Method for detecting pediatric exogenous wind heat composition

Abstract

The application relates to the technical field of drug detection, in particular to a detection method of a pediatric external wind-heat composition. The detection method comprises the following steps: adopting liquid chromatography to detect a test sample solution, and the test sample solution comprises the pediatric external wind-heat composition. In the liquid chromatography detection, a mobile phase comprises mobile phase A and mobile phase B, the mobile phase A is acetonitrile, the mobile phase B is a 0.05%-0.15% formic acid solution, and the elution is carried out in the following gradient elution mode. The detection method realizes good separation among characteristic components in the pediatric external wind-heat composition through a specific gradient elution procedure, the separation degree among the characteristic peaks is good, the detection of the pediatric external wind-heat composition is effectively realized, and the detection has high precision, good repeatability and high sensitivity.

Description

Technical Field

[0001] This application belongs to the field of drug detection technology, and in particular relates to a method for detecting a pediatric exogenous wind-heat composition. Background Technology

[0002] Common cold due to wind-heat is a common childhood illness, primarily characterized by fever. Currently, various traditional Chinese medicine (TCM) combinations are used clinically to treat common cold due to wind-heat in children. Among them, the pediatric exogenous wind-heat combination is a TCM combination with good efficacy for treating common cold due to wind-heat in children.

[0003] However, the composition of pediatric exogenous wind-heat syndrome compositions contains extracts of various traditional Chinese medicines such as peppermint, silkworm pupae, gypsum, anemarrhena, scutellaria, artemisia, scrophularia, and lycium bark, resulting in a complex composition that greatly increases the difficulty of detection and identification. Although the Chinese Pharmacopoeia includes methods for determining the content of some raw materials or extracts of pediatric exogenous wind-heat syndrome compositions, such as methods for determining the content of baicalin in scutellaria extract and mangiferin in anemarrhena, these methods are not applicable to the detection of pediatric exogenous wind-heat syndrome compositions due to their more complex composition. Therefore, there is an urgent need to provide a detection method for pediatric exogenous wind-heat syndrome compositions. Summary of the Invention

[0004] The purpose of this application is to provide a method for detecting pediatric exogenous wind-heat composition, so as to solve the technical problem that the existing methods for detecting raw materials of pediatric exogenous wind-heat composition cannot detect pediatric exogenous wind-heat composition.

[0005] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows:

[0006] In a first aspect, embodiments of this application provide a method for detecting a pediatric exogenous wind-heat composition. The detection method of this application includes the following steps:

[0007] The test solution, which included a pediatric exogenous wind-heat syndrome composition, was detected by liquid chromatography.

[0008] In the liquid chromatography detection, the mobile phase includes mobile phase A and mobile phase B. Mobile phase A is acetonitrile, and mobile phase B is a 0.05%–0.15% formic acid solution. Elution is performed using the following gradient elution method:

[0009]

[0010] The detection method of the embodiment of the present application realizes good separation between the characteristic components in the pediatric exogenous wind-heat composition through a specific gradient elution procedure, the peak shape of the characteristic peaks in the chromatogram is good, and the separation degree between adjacent characteristic peaks is good, effectively realizing the detection of the pediatric exogenous wind-heat composition, and the detection has high precision, good repeatability and high sensitivity. The detection method of the embodiment of the present application can not only be used for qualitative detection of the pediatric exogenous wind-heat composition, but also be used for quantitative detection of the characteristic components in the pediatric exogenous wind-heat composition. BRIEF DESCRIPTION OF DRAWINGS

[0011] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments or prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0012] Figure 1 The chromatogram of the sample solution of the pediatric exogenous wind-heat composition granules provided for Example 1 is shown in Figure 1.

[0013] Figure 2 The standard characteristic chromatogram of the pediatric exogenous wind-heat composition granules provided for Example 2 is shown in Figure 2.

[0014] Figure 3 The standard characteristic chromatogram of the pediatric exogenous wind-heat composition granules and the chromatogram of the sample of three batches of pediatric exogenous wind-heat composition granules are shown in Figure 3.

[0015] Figure 4 The chromatogram of the sample solution of the pediatric exogenous wind-heat composition granules provided for Comparative Example 1 is shown in Figure 4.

[0016] Figure 5 The chromatogram of the sample solution of the pediatric exogenous wind-heat composition granules provided for Comparative Example 2 is shown in Figure 5. DETAILED DESCRIPTION

[0017] In order to make the technical problems, technical solutions and beneficial effects of the present application more clearly understood, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and not to limit the present application.

[0018] In the present application, the term "and / or" describes the association relationship of the associated objects, which means that there can be three kinds of relationships, for example, A and / or B can mean that A exists alone, A and B exist together, and B exists alone. Wherein A and B can be singular or plural. The character " / " generally represents an "or" relationship between the associated objects before and after it.

