of 60°C to 90°C; preferably, the petroleum ether is a petroleum ether with a boiling point of 30°C to 60°C.

[0078] Understandably, the color development step of the color developer includes: adding the color developer to a thin-layer plate and heating it at a certain temperature until the spots are clear.

[0079] In some embodiments, the temperature of the color development treatment is 100°C to 120°C.

[0080] Understandably, the temperature of the color development treatment includes, but is not limited to, 100℃, 102℃, 104℃, 105℃, 108℃, 110℃, 115℃, and 120℃; preferably, the temperature of the color development treatment is 105℃.

[0081] In some embodiments, the thin-layer chromatography plate is a silica gel G thin-layer plate.

[0082] Understandably, the silicone G thin film includes, but is not limited to, the silicone G film from Merck GmbH, Germany, the silicone G film from Yantai Huayang New Material Technology Co., Ltd. (Yinlong), and the silicone G film from Yantai Chemical Industry Research Institute.

[0083] Understandably, in the thin-layer chromatography detection method for Bupleurum chinense, after developing with a developing solvent, the thin-layer chromatography plate is dried, and then a colorimetric reagent is added for color development before the first inspection.

[0084] Understandably, in the thin-layer chromatography detection method for Angelica dahurica, after development with the developing solvent, the thin-layer chromatography plate is dried and directly subjected to a second inspection without the need for additional colorimetric reagent.

[0085] In some embodiments, the first light source for inspection is a fluorescent lamp or natural light, and the second light source for inspection is a 365nm ultraviolet lamp.

[0086] Understandably, the fluorescent lamps include, but are not limited to, conventional incandescent lamps.

[0087] In some embodiments, the preparation of the Bupleurum chinense test solution includes the following steps:

[0088] The pediatric safety preparation, water, and water-saturated n-butanol were mixed and extracted to obtain an organic phase solution. The organic phase was washed with an alkaline solution, and the organic phase solution was concentrated and diluted to prepare the Bupleurum chinense test solution.

[0089] The preparation of the Bupleurum reference solution includes the following steps:

[0090] Take saikosaponin a reference standard and saikosaponin d reference standard, and dissolve them in the first organic solvent to obtain saikosaponin a reference standard solution and saikosaponin d reference standard solution, respectively.

[0091] In some embodiments, the extraction method is to mix the pediatric safety preparation, water, and water-saturated n-butanol for extraction, and the number of extractions and the number of washings with the alkaline solution are each 1-5 times.

[0092] Understandably, the number of extractions can be 1, 2, 3, 4, or 5 times; preferably, the number of extractions is 1.

[0093] Understandably, the number of times the washing is performed can be 1, 2, 3, 4, or 5 times; preferably, the number of times the washing is performed is 1.

[0094] Understandably, the pediatric safety preparation, water, and water-saturated n-butanol are mixed for extraction. Specifically, water is added to the pediatric safety preparation to dissolve it, and then water-saturated n-butanol is added for extraction.

[0095] Preferably, the ratio of the amount of water-saturated n-butanol added to the pediatric safety preparation is 1g:(15-25)mL; more preferably, the ratio of the amount of water-saturated n-butanol added to the pediatric safety preparation is 1g:20mL.

[0096] Understandably, the alkaline solution includes, but is not limited to, sodium hydroxide solution, potassium hydroxide solution, and ammonia solution; preferably, the alkaline solution is an ammonia solution.

[0097] Understandably, the concentration specifically involves evaporating the organic phase layer to dryness, yielding a residue.

[0098] Understandably, the dilution specifically involves dissolving the concentrated residue in a first organic solvent.

[0099] In some embodiments, the first organic solvent is one or more of methanol, ethanol, and ethyl acetate.

[0100] In some embodiments, the preparation of the Peucedanum praeruptorum test solution includes the following steps:

[0101] The pediatric safety preparation is mixed with a second organic solvent for extraction to obtain an extract, which is then concentrated, diluted, and used to prepare the Peucedanum praeruptorum test solution.

[0102] The preparation of the Peucedanum praeruptorum reference solution includes the following steps:

[0103] Dissolve the white peucedanin reference standard in a third organic solvent to obtain the white peucedanin reference standard solution.

[0104] Preferably, in the extraction step of mixing the pediatric safety preparation with the second organic solvent, the ratio of the amount of the second organic solvent added to the pediatric safety preparation is 1g:(3-6)mL.

[0105] In some embodiments, the pediatric safety preparation is extracted by mixing it with a second organic solvent using ultrasonic extraction, and the ultrasonic extraction time is 10-40 minutes.

[0106] Understandably, the ultrasound time includes, but is not limited to, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, and 40 minutes; preferably, the ultrasound time is 20 minutes.

[0107] Understandably, the concentration step involves filtering the extract, evaporating the filtrate to dryness, and obtaining residue.

[0108] Understandably, the filtration includes, but is not limited to, conventional filtration methods, such as filter paper filtration.

[0109] Understandably, the dilution step involves dissolving the concentrated residue in a first organic solvent.

[0110] Understandably, before the pediatric safety preparation is mixed with the second organic solvent for extraction, the process may further include a step of mixing and grinding the pediatric safety preparation with diatomaceous earth.

[0111] In some embodiments, the second organic solvent is one or more of ethyl acetate, chloroform, diethyl ether, and dichloromethane; and the third organic solvent is one or more of methanol, ethanol, and ethyl acetate.