[0019] In this application, "at least one" means one or more, and "multiple" means two or more. "At least one of the following" or similar expressions means any combination of the items, including a single item or any combination of multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can mean a, b, c, a-b (i.e. a and b), a-c, b-c, or a-b-c, where a, b, and c can be single or multiple.

[0020] It should be understood that the size of the sequence number of the above-mentioned processes in various embodiments of the present application does not mean the order of execution, and part or all of the steps can be executed in parallel or in sequence, and the execution order of the processes should be determined according to their functions and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0021] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a" and "the" used in the embodiments of the present application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0022] The weight of the related components mentioned in the embodiments of the present application can not only refer to the specific content of each component, but also represent the weight ratio relationship between each component, therefore, as long as the content of the related components in the embodiments of the present application is enlarged or reduced in proportion, it is within the scope disclosed in the embodiments of the present application. Specifically, the mass in the embodiments of the present application can be μg, mg, g, kg and other mass units commonly known in the chemical field.

[0023] The terms "first", "second" are only for the purpose of description, used to distinguish objects such as substances from each other, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX can also be called the second XX, and similarly, the second XX can also be called the first XX. Therefore, the features limited by "first" and "second" can explicitly or implicitly include one or more of the features.

[0024] To solve the technical problem that the detection method for the raw materials of the pediatric exogenous wind-heat composition in the prior art cannot realize the detection of the pediatric exogenous wind-heat composition, the present application proposes the following technical solutions.

[0025] In a first aspect, the embodiments of the present application provide a detection method for a pediatric exogenous wind-heat composition. The detection method of the embodiments of the present application comprises the following steps:

[0026] The test solution is detected by liquid chromatography, and the test solution includes the pediatric external wind-heat composition;

[0027] In the liquid chromatography detection, the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is a 0.05% to 0.15% formic acid solution. The elution is performed in the following gradient elution manner:

[0028]

[0029] The detection method of the embodiments of the present application can achieve good separation between the characteristic components in the pediatric external wind-heat composition through a specific gradient elution program, the separation degree between the characteristic peaks is good, the detection of the pediatric external wind-heat composition is effectively achieved, and the detection has high precision, good repeatability, and high sensitivity.

[0030] In some embodiments, the liquid chromatography elution program is in the time range of 70 to 80 min, and the proportion of the mobile phase can be the same as that of the mobile phase at 70 min, that is, in the time range of 70 to 80 min, the proportion of the mobile phase A can be 17% to 19%, and the proportion of the mobile phase B can be 81% to 83%.

[0031] In some embodiments, the test solution detected by the detection method of the embodiments of the present application is the pediatric external wind-heat composition containing a traditional Chinese medicine extract. The extraction raw materials of the traditional Chinese medicine extract include peppermint, silkworm chrysalis, raw gypsum, anemarrhena, scutellaria, artemisia, figwort, and psoralea. In further embodiments, the extraction raw materials of the traditional Chinese medicine extract include 3 to 11 parts of peppermint, 3 to 11 parts of silkworm chrysalis, 15 to 25 parts of raw gypsum, 7 to 17 parts of anemarrhena, 10 to 14 parts of scutellaria, 10 to 14 parts of artemisia, 10 to 14 parts of figwort, and 10 to 14 parts of psoralea.

[0032] In some embodiments, the traditional Chinese medicine extract can be obtained by decocting and extracting the extraction raw materials and concentrating. In an exemplary embodiment, the extraction method of the traditional Chinese medicine extract can include the following steps: mixing the traditional Chinese medicinal materials, immersing the mixed traditional Chinese medicinal materials in water, soaking and decocting, filtering out the first filtrate and the filter residue; mixing the filter residue with water for decocting, filtering out the second filtrate, combining the first filtrate and the second filtrate, obtaining the extraction liquid, and concentrating the extraction liquid to obtain the extract, which contains the traditional Chinese medicinal material extract. The relative density of the extract can be 1.2 to 1.3.

[0033] In some embodiments, the pediatric external wind-heat composition can further include an excipient. In further embodiments, the content of the traditional Chinese medicine extract can be 20% to 30%, and the content of the excipient can be 80% to 70%. In an exemplary embodiment, the pediatric external wind-heat composition can be a granule, which includes the extract and the excipient dextrin mentioned above, and the mass ratio of the extract to the excipient dextrin can be 1:3.

[0034] In some embodiments, the detection method of the present application can be used for qualitative detection of the pediatric exogenous wind-heat composition, and can also be used for quantitative detection of the pediatric exogenous wind-heat composition. In exemplary examples, the detection method of the present application can be used for identification of the pediatric exogenous wind-heat composition, and can also be used for establishing the fingerprint of the pediatric exogenous wind-heat composition, and can also be used for content detection of the characteristic ingredients in the pediatric exogenous wind-heat composition, which is not limited in particular.