[0112] Understandably, the pediatric safety preparation of the present invention comprises the following raw materials in parts by weight: 1-3 parts of processed Pinellia ternata, 1-3 parts of processed Gastrodia elata, 1-3 parts of soaked Atractylodes lancea, 1-3 parts of Pogostemon cablin, 1-3 parts of Notopterygium incisum, 1-3 parts of roasted malt, 1-3 parts of Areca catechu peel, 1-2 parts of Aucklandia lappa, 1-3 parts of de-oiled bitter almond, 1-3 parts of Platycodon grandiflorus, 1-3 parts of Uncaria rhynchophylla, and 1-2 parts of pearl powder. 1 part, cinnabar 3-5 parts by weight, saposhnikovia root 1-3 parts by weight, peppermint 1-3 parts by weight, poria cocos 1-3 parts by weight, magnolia bark (stir-fried with ginger juice) 1-3 parts by weight, silkworm (processed) 1-3 parts by weight, coptis chinensis 1-3 parts by weight, tangerine peel 1-3 parts by weight, medicated leaven 1-3 parts by weight, asarum 1-2 parts by weight, cinnamon twig 1-2 parts by weight, angelica dahurica 2-4 parts by weight, bupleurum chinense 3-5 parts by weight, amber 1-3 parts by weight, borneol 2-4 parts by weight, licorice root 1-2 parts by weight.

[0113] The raw materials of the pediatric safety preparation also include pharmaceutically acceptable excipients.

[0114] The preparation method of the pediatric safety preparation is as follows: 2 parts by weight of processed Pinellia ternata, 2 parts by weight of processed Gastrodia elata, 2 parts by weight of soaked Atractylodes lancea, 2 parts by weight of Pogostemon cablin, 2 parts by weight of Notopterygium incisum, 2 parts by weight of stir-fried malt, 2 parts by weight of Areca catechu peel, 1 part by weight of Aucklandia lappa, 2 parts by weight of de-oiled bitter almond, 2 parts by weight of Platycodon grandiflorus, 2 parts by weight of Uncaria rhynchophylla, 1 part by weight of pearl, 4 parts by weight of cinnabar, 2 parts by weight of Saposhnikovia divaricata, 2 parts by weight of Mentha haplocalyx, 2 parts by weight of Poria cocos, 2 parts by weight of stir-fried Magnolia officinalis (with ginger juice), 2 parts by weight of processed Bombyx mori, 2 parts by weight of Coptis chinensis, 2 parts by weight of Citrus reticulata peel, 2 parts by weight of Shenqu (medicated leaven), 1 part by weight of Asarum heterotropoides, 1 part by weight of Cinnamomum cassia, 3 parts by weight of Peucedanum praeruptorum, 4 parts by weight of Bupleurum chinense, 2 parts by weight of amber, 3 parts by weight of borneol, and 1 part by weight of licorice. Of the 28 medicinal materials mentioned above, pearl, amber, and cinnabar were water-milled into extremely fine powder, and borneol was ground into fine powder. The remaining 24 materials were pulverized into fine powder, sieved, mixed, and mixed with conventional excipients. Following conventional processes, they were made into a clinically acceptable pharmaceutical preparation.

[0115] The dosage form of the pediatric safety preparation can be selected from one of the following: granules, powders, tablets, capsules, or pills.

[0116] The above-mentioned thin-layer chromatography method can identify the two medicinal materials, Bupleurum chinense and Peucedanum praeruptorum, in the pediatric safety preparation. The corresponding thin-layer chromatographic spots are clear and there is no interference from the negative control, which improves the accuracy and comprehensiveness of the quality control of the pediatric safety preparation and is more conducive to the establishment and overall evaluation of the quality standards of the pediatric safety preparation.

[0117] The following description, in conjunction with specific embodiments, further illustrates the point:

[0118] 1. Instruments and reagents

[0119] Pediatric safety preparations D00001M, D00002M, D00003M (self-made);

[0120] Saikosaponin a (batch number: 110777-200406, National Institutes for Food and Drug Control, China);

[0121] Saikosaponin D (batch number: 110778-200505, National Institutes for Food and Drug Control, China);

[0122] White Peucedanum praeruptorum glycoside reference standard (batch number: 111711-200501, National Institutes for Food and Drug Control, China);

[0123] Bupleurum reference material (batch number 120992-201509, China National Institutes for Food and Drug Control);

[0124] Bupleurum negative sample (self-made);

[0125] Negative sample of Peucedanum praeruptorum (self-made);

[0126] Silicone G thin film: Merck AG silicone G film, Yantai Huayang New Material Technology Co., Ltd. (Yinlong) silicone G film, Yantai Chemical Industry Research Institute silicone G film;

[0127] All other reagents used were of analytical grade.

[0128] 2. Drug testing

[0129] 2.1 Preparation of Pediatric Safety Preparations

[0130] Weigh out 300g of processed Pinellia ternata, 300g of processed Gastrodia elata, 300g of soaked Atractylodes lancea, 300g of Pogostemon cablin, 300g of Notopterygium incisum, 300g of stir-fried malt, 300g of Areca catechu peel, 150g of Aucklandia lappa, 300g of dried bitter almond, 300g of Platycodon grandiflorus, 300g of Uncaria rhynchophylla, 150g of pearl, 600g of cinnabar, 300g of Saposhnikovia divaricata, 300g of Mentha haplocalyx, 300g of Poria cocos, 300g of stir-fried Magnolia officinalis (with ginger juice), 300g of processed Bombyx mori, 300g of Coptis chinensis, 300g of Citrus reticulata peel, 300g of Shenqu (medicated leaven), 150g of Asarum heterotropoides, 150g of Cinnamomum cassia, 450g of Peucedanum praeruptorum, 600g of Bupleurum chinense, 300g of amber, 450g of borneol, and 150g of Glycyrrhiza uralensis.

[0131] Of the 28 medicinal materials listed above, pearl, amber, and cinnabar were water-milled into extremely fine powder, and borneol was ground into fine powder. The remaining 24 materials were pulverized into fine powder, sieved, mixed, and then mixed with refined honey to make large honey pills.

[0132] 2.2 Preparation of Pediatric Safety Powder

[0133] Weigh out 300g of processed Pinellia ternata, 300g of processed Gastrodia elata, 300g of soaked Atractylodes lancea, 300g of Pogostemon cablin, 300g of Notopterygium incisum, 300g of stir-fried malt, 300g of Areca catechu peel, 150g of Aucklandia lappa, 300g of dried bitter almond, 300g of Platycodon grandiflorus, 300g of Uncaria rhynchophylla, 150g of pearl, 600g of cinnabar, 300g of Saposhnikovia divaricata, 300g of Mentha haplocalyx, 300g of Poria cocos, 300g of stir-fried Magnolia officinalis (with ginger juice), 300g of processed Bombyx mori, 300g of Coptis chinensis, 300g of Citrus reticulata peel, 300g of Shenqu (medicated leaven), 150g of Asarum heterotropoides, 150g of Cinnamomum cassia, 450g of Peucedanum praeruptorum, 600g of Bupleurum chinense, 300g of amber, 450g of borneol, and 150g of Glycyrrhiza uralensis.

[0134] Of the 28 medicinal materials listed above, pearl, amber, and cinnabar were water-milled into extremely fine powder, and borneol was ground into the finest powder. The remaining 24 materials were pulverized into the finest powder, sieved, mixed, and made into a powder.

[0135] 3. Testing

[0136] 3.1 Thin-layer chromatographic identification of Bupleurum chinense

[0137] 3.1.1 Preparation of Bupleurum chinense test solution

[0138] Take 1.5g of the pediatric safety preparation, add 10ml of water to dissolve it, extract it once with 30ml of water-saturated n-butanol, wash the n-butanol layer once with 20ml of ammonia solution, discard the aqueous liquid, evaporate the n-butanol liquid to dryness, dissolve the residue in 1ml of methanol, and use it as the test solution.

[0139] 3.1.2 Preparation of Bupleurum reference solution

[0140] Take saikosaponin a reference standard and saikosaponin d reference standard, add methanol to prepare a mixed solution containing 0.5 mg of each per 1 ml, and use it as the reference solution.

[0141] 3.1.3 Preparation of Bupleurum negative control solution

[0142] Take 0.8g of the pediatric safety preparation Bupleurum negative control powder, add 0.7g of refined honey, add 10ml of water to dissolve, then add 30ml of water and saturated n-butanol to extract once, wash the n-butanol layer once with 20ml of ammonia solution, evaporate the n-butanol layer to dryness, add 1ml of methanol to dissolve the residue, and use it as a negative control solution.

[0143] 3.1.4 Perform the test according to the thin-layer chromatography method (General Rule 0502).

[0144] Take 10 μL each of the above-mentioned Bupleurum chinense test solution, Bupleurum chinense reference solution, and Bupleurum chinense negative control solution, and spot them separately on the same silica gel G thin-layer plate. Develop the plate using ethyl acetate-ethanol-water (8:2:1) as the developing solvent. Remove the plate, air dry, spray with a 2% p-dimethylaminobenzaldehyde solution in 10% sulfuric acid ethanol, and heat at 105℃ until the spots are clearly visible. Examine under fluorescent light. In the chromatogram of the Bupleurum chinense test solution, spots of the same color appear at the corresponding positions as in the chromatogram of the reference solution.

[0145] Figure 1 shows the thin-layer chromatogram of Bupleurum in the pediatric safety preparation, where 1, 2, 3, 4, and 5 are respectively the mixed reference standard of saikosaponin a and d, the negative control solution of Bupleurum in the pediatric safety preparation, pediatric safety preparation D00001M, pediatric safety preparation D00002M, and pediatric safety preparation D00003M.

[0146] 3.2 Identification of Peucedanum praeruptorum by thin-layer chromatography

[0147] 3.2.1 Preparation of Peucedanum praeruptorum test solution

[0148] Take 6g of the pediatric safety preparation, add 20ml of ethyl acetate, sonicate for 20 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of methanol, and use it as the test solution.

[0149] 3.2.2 Preparation of Peucedanum praeruptorum reference solution

[0150] Take the white peucedanum praeruptorum reference standard and add methanol to prepare a solution containing 0.5 mg per 1 ml, which is used as the reference solution.

[0151] 3.2.3 Preparation of the negative control solution of the pediatric safety preparation, Peucedanum praeruptorum.

[0152] Take 2.5g of the pediatric safety preparation, Peucedanum praeruptorum negative control powder, add 3.5g of refined honey and mix well, add 20ml of ethyl acetate, sonicate for 20 minutes, filter, evaporate the filtrate to dryness, add 1ml of methanol to dissolve the residue, and use it as a negative control solution.

[0153] 3.2.4 Perform the test according to the thin-layer chromatography method (General Rule 0502).