[0035] In some embodiments, the volume concentration of formic acid in the mobile phase B can be 0.06% to 0.14%, which can be selected as 0.07% to 0.13%, 0.08% to 0.12%, 0.09% to 0.11%. In exemplary examples, the volume concentration of formic acid in the mobile phase B can be 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15% and the like typical but non-limiting size, or any size between any two numerical ranges. By controlling the concentration of formic acid in the mobile phase B within the range, in combination with the elution program, column temperature and other chromatographic conditions, the separation degree of the characteristic ingredients in the pediatric exogenous wind-heat composition is further improved.

[0036] In some embodiments, the mobile phase B of the detection method of the present application can be a formic acid solution with a volume concentration of 0.1%, and the gradient elution profile can be as follows:

[0037]

[0038] By using a formic acid solution with a volume concentration of 0.1% as the mobile phase B and controlling the gradient elution program within the range, the separation effect between the characteristic ingredients in the pediatric exogenous wind-heat composition is further improved, the separation degree between the characteristic peaks in the chromatogram of the test solution is improved, the peak shape of the characteristic peak is improved, and the detection sensitivity and precision are improved.

[0039] In some embodiments, the solvent of the test solution can include water. When the extraction method of the traditional Chinese medicine extract in the pediatric exogenous wind-heat composition is water decoction extraction, using water as the solvent effectively improves the solubility of the ingredients in the pediatric exogenous wind-heat composition in the solvent, further improves the accuracy of the detection method, and reduces the difficulty of the operation of the detection method.

[0040] In some embodiments, the concentration of the test sample solution can be 8-12 mg / mL, optionally 9 mg / mL or 10 mg / mL, based on the pediatric exogenous wind-heat composition. In exemplary embodiments, the concentration of the test sample solution can be 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, or the like typical but non-limiting size, based on the pediatric exogenous wind-heat composition. In an exemplary embodiment, 0.4-0.6 g of the test sample can be dissolved and diluted with water to 50 mL. Controlling the concentration of the test sample solution in this range can further improve the detection sensitivity while improving the peak shape of the characteristic peak and increasing the separation degree between adjacent characteristic peaks.

[0041] In some embodiments, the chromatographic column used in the liquid chromatography detection can be a chromatographic column with octadecylsilane-bonded silica gel as the filler. In further embodiments, the chromatographic column can be a YMC-Triart C18 chromatographic column, and the specification of the chromatographic column can be 4.6 x 250 mm, 5 μm. Using this chromatographic column for liquid chromatography detection can further improve the separation degree and peak shape of the characteristic peak in the chromatogram of the test sample solution and improve the sensitivity and accuracy of the detection.

[0042] In some embodiments, the flow rate of the liquid chromatography detection can be 0.8-1.2 mL / min, optionally 0.9-1.1 mL / min. In exemplary embodiments, the flow rate of the liquid chromatography detection can be 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, 1.2 mL / min, or the like typical but non-limiting size, or any flow rate between any two numerical ranges.

[0043] In some embodiments, the column temperature of the liquid chromatography detection can be 25-30°C, optionally 28-32°C. In exemplary embodiments, the column temperature of the liquid chromatography detection can be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, or the like typical but non-limiting size, or any temperature between any two numerical ranges.

[0044] Controlling the flow rate and column temperature in this range, combined with the gradient elution program and the flow phase of the chromatographic conditions, can further improve the separation effect of the index components in the test sample solution, increase the separation degree between the characteristic peaks in the chromatogram of the test sample solution, improve the accuracy of the detection, and improve the repeatability of the detection results.

[0045] In some embodiments, the injection volume for liquid chromatography detection can be 8-12 μL, optionally 9-11 μL. In exemplary embodiments, the injection volume can be 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, or the like typical but non-limiting size, or any size between any two ranges. Controlling the injection volume within this range, in combination with the conditions such as the concentration of the sample solution, the gradient elution program, and the detection wavelength, further improves the peak shape of the characteristic peak of the sample solution, reduces the occurrence of flat peaks due to overloading, and improves the sensitivity of detection.

[0046] In some embodiments, the detection wavelength can be 255-261 nm, optionally 256-260 nm. In exemplary embodiments, the detection wavelength can be 255 nm, 256 nm, 257 nm, 258 nm, 259 nm, 260 nm, 261 nm, or the like typical but non-limiting size, or any wavelength size between any two wavelengths. Controlling the detection wavelength within this range further improves the response value of the characteristic component in the sample solution, and improves the detection sensitivity.

[0047] In some embodiments, the sample solution chromatogram can contain at least 9 characteristic peaks, sorted in ascending order of relative retention time, and the 9 characteristic peaks are peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, and peak 9. In further embodiments, taking peak 9 as the reference peak, the relative retention times of peaks 1 to 8 are as follows:

[0048] The relative retention time of peak 1 is 0.126±5%;

[0049] The relative retention time of peak 2 is 0.293±5%;

[0050] The relative retention time of peak 3 is 0.313±5%;

[0051] The relative retention time of peak 4 is 0.407±5%;

[0052] The relative retention time of peak 5 is 0.444±5%;

[0053] The relative retention time of peak 6 is 0.793±5%;

[0054] The relative retention time of peak 7 is 0.877±5%;

[0055] The relative retention time of peak 8 is 0.929±5%.

[0056] In some embodiments, the relative retention time of peak 1 can be 0.126 ± 4%, 0.126 ± 3%, 0.126 ± 2%, 0.126 ± 1%, 0.126 ± 0.5%, and the like typical but non-limiting size. The relative retention time of peak 2 can be 0.293 ± 4%, 0.293 ± 3%, 0.293 ± 2%, 0.293 ± 1%, 0.293 ± 0.5%, and the like typical but non-limiting size. The relative retention time of peak 3 can be 0.313 ± 4%, 0.313 ± 3%, 0.313 ± 2%, 0.313 ± 1%, 0.313 ± 0.5%, and the like typical but non-limiting size. The relative retention time of peak 4 can be 0.407 ± 4%, 0.407 ± 3%, 0.407 ± 2%, 0.407 ± 1%, 0.407 ± 0.5%, and the like typical but non-limiting size. The relative retention time of peak 5 can be 0.444 ± 4%, 0.444 ± 3%, 0.444 ± 2%, 0.444 ± 1%, 0.444 ± 0.5%, and the like typical but non-limiting size. The relative retention time of peak 6 can be 0.793 ± 4%, 0.793 ± 3%, 0.793 ± 2%, 0.793 ± 1%, 0.793 ± 0.5%, and the like typical but non-limiting size. The relative retention time of peak 7 can be 0.877 ± 4%, 0.877 ± 3%, 0.877 ± 2%, 0.877 ± 1%, 0.877 ± 0.5%, and the like typical but non-limiting size. The relative retention time of peak 8 can be 0.929 ± 4%, 0.929 ± 3%, 0.929 ± 2%, 0.929 ± 1%, 0.929 ± 0.5%, and the like typical but non-limiting size.

[0057] The pediatric exogenous wind-heat composition is identified by the nine characteristic peaks, which has good specificity and is fast and convenient to detect. The identification of the pediatric exogenous wind-heat composition containing multiple traditional Chinese medicine extracts can be completed at one time. In some embodiments, peaks 1 to 3 belong to Anemarrhena asphodeloides, and peaks 4 to 9 belong to Scutellaria extract. The detection method of the embodiments of the present application can control the liquid chromatogram conditions to complete the detection of the characteristic components of Anemarrhena asphodeloides and Scutellaria in the pediatric exogenous wind-heat composition at one time, and has high detection efficiency.

[0058] In some embodiments, the detection method of the embodiments of the present application further comprises detecting the control solution by liquid chromatography. The control solution contains Scutellaria extract and Mango glycoside control. Anemarrhena asphodeloides contains Mango glycoside, and Scutellaria contains Scutellaria extract. The characteristic peaks of Mango glycoside and Scutellaria extract in the chromatogram of the test solution are located by detecting the control solution. It can be known that the relative retention time of the characteristic peak of Mango glycoside matches the relative retention time of peak 2, and the relative retention time of the characteristic peak of Scutellaria extract matches the relative retention time of peak 6, with peak 9 in the chromatogram of the test solution as a reference. That is, peak 2 in the chromatogram of the test solution is the characteristic peak of Mango glycoside, and peak 6 is the characteristic peak of Scutellaria extract.

[0059] In some embodiments, the fingerprint spectrum of the pediatric exogenous wind-heat composition can be constructed by using the detection method of the embodiments of the present application. When the fingerprint spectrum of the pediatric exogenous wind-heat composition is constructed by using the detection method of the embodiments of the present application, the following steps can be included:

[0060] The test sample solution and the control sample solution of the pediatric exogenous wind-heat composition that have passed the detection are detected by using the liquid chromatography method in the above, the chromatogram is recorded, and the test sample solution chromatogram and the control sample solution are obtained;

[0061] The test sample solution chromatogram and the control sample solution chromatogram are introduced into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System", and the fingerprint spectrum of the pediatric exogenous wind-heat composition is developed.

[0062] Through the fingerprint spectrum of the pediatric exogenous wind-heat composition, the comparison can be quickly performed when the pediatric exogenous wind-heat composition is identified and detected, it is confirmed whether the chromatogram of the sample to be detected matches the fingerprint spectrum of the pediatric exogenous wind-heat composition, and it is confirmed whether the identification and detection items of the pediatric exogenous wind-heat composition are qualified.