[0154] Take 10 μL of the above-mentioned Peucedanum praeruptorum test solution, 2 μL each of the Peucedanum praeruptorum reference solution and the Peucedanum praeruptorum negative control solution, and spot them separately on the same silica gel G thin-layer plate. Develop the plate using petroleum ether (30–60℃)-ethyl acetate (3:1) as the developing solvent. Remove the plate, air dry it, and examine it under ultraviolet light (365 nm). In the chromatogram of the Peucedanum praeruptorum test solution, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference solution.

[0155] Figure 2 shows the thin-layer chromatogram of Peucedanum praeruptorum in the pediatric safety preparation. Among them, 1, 2, 3, 4 and 5 are respectively the reference standard of Peucedanum praeruptorum A, the negative control solution of Peucedanum praeruptorum in the pediatric safety preparation, the pediatric safety preparation D00001M, the pediatric safety preparation D00002M and the pediatric safety preparation D00003M.

[0156] 4. Thin-layer chromatographic identification study of Bupleurum chinense

[0157] 4.1 Method Optimization

[0158] 4.1.1 Investigation into the preparation method of Bupleurum chinense test solution

[0159] (1) Investigation of the number of n-butanol extractions and ammonia washings

[0160] Preparation of Bupleurum chinense test solution

[0161] Take 1.5g of the pediatric safety preparation, add 10ml of water to dissolve it, and prepare test solution 1, test solution 2 and test solution 3 according to the preparation method of Bupleurum test solution under section 3.1.1.

[0162] Specifically, test solution 1 is extracted three times with water-saturated n-butanol, with 30 mL of water-saturated n-butanol added each time; then the n-butanol layer is washed three times with ammonia solution, with 20 mL of ammonia solution added each time; test solution 2 is extracted three times with water-saturated n-butanol, with 30 mL of water-saturated n-butanol added each time; the n-butanol layer is washed once with 20 mL of ammonia solution; test solution 3 is extracted once with 30 mL of water-saturated n-butanol, and the n-butanol layer is washed once with 20 mL of ammonia solution.

[0163] Preparation of Bupleurum reference solution

[0164] Take saikosaponin a reference standard and saikosaponin d reference standard, add methanol to prepare a mixed solution containing 0.5 mg of each per 1 ml, and use it as the reference solution.

[0165] Preparation of Bupleurum reference herb solution

[0166] Take 0.5g of Bupleurum reference material, add 20ml of methanol, sonicate for 30 minutes, filter, and concentrate the filtrate to 2ml as the reference material solution.

[0167] Test according to thin-layer chromatography (General Rule 0502)

[0168] Take 10 μL of the Bupleurum chinense test sample, Bupleurum chinense reference standard, and Bupleurum chinense reference herb solution, and spot them separately on the same silica gel G thin-layer plate. Develop the plate using ethyl acetate-ethanol-water (8:2:1) as the developing solvent. Remove the plate, air dry, and spray with a 2% p-dimethylaminobenzaldehyde solution in 10% sulfuric acid ethanol. Heat at 105℃ until the spots are clearly visible and examine under fluorescent light. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference standard.

[0169] Figure 3 shows the thin-layer chromatograms of Bupleurum chinense after different extraction times with water-saturated n-butanol and washing times with ammonia solution. In the figure, 1, 2, 3, 4 and 5 are respectively the mixed reference standard of saikosaponins a and d, Bupleurum chinense reference medicinal material, test solution 1, test solution 2 and test solution 3.

[0170] The results showed that all three combinations of n-butanol extraction and ammonia solution washing could produce spots of the same color. To reduce sample preparation time and save reagents, one n-butanol extraction and one ammonia solution washing were selected.

[0171] (2) Investigation on the amount of ammonia solution used

[0172] Preparation of Bupleurum chinense test solution

[0173] Take 1.5g of the pediatric safety preparation, add 10ml of water to dissolve it, and prepare test solution 1, test solution 2 and test solution 3 according to the test solution preparation method under section 3.1.1.

[0174] Specifically, test solution 1 was extracted once with 30 mL of water-saturated n-butanol, and the n-butanol layer was washed once with 20 mL of ammonia solution; test solution 2 was extracted once with 30 mL of water-saturated n-butanol, and the n-butanol layer was washed once with 40 mL of ammonia solution; test solution 3 was extracted three times with 30 mL of water-saturated n-butanol, and the n-butanol layer was washed three times with 40 mL of ammonia solution each time.

[0175] Preparation of reference solution

[0176] Take saikosaponin a reference standard and saikosaponin d reference standard, add methanol to prepare a mixed solution containing 0.5 mg of each per 1 ml, and use it as the reference solution.

[0177] Test according to thin-layer chromatography (General Rule 0502)

[0178] Take 10 μL each of the Bupleurum chinense test solution, Bupleurum chinense reference solution, and Bupleurum chinense reference herb solution, and spot them separately on the same silica gel G thin-layer plate. Develop the plate using ethyl acetate-ethanol-water (8:2:1) as the developing solvent. Remove the plate, air dry, and spray with a 2% p-dimethylaminobenzaldehyde solution in 10% sulfuric acid ethanol. Heat the plate at 105℃ until the spots are clearly visible and examine them under fluorescent light. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0179] Figure 4 shows the thin-layer chromatograms of Bupleurum chinense with different amounts of ammonia solution. Among them, 1, 2, 3, and 4 are respectively the mixed reference standard of Bupleurum saponins a and d, extracted once with water-saturated n-butanol, washed once with 20 ml of ammonia solution, extracted once with water-saturated n-butanol, washed once with 40 ml of ammonia solution, extracted three times with water-saturated n-butanol, and washed three times with ammonia solution, 40 ml each time.

[0180] The results showed that there was no significant difference in washing effect between 20 mL ammonia solution and 40 mL ammonia solution. To save reagents, 20 mL ammonia solution was chosen for washing.

[0181] (3) Validation of the test solution preparation method

[0182] In summary, the preparation method of Bupleurum chinense test solution is as follows: Take 1.5g of the pediatric safety preparation, add 10ml of water to dissolve it, extract it once with 30ml of water-saturated n-butanol, wash the n-butanol layer once with 20ml of ammonia solution, discard the aqueous liquid, evaporate the n-butanol liquid to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution.

[0183] 4.1.2 Thin-layer chromatographic detection conditions for Bupleurum chinense

[0184] (1) Investigation of different developing solvent ratios

[0185] Prepare the test solution and reference solution according to the methods described in sections 3.1.1-3.1.2, respectively, with the difference being that the developing solvent ratios in section 3.1.4 are 8:2:1, 8:3:1, 8:4:1, and 7:2:1, respectively, while keeping other conditions unchanged. In the chromatogram of the test solution, spots of the same color appear at the corresponding positions as in the chromatogram of the reference solution.

[0186] Figure 5 shows the thin-layer chromatograms of Bupleurum chinense with different developing solvent ratios, where A is 8:2:1, B is 8:3:1, C is 8:4:1, D is 7:2:1, and 1, 2, 3, and 4 are respectively the mixed reference standard of saikosaponins a and d, pediatric safety preparation D00001M, pediatric safety preparation D00002M, and pediatric safety preparation D00003M.

[0187] The results showed that all four ratios of developing solvent had good effects, and the ratio of 8:2:1 was selected for this experiment.

[0188] (2) Investigation of different colorimetric reagents

[0189] Prepare the test solution and reference solution according to the methods described in sections 3.1.1-3.1.2, respectively. The difference lies in the colorimetric reagents used in section 3.1.4: a 10% sulfuric acid ethanol solution of 2% p-dimethylaminobenzaldehyde and a 40% sulfuric acid solution of 2% p-dimethylaminobenzaldehyde, respectively, while keeping other conditions unchanged. In the chromatogram of the test solution, spots of the same color appear at the corresponding positions as in the chromatogram of the reference solution.

[0190] Figure 6 shows the thin-layer chromatograms of Bupleurum chinense under different colorimetric reagents. In the figure, A is a 2% p-dimethylaminobenzaldehyde solution in 10% sulfuric acid ethanol, B is a 2% p-dimethylaminobenzaldehyde solution in 40% sulfuric acid, and 1, 2, 3, and 4 are respectively the mixed reference standard of saikosaponin a and d, pediatric safety preparation D00001M, pediatric safety preparation D00002M, and pediatric safety preparation D00003M.

[0191] The results showed that the color development effects of the two colorimetric reagents were not significantly different. In this experiment, a 2% p-dimethylaminobenzaldehyde solution in 10% sulfuric acid ethanol was selected.

[0192] 4.2 Methodological Examination

[0193] 4.2.1 Specificity Experiment

[0194] Three test solutions of pediatric safety preparations D00001M, D00002M, and D00003M were prepared according to the method described in section 3.1.1.

[0195] Prepare the Bupleurum reference solution and Bupleurum negative control solution according to the methods in sections 3.1.2-3.1.3. Then, take 10 μL of each of the Bupleurum test solution, Bupleurum reference solution, and Bupleurum negative control solution and spot them on the same Merck silica gel G plate for development. Perform the thin-layer chromatography test (General Rule 0502) according to the method in section 3.1.4.

[0196] Figure 7 shows the thin-layer chromatogram of Bupleurum chinense obtained from the specificity test. In the chromatogram, 1 is the mixed reference standard of saikosaponins a and d, 2 is the negative control solution of Bupleurum chinense, 3 is the pediatric safety preparation D00001M, 4 is the pediatric safety preparation D00002M, and 5 is the pediatric safety preparation D00003M. The results show that the test sample chromatogram shows spots of the same color at the corresponding positions as the reference standard chromatogram, and there is no interference from the negative control.

[0197] 4.2.2 Thin-layer plate durability test

[0198] Three test solutions of pediatric safety preparations D00001M, D00002M, and D00003M were prepared according to the method described in section 3.1.1.

[0199] Prepare Bupleurum reference solution and Bupleurum negative control solution according to the methods described in sections 3.1.2-3.1.3. Then, perform thin-layer chromatography (TLC) tests according to the methods described in section 3.1.4 using Merck AG silica gel G thin-layer plates and Yantai Huayang New Material Technology Co., Ltd. (Yinlong) silica gel G thin-layer plates. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0200] Figure 8 shows the thin-layer chromatograms of Bupleurum chinense on different silica gel G plates, where A is the German Merck plate, B is the Silver Dragon plate, and 1, 2, 3, 4, and 5 are the mixed reference standard of saikosaponin a and d, the negative control solution of Bupleurum chinense, and the pediatric safety preparations D00001M, D00002M, and D00003M, respectively.

[0201] The results showed that both types of thin-layer chromatography silica gel plates could achieve good separation effects.