[0063] In some embodiments, the pediatric exogenous wind-heat composition can also be identified by using the detection method of the embodiments of the present application. When the pediatric exogenous wind-heat composition is identified by using the detection method of the embodiments of the present application, the following steps can be included:

[0064] The test sample solution is detected by using the liquid chromatography, and the test sample solution chromatogram is recorded;

[0065] The relative retention time of the characteristic peak in the test sample solution chromatogram is compared with the relative retention time of the peak 1 to the peak 9, if the test sample solution chromatogram contains the characteristic peak corresponding to the relative retention time of the peak 1 to the peak 9, the identification result is qualified.

[0066] In some other embodiments, when the pediatric exogenous wind-heat composition is identified by using the detection method of the embodiments of the present application, the following steps can be included:

[0067] The test sample solution is detected by using the liquid chromatography, and the test sample solution chromatogram is recorded;

[0068] The test sample solution chromatogram is introduced into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" and compared with the standard fingerprint spectrum, the similarity is calculated, and whether the identification result of the test sample is qualified is confirmed according to the similarity.

[0069] In some embodiments, the pediatric exogenous wind-heat composition can also be quantitatively detected by using the detection method of the embodiments of the present application. When the pediatric exogenous wind-heat composition is quantitatively detected by using the detection method of the embodiments of the present application, the following steps can be included:

[0070] The standard curve is obtained by detecting different concentrations of the control solution by liquid chromatography, and according to the measured peak area and the concentration of the corresponding control solution, a regression equation is obtained. In the standard curve, the peak area or peak height is taken as the vertical coordinate, and the concentration of the control solution is taken as the horizontal coordinate. The control solution can contain the control of mangiferin and / or the control of baicalin;

[0071] The concentration of the target component in the test sample solution is calculated by bringing the peak area or peak height of the characteristic peak corresponding to the control solution in the chromatogram of the test sample solution into the regression equation, so as to detect the content of baicalin and / or mangiferin in the pediatric exogenous wind-heat composition.

[0072] The detection method of the embodiment of the present application has good separation effect on the characteristic components in the pediatric exogenous wind-heat composition. Therefore, the quantitative detection of the pediatric exogenous wind-heat composition by the detection method of the embodiment of the present application can complete the detection of multiple components at one time, and has high detection efficiency.

[0073] In order to enable the above-mentioned implementation details and operations of the present application to be clearly understood by those skilled in the art, and the performance of the detection method of the pediatric exogenous wind-heat composition of the embodiment of the present application to be significantly embodied, the above-mentioned technical solutions are illustrated by multiple embodiments as follows.

[0074] Embodiment 1

[0075] The embodiment provides a detection method of a pediatric exogenous wind-heat composition. The detection method of the embodiment is to detect a test sample solution by using a high-performance liquid chromatograph.

[0076] The chromatographic conditions of the detection method of the embodiment are as follows: a chromatographic column: YMC-Triart C18 (4.6*250 mm, 5 μm) chromatographic column; column temperature: 30°C; detector: VWD detector; detection wavelength: 258 nm; mobile phase: binary gradient elution of acetonitrile (A)-0.1% formic acid (B), and the gradient elution program is shown in Table 1; injection volume: 10 μL; flow rate: 1.0 mL / min.

[0077] Table 1

[0078] Time / min Acetonitrile / % 0.1% formic acid in water / % 0 18 82 10 18 82 15 21 79 20 25 75 40 25 75 50 32 68 65 32 68 70 18 82 80 18 82

[0079] The detection method of the embodiment includes the following steps:

[0080] Step S1: Preparation of test sample solution. 0.5 g of the pediatric exogenous wind-heat composition granules was precisely weighed into a 50 mL volumetric flask, 25 mL of water was added, and ultrasonic dissolution was performed for 30 min. After cooling, water was added to the calibration line, shaken well, and filtered to obtain the test sample solution. The preparation method of the pediatric exogenous wind-heat composition granules used in this example is the same as that of the pediatric exogenous wind-heat composition granules in Example 2.

[0081] Step S2: The test sample solution was injected into the high performance liquid chromatograph, and the chromatogram was recorded, as shown in Figure 1

[0082] As shown in Figure 1 , the test sample solution chromatogram of the detection method of this example had 9 chromatographic peaks in the range of about 8 min to 64 min, and the separation degree of the 9 chromatographic peaks was good. The test sample solution was continuously injected multiple times, and the retention time and peak area of the chromatographic peaks in the obtained chromatograms were relatively stable.

[0083] Example 2

[0084] This example provides a detection method for a pediatric exogenous wind-heat composition. The detection method of this example is to detect the test sample solution by using a high performance liquid chromatograph.

[0085] The chromatographic conditions of the detection method of this example are as follows: chromatographic column: YMC-Triart C18 (4.6 x 250 mm, 5 μm) chromatographic column; column temperature: 30°C; detector: VWD detector; detection wavelength: 258 nm; mobile phase: binary gradient elution of acetonitrile (A)-0.1% formic acid (B), and the gradient elution program is shown in Table 2; injection volume: 10 μL; flow rate: 1.0 mL / min.

[0086] Table 2

[0087] Time / min Acetonitrile / % 0.1% formic acid in water / % 0 18 82 10 18 82 15 21 79 20 25 75 40 25 75 50 32 68 65 32 68 70 18 82 80 18 82

[0088] I. Labeling and attribution of characteristic peaks

[0089] (1) Preparation of experimental sample

[0090] 1) Preparation of test sample solution:

[0091] 0.5 g of the pediatric exogenous wind-heat composition granules was precisely weighed into a 50 mL volumetric flask, 25 mL of water was added, and ultrasonic dissolution was performed for 30 min. After cooling, water was added to the calibration line, shaken well, and filtered to obtain the test sample solution.

[0092] The preparation method of the pediatric exogenous wind-heat composition granules used in the detection method of this example is as follows:

[0093] ​Step G1: Chinese medicine extract preparation. Mix mint 9 parts, can 9 parts, raw gypsum 18 parts, jiaohuang 10 parts, huangqi 12 parts, qinghao 12 parts, xuan 12 parts and diben 10 parts, and immerse the mixed Chinese medicinal materials in water for 15 min, filter out the first filtrate and filter residue after decocting for 30 min, mix the filter residue with water according to a mass ratio of 1:6, filter out the second filtrate after decocting for 30 min, and combine the first filtrate and the second filtrate to obtain an extract. Concentrate the extract under the condition of a temperature of not more than 70 DEG C and a vacuum degree of-0.07 MPa to obtain a Chinese medicine extract paste with a relative density of 1.2.

[0094] Step G2: Chinese medicine composition fine powder preparation. After the drying air is dried by a rotary dehumidifier, the dried inlet air is heated to 60 DEG C by a primary filter and a heater, and then enters a fluidized bed from the bottom of the fluidized bed for preheating for 30 min. Then, the extract paste is dried and passed through a 100-mesh sieve, mixed with excipients (dextrin) according to a mass ratio of 1:3 to obtain a Chinese medicine composition fine powder.

[0095] Step G3: Granule preparation. The Chinese medicine composition fine powder is in a fluidized state driven by hot air, water is added according to a mass ratio of 1:3 with the Chinese medicine composition fine powder, and a peristaltic pump is used to deliver the water to a spray gun pipe. The water is atomized into small droplets by 1 bar of compressed air, and dispersed in the fluidized bed layer at a speed of 5.4 g / min / kg to collide with the Chinese medicine composition fine powder to form granules. After the inlet air continues for 5 min, the granules are prepared, passed through a 14-mesh sieve, and a pediatric exogenous wind-heat composition granule for treating pediatric exogenous wind-heat is obtained.

[0096] 2) Preparation of reference solution:

[0097] An appropriate amount of mangiferin reference substance and baicalin reference substance was taken, 80% methanol was added to prepare a reference solution with a content of 0.1 mg / mL.

[0098] 3) Preparation of positive sample solution:

[0099] The single herb dry extract powder of each medicinal ingredient was prepared according to the extraction, concentration and drying process conditions. The dry extract preparation method is as follows:

[0100] Each Chinese medicinal material was immersed in water, soaked for 15 min, and filtered to obtain a first filtrate and residue after decocting for 30 min. The residue was mixed with water at a mass ratio of 1:6, decocted for 30 min, and filtered to obtain a second filtrate. The first filtrate and the second filtrate were combined to obtain an extract. The extract was concentrated under the conditions of a temperature of not more than 70 °C and a vacuum degree of -0.07 MPa to obtain a Chinese medicinal extract paste with a relative density of 1.2. After the drying air was dehumidified by a rotary dehumidifier, the dried inlet air was heated to 60 °C by a primary filter and a heater, and then entered a fluidized bed from the bottom of the fluidized bed to be preheated for 30 min. After the extract paste was dried and passed through a 100-mesh sieve, the dried paste was mixed with excipients (dextrin) at a mass ratio of 1:3 to obtain a single-herb dry extract powder of each medicinal ingredient.

[0101] 0.4 g of each single-herb dry extract powder of each medicinal ingredient prepared was treated according to the preparation method of the test sample solution to obtain a positive sample solution of each single herb.

[0102] 4) Preparation of a negative sample solution

[0103] Each single-herb dry extract powder of each medicinal ingredient was prepared according to the extraction, concentration, and drying process conditions of the single-herb dry extract powder described above according to the prescription proportion of each missing single herb in the pediatric exogenous wind-heat composition granules. 0.4 g of each single-herb dry extract powder was treated according to the preparation method of the test sample solution to obtain a negative sample solution of each single herb.