[0202] 4.2.3 Investigation at different humidity levels

[0203] Three test solutions of pediatric safety preparations D00001M, D00002M, and D00003M were prepared according to the method described in section 3.1.1.

[0204] Prepare the Bupleurum reference solution according to the method described in section 3.1.2. Take the spotted Merck silica gel G thin-layer plate according to the method described in section 3.1.4, and perform thin-layer chromatography (General Rule 0502) at 40% and 70% conditions. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0205] Figure 9 shows the thin-layer chromatograms of Bupleurum chinense under different relative humidity, where A represents 40% relative humidity, B represents 70% relative humidity, and 1, 2, 3, and 4 represent the mixed reference standard of saikosaponins a and d, pediatric safety preparation D00001M, pediatric safety preparation D00002M, and pediatric safety preparation D00003M, respectively.

[0206] The results showed that when the method was developed at relative humidity of 40% and 70%, the test sample chromatogram showed spots of the same color at the corresponding positions as the reference sample chromatogram.

[0207] 4.2.4 Investigation of different development temperatures

[0208] Three test solutions of pediatric safety preparations D00001M, D00002M, and D00003M were prepared according to the method described in section 3.1.1.

[0209] Prepare the Bupleurum reference solution according to the method described in section 3.1.2. Following the method described in section 3.1.4, take the spotted Merck silica gel G plate and develop it at 4℃ and 32℃ respectively, performing the thin-layer chromatography test (General Rule 0502). In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0210] Figure 10 shows the thin-layer chromatograms of Bupleurum at different temperatures, where A is 4℃, B is 32℃, and 1, 2, 3, and 4 are respectively the mixed reference standard of saikosaponin a and d, pediatric safety preparation D00001M, pediatric safety preparation D00002M, and pediatric safety preparation D00003M.

[0211] The results showed that when the method was developed at 4℃ and 32℃, the test sample chromatogram showed spots of the same color at the corresponding positions as the reference sample chromatogram.

[0212] 5. Thin-layer chromatographic identification study of Peucedanum praeruptorum

[0213] 5.1 Method Optimization

[0214] 5.1.1 Investigation of the preparation method of the test solution

[0215] (1) Examination of different extraction methods

[0216] Preparation of test solutions: Take 6g of pediatric protective preparation, add 2.5g of diatomaceous earth and grind evenly, add a certain volume of organic solvent, and prepare Qianhu test solution 1, Qianhu test solution 2, Qianhu test solution 3, and Qianhu test solution 4 according to the preparation method of Qianhu test solution under section 3.2.1;

[0217] Specifically, 20 ml of ethyl acetate was added to the Peucedanum praeruptorum test solution 1 as the extraction solvent and sonicated for 20 minutes; 40 ml of ethyl acetate was added to the Peucedanum praeruptorum test solution 2 as the extraction solvent and sonicated for 20 minutes; 40 ml of ethyl acetate was added to the Peucedanum praeruptorum test solution 3 as the extraction solvent and soaked overnight; 40 ml of ethyl acetate was added to the Peucedanum praeruptorum test solution 4 as the extraction solvent and shaken for 40 minutes.

[0218] Preparation of reference solution: Prepare the reference solution of white peucedanum praeruptorum glycoside according to the method described in 3.2.2.

[0219] Perform the thin-layer chromatography test (General Rule 0502) according to the method described in section 3.2.4. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0220] Figure 11 shows the thin-layer chromatograms of Peucedanum praeruptorum extracted by different methods, where 1, 2, 3, 4, and 5 are Peucedanum praeruptorum A, test solution 1, test solution 2, test solution 3, and test solution 4, respectively.

[0221] The results showed that the spots were lightest after shaking the sample for 40 minutes. The spot colors of the chromatograms after sonication for 20 minutes and 40 minutes were not significantly different. Although the spots were darker after soaking overnight, the 20-minute sonication was chosen because it took longer.

[0222] (2) Investigation of different extraction solvents

[0223] Preparation of Peucedanum praeruptorum test solution: Take 6g of pediatric protective preparation, add 2.5g of diatomaceous earth and grind evenly, add 20mL of organic solvent and sonicate, and continue to refer to the preparation method of Peucedanum praeruptorum test solution under section 3.2.1 to prepare Peucedanum praeruptorum test solution 1, Peucedanum praeruptorum test solution 2, Peucedanum praeruptorum test solution 3 and Peucedanum praeruptorum test solution 4 respectively.

[0224] Specifically, 20 ml of chloroform was added to Peucedanum praeruptorum test solution 1 as the extraction solvent, 20 ml of ethyl acetate was added to Peucedanum praeruptorum test solution 2 as the extraction solvent, 20 ml of diethyl ether was added to Peucedanum praeruptorum test solution 3 as the extraction solvent, and 20 ml of dichloromethane was added to Peucedanum praeruptorum test solution 4 as the extraction solvent.

[0225] Preparation of reference solution: Prepare the white peucedanum praeruptorum reference solution according to the method described in 3.2.2;

[0226] Perform the thin-layer chromatography test (General Rule 0502) according to the method described in section 3.2.4. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0227] Figure 12 shows the thin-layer chromatograms of Peucedanum praeruptorum extracted with different solvents, where 1, 2, 3, 4, and 5 are Peucedanum praeruptorum A, test solution 1, test solution 2, test solution 3, and test solution 4, respectively.

[0228] The results showed that the spots of the Peucedanum praeruptorum sample were more obvious after extraction with ethyl acetate and dichloromethane. Due to the high toxicity of dichloromethane, ethyl acetate was chosen as the extraction solvent.

[0229] (3) Examination of different pretreatment methods

[0230] Preparation of test solution: Take 6g of pediatric protective preparation, cut it into small pieces, add 2.5g of diatomaceous earth and grind it evenly, add 20ml of ethyl acetate, sonicate for 20 minutes, and continue to refer to the preparation method of Qianhu test sample under section 3.2.1 to prepare Qianhu test sample solution 1;

[0231] Take 6g of the pediatric protective preparation, cut it into small pieces, add 20ml of ethyl acetate, sonicate for 20 minutes, and continue to prepare the Peucedanum test sample solution 2 according to the preparation method of Peucedanum test sample under section 3.2.1;

[0232] Preparation of reference solution: Prepare the reference solution of white peucedanum praeruptorum glycoside according to the method described in 3.2.2.

[0233] Perform the thin-layer chromatography test (General Rule 0502) according to the method described in section 3.2.4, wherein for test solution 1, 10 μL and 15 μL are respectively taken for thin-layer chromatography experiments.

[0234] In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0235] Figure 13 shows the thin-layer chromatograms of Peucedanum praeruptorum obtained by different pretreatment methods. In the figure, 1, 2, 3, and 4 are Peucedanum praeruptorum A, shredded and unground sample, shredded and ground sample (10 μL), and shredded and ground sample (15 μL), respectively.

[0236] The results showed that the spots developed a better color after grinding.

[0237] 5.2 Methodological Examination

[0238] (1) Specificity examination

[0239] Preparation of test solutions: Take 4 pills each of the pediatric safety preparations D00001M, D00002M, and D00003M, add 2.5g of diatomaceous earth and grind them evenly, add 20ml of ethyl acetate to dissolve them, and sonicate for 20 minutes. Filter, evaporate the filtrate to dryness, and dissolve the residue in 1ml of methanol to obtain test solutions 1, 2, and 3.

[0240] Prepare the white angelica sinensis control solution and the angelica sinensis negative control solution according to the methods in sections 3.2.2-3.2.3.

[0241] Take 10 μL of each test solution, 2 μL of each reference solution and negative control solution, and spot them separately on the same Merck silica gel G plate for development. Perform thin-layer chromatography (General Rule 0502) according to the method described in section 3.2.4. Figure 14 is the thin-layer chromatogram of Peucedanum praeruptorum obtained from the specificity test. 1 is Peucedanum praeruptorum A, 2 is Peucedanum praeruptorum negative control, 3 is pediatric safety preparation D00001M, 4 is pediatric safety preparation D00002M, and 5 is pediatric safety preparation D00003M.

[0242] The results showed that the test sample chromatogram showed fluorescent spots of the same color at the corresponding positions as the reference sample chromatogram, and the negative control showed no interference.

[0243] (2) Test of the durability of thin-layer plates

[0244] Three test solutions of pediatric safety preparations D00001M, D00002M, and D00003M were prepared according to the method described in section 3.2.1.

[0245] Prepare the Peucedanum praeruptorum reference solution and the Peucedanum praeruptorum negative control solution according to the methods described in sections 3.1.2-3.1.3, and then perform thin-layer chromatography (General Rule 0502) using silica gel G plates from Merck AG (Germany) and Yantai Chemical Industry Research Institute, respectively, according to the method described in section 3.2.4. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0246] Figure 15 shows the thin-layer chromatograms of Peucedanum praeruptorum on different silica gel G plates, where A is the German Merck plate, B is the Yantai plate, and 1, 2, 3, 4, and 5 are Peucedanum praeruptorum A, Peucedanum praeruptorum negative control solution, pediatric safety preparation D00001M, pediatric safety preparation D00002M, and pediatric safety preparation D00003M, respectively.

[0247] The results showed that both types of thin-layer chromatography silica gel plates could achieve good separation effects.

[0248] (3) Investigation under different humidity levels

[0249] Three test solutions of pediatric safety preparations D00001M, D00002M, and D00003M were prepared according to the method described in section 3.2.1.

[0250] Prepare the Peucedanum praeruptorum reference solution and the Peucedanum praeruptorum negative control solution according to the methods described in sections 3.2.2-3.2.4, and develop the sample onto Merck silica gel G plates at 40% and 76% ppm respectively using thin-layer chromatography (General Rule 0502). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0251] Figure 16 shows the thin-layer chromatograms of Peucedanum praeruptorum under different relative humidity. A represents 40% relative humidity, B represents 76% relative humidity, and 1, 2, 3, 4, and 5 represent Peucedanum praeruptorum A, Peucedanum praeruptorum negative control solution, pediatric safety preparation D00001M, pediatric safety preparation D00002M, and pediatric safety preparation D00003M, respectively.

[0252] The results showed that when the method was carried out under humidity conditions of 40% and 76%, the test sample chromatogram showed fluorescent spots of the same color at the corresponding positions as the reference sample chromatogram.

[0253] (4) Investigation at different development temperatures

[0254] Three test solutions of pediatric safety preparations D00001M, D00002M, and D00003M were prepared according to the method described in section 3.2.1.