[0104] The test sample solution, the control sample solution, the positive sample solution, and the negative sample solution were injected into a liquid chromatograph, respectively, for analysis. Three batches of the pediatric exogenous wind-heat composition granules of a pilot scale were introduced into the "Traditional Chinese Medicine Fingerprint Similarity Evaluation System 2004A Edition" to form a standard characteristic spectrum, as shown in Figure 2 Figure 2 The standard characteristic spectrum of the pediatric exogenous wind-heat composition granules is shown in the figure, which presents nine characteristic peaks. Peak No. 9 is a reference peak (S), and the relative retention times (tR) of the remaining characteristic peaks are 0.126 (peak 1), 0.293 (peak 2), 0.313 (peak 3), 0.407 (peak 4), 0.444 (peak 5), 0.793 (peak 6), 0.877 (peak 7), and 0.929 (peak 8). The control sample solution was used for calibration, and the characteristic peaks were identified by comparison with the positive sample solution and the negative sample solution. Peaks 1-3 belong to Anemarrhena asphodeloides Bunge, and peaks 4-9 belong to Scutellaria baicalensis Georgi. Two characteristic peaks were calibrated, in which peak 2 is a characteristic peak of mangiferin, and peak 6 is a characteristic peak of baicalin.

[0105] II. Methodology investigation

[0106] (1) Precision investigation

[0107] ​Take the same batch of pediatric exogenous wind heat composition particles, prepare the test solution, according to the above chromatographic conditions, 6 times, determine 9 common peaks, take peak 9 as the reference peak, and calculate the relative retention time and relative peak area of the remaining characteristic peaks according to the following formula (1) and formula (2).

[0108] Relative retention time t = retention time t of characteristic peak / retention time t of reference peak s Formula (1)

[0109] Relative peak area A = peak area A of characteristic peak / peak area A of reference peak s Formula (2)

[0110] The results of relative retention time and relative peak area are shown in Tables 3 and 4, wherein Table 3 is the relative retention time investigation results of precision experiment, and Table 4 is the relative retention peak area investigation results of precision experiment.

[0111] Table 3

[0112]

[0113]

[0114] Table 4

[0115] Serial number S1 S2 S3 S4 S5 S6 S7 S8 S9 (reference) 1 0.344 0.557 0.064 0.158 0.141 3.551 0.201 0.678 1.000 2 0.342 0.550 0.066 0.163 0.142 3.550 0.199 0.677 1.000 3 0.345 0.560 0.064 0.158 0.141 3.555 0.198 0.675 1.000 4 0.352 0.557 0.064 0.163 0.142 3.550 0.200 0.675 1.000 5 0.345 0.571 0.065 0.157 0.142 3.552 0.200 0.676 1.000 6 0.350 0.561 0.063 0.157 0.142 3.561 0.204 0.677 1.000 Average 0.346 0.559 0.064 0.159 0.142 3.553 0.200 0.676 1.000 RSD / % 1.11 1.23 1.61 1.80 0.36 0.12 1.03 0.18 /

[0116] As shown in Tables 3 and 4, in the precision investigation experiment, the relative retention time RSD value and relative peak area RSD of each chromatographic peak of peaks 1 to 9 of the chromatogram are less than 2%, indicating that the precision of the detection method of the present embodiment is good

[0117] (2) Repeatability investigation

[0118] Take an appropriate amount of the same batch of pediatric exogenous wind heat composition particles, respectively, and parallelly prepare 6 test solution, according to the above chromatographic conditions, respectively, and sample for determination. Take peak 9 as the reference peak, and calculate the relative retention time and relative peak area of the remaining characteristic peaks according to formula (1) and formula (2). The results are shown in Tables 5 and 6, wherein Table 5 is the relative retention time investigation results of repeatability experiment, and Table 6 is the relative retention peak area investigation results of repeatability experiment.

[0119] Table 5

[0120]

[0121]

[0122] Table 6

[0123] Serial number S1 S2 S3 S4 S5 S6 S7 S8 S9 (reference) 1 0.344 0.561 0.069 0.157 0.144 3.553 0.201 0.675 1.000 2 0.342 0.560 0.068 0.157 0.140 3.566 0.199 0.679 1.000 3 0.343 0.558 0.068 0.156 0.143 3.553 0.200 0.677 1.000 4 0.351 0.560 0.069 0.157 0.140 3.553 0.201 0.707 1.000 5 0.346 0.560 0.070 0.158 0.142 3.552 0.209 0.682 1.000 6 0.355 0.570 0.071 0.163 0.140 3.556 0.199 0.691 1.000 Average 0.347 0.562 0.069 0.158 0.142 3.556 0.202 0.685 1.000 RSD / % 1.47 0.76 1.69 1.60 1.24 0.15 1.88 1.76 /

[0124] As shown in Table 5 and Table 6, in the repeatability investigation experiment, the relative retention time RSD value and the relative peak area RSD of each chromatographic peak of peaks 1 to 9 of the chromatogram were less than 2%, indicating that the detection method of the present example had good repeatability

[0125] (3) Stability investigation

[0126] An appropriate amount of the same batch number of the pediatric exogenous wind-heat composition granules was taken to prepare a test solution, which was sampled at 0, 2, 6, 12, 18, and 24 hours. Peak 9 was taken as the reference peak, and the relative retention time and the relative peak area were calculated according to Formulas (1) and (2), and the results are shown in Table 7 and Table 8. Table 7 is the relative retention time investigation results of the stability experiment, and Table 8 is the relative peak area investigation results of the stability experiment.