[0255] Prepare the Peucedanum praeruptorum reference solution and the Peucedanum praeruptorum negative control solution according to the methods described in sections 3.2.2-3.2.4, and take the spotted Merck silica gel G plates for thin-layer chromatography (General Rule 0502) at 4℃ and 32℃ respectively. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0256] Figure 17 shows the thin-layer chromatograms of Peucedanum praeruptorum at different temperatures. A represents 4℃, B represents 32℃, and 1, 2, 3, 4, and 5 represent Peucedanum praeruptorum A, Peucedanum praeruptorum negative control, and pediatric safety preparation D00001M, pediatric safety preparation D00002M, and pediatric safety preparation D00003M, respectively.

[0257] The results showed that when the method was developed at 4℃ and 32℃, the test sample chromatogram showed fluorescent spots of the same color at the corresponding positions as the reference sample chromatogram.

[0258] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0259] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A thin-layer chromatography detection method for a pediatric safety preparation, characterized in that, The raw materials of the pediatric safety preparation include: Pinellia ternata, Gastrodia elata, Atractylodes lancea, Pogostemon cablin, Notopterygium incisum, Hordeum vulgare, Areca catechu peel, Aucklandia lappa, Prunus armeniaca, Platycodon grandiflorus, Uncaria rhynchophylla, Pearl, Cinnamon, Saposhnikovia divaricata, Mentha haplocalyx, Poria cocos, Magnolia officinalis, Bombyx batryticatus, Coptis chinensis, Citrus reticulata peel, Massa fermentata, Asarum heterotropoides, Cinnamomum cassia, Peucedanum praeruptorum, Bupleurum chinense, Amber, Borneol, and Glycyrrhiza uralensis; the thin-layer chromatography detection method of the pediatric safety preparation includes identifying Bupleurum chinense and Peucedanum praeruptorum in the pediatric safety preparation using thin-layer chromatography; wherein, the thin-layer chromatography detection method of Bupleurum chinense includes the following steps: preparing Bupleurum chinense test solution and Bupleurum chinense test solution respectively. For the reference solution, spot the test solution and the reference solution onto the same thin-layer chromatography plate, and develop with a mixture of ethyl acetate, ethanol, and water in a volume ratio of 8:2:1 as the developing solvent. After color development with the colorimetric reagent, perform the first inspection. The colorimetric reagent is a mixture of p-dimethylaminobenzaldehyde and sulfuric acid in ethanol, or a mixture of p-dimethylaminobenzaldehyde and sulfuric acid in aqueous solution. The mass-to-volume ratio of p-dimethylaminobenzaldehyde to the sulfuric acid in ethanol is (1-5) g:100 mL, and the volume percentage of sulfuric acid in the sulfuric acid in the ethanol solution is 9%- 11%; the mass-to-volume ratio of p-dimethylaminobenzaldehyde to the sulfuric acid aqueous solution is (1-5) g: 100 mL, and the volume percentage of sulfuric acid in the sulfuric acid aqueous solution is 30%-50%; the preparation method of the Bupleurum chinense test solution is as follows: take 1.5 g of the pediatric safety preparation, add 10 mL of water to dissolve it, extract it once with 30 mL of water-saturated n-butanol, wash the n-butanol layer once with 20 mL of ammonia solution, discard the aqueous solution, evaporate the n-butanol solution to dryness, and dissolve the residue in 1 mL of methanol; the thin-layer chromatography detection method of the Peucedanum praeruptorum includes the following steps Step: Prepare the Peucedanum praeruptorum test solution and the Peucedanum praeruptorum reference solution separately. Spot the test solution and the reference solution on the same thin-layer chromatography plate and develop with a mixture of petroleum ether and ethyl acetate in a volume ratio of 3:1 as the developing solvent for the second inspection. The preparation method of the Peucedanum praeruptorum test solution is as follows: take 6g of pediatric protective preparation, cut it into small pieces, add 2.5g of diatomaceous earth and grind it evenly, add 20mL of ethyl acetate, sonicate for 20 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 1mL of methanol to obtain the test solution. The thin-layer chromatography plate is a silica gel G thin-layer plate.

2. The thin-layer chromatography detection method for pediatric safety preparations according to claim 1, characterized in that, The preparation of the Bupleurum reference solution includes the following steps: taking Bupleurum saponin a reference standard and Bupleurum saponin d reference standard, dissolving them respectively in a first organic solvent to obtain Bupleurum saponin a reference solution and Bupleurum saponin d reference solution.

3. The thin-layer chromatography detection method for pediatric safety preparations according to claim 2, characterized in that, The first organic solvent is one or more of methanol, ethanol and ethyl acetate.

4. The thin-layer chromatography detection method for pediatric safety preparations according to claim 1, characterized in that, The preparation of the Peucedanum reference solution includes the following steps: take the Peucedanum praeruptorum A reference standard, add a third organic solvent to dissolve it, and the Peucedanum praeruptorum A reference solution is obtained.

5. The thin-layer chromatography detection method for pediatric safety preparations according to claim 4, characterized in that, The third organic solvent is one or more of methanol, ethanol and ethyl acetate.

6. The thin-layer chromatography detection method for the pediatric safety preparation according to claim 1, characterized in that, The temperature for the color development treatment is 100℃-120℃.

7. The thin-layer chromatography detection method for pediatric safety preparations according to any one of claims 1-6, characterized in that, The first light source for inspection is fluorescent light or natural light, and the second light source for inspection is a 365nm ultraviolet lamp.

8. The thin-layer chromatography detection method for pediatric safety preparations according to any one of claims 1-6, characterized in that, The dosage form of the pediatric safety preparation is selected from one of the following: granules, powders, tablets, capsules, or pills.

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