[0127] Table 7

[0128]

[0129]

[0130] Table 8

[0131]

[0132] As shown in Table 7 and Table 8, in the stability investigation experiment, the relative retention time RSD value and the relative peak area RSD of each chromatographic peak of peaks 1 to 9 of the chromatogram were less than 2%, indicating that the test solution was stable within 24 hours.

[0133] (4) Durability investigation

[0134] An appropriate amount of the same batch number of the pediatric exogenous wind-heat composition granules was taken to prepare a test solution. The column temperature, flow rate, and durability between different chromatographic columns in the chromatographic conditions of Example 1 were investigated, and the durability chromatographic condition variation parameters are shown in Table 9, and the experimental results are shown in Table 10 to Table 12. Table 10 is the relative retention time investigation results in the column temperature durability test, Table 11 is the relative retention time investigation results in the flow rate durability test, and Table 12 is the relative retention time investigation results in the chromatographic column durability test.

[0135] Table 9

[0136]

[0137] Table 10

[0138]

[0139] Table 11

[0140]

[0141] Table 12

[0142]

[0143] As can be seen from Tables 10-12, the RSD of the relative retention time of each characteristic peak to the S peak is less than 2.0% under different column temperatures, flow rates, and chromatographic columns, indicating that the method has good robustness.

[0144] (5) Detection of characteristic chromatograms of 3 batches of samples

[0145] Three batches of samples (batch numbers: 20230901, 20231001, and 20231101) of the pediatric exogenous wind-heat composition granules were detected, and the chromatograms of the test sample solutions of the three batches of the pediatric exogenous wind-heat composition granules are shown in Figure 3 . Figure 3 In the formula, R is the generated reference chromatogram, i.e., the standard characteristic chromatogram, S1 is the chromatogram of the 20230901 batch, S2 is the chromatogram of the 20231001 batch, and S3 is the chromatogram of the 20231101 batch. The chromatogram of each test sample solution takes peak No. 9 as the reference peak, and the relative retention time and the relative peak area are calculated according to formula (1) and formula (2). The average relative retention time and the average relative peak area are shown in Table 13. The similarity between the chromatogram of each batch of granules and the standard characteristic chromatogram was calculated and compared using the “Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System” software developed by the National Pharmacopoeia Committee.

[0146] Table 13

[0147]

[0148] As shown in Table 13, the average relative retention time of the chromatogram of the test sample solution of the three batches of the pediatric exogenous wind-heat composition granules is similar to that of the standard characteristic chromatogram, and there is no obvious difference in the relative peak area of the chromatogram of the test sample solution of the three batches of the pediatric exogenous wind-heat composition granules. The similarity between the chromatogram of the test sample solution of each batch of the pediatric exogenous wind-heat composition granules and the standard characteristic chromatogram is 0.986, 0.992, and 0.989, respectively, indicating good similarity and good quality uniformity and stability among batches.

[0149] Comparative Example 1

[0150] This comparative example provides a detection method for the pediatric exogenous wind-heat composition. The detection method of this comparative example is to detect the test sample solution using a high-performance liquid chromatograph, and the chromatogram of the test sample solution is shown in Figure 4 .

[0151] The preparation method of the test solution for this comparative method is the same as that for Example 1. The chromatographic conditions for this comparative method are as follows: column: YMC-Triart C18 (4.6×250mm, 5μm) column; column temperature: 30℃; detector: VWD detector; detection wavelength: 258nm; injection volume: 10μL; flow rate: 1.0mL / min.

[0152] The mobile phase was acetonitrile, and mobile phase B was a 0.1% formic acid solution. The elution gradient is shown in Table 14.

[0153] Table 14

[0154] Time / min Acetonitrile / % 0.1% formic acid in water / % 0 5 95 100 95 5

[0155] like Figure 4 As shown, in the chromatogram of the test sample solution in this comparative example, there were no chromatographic peaks after a retention time of 44 min, and the resolution between the chromatographic peaks was poor.

[0156] Comparative Example 2

[0157] This comparative example provides a method for detecting a pediatric exogenous wind-heat syndrome composition. The detection method in this comparative example involves using high-performance liquid chromatography (HPLC) to detect the sample solution. The chromatogram of the sample solution is shown below. Figure 5 As shown.

[0158] The test solution of this comparative example was prepared in the same manner as the test solution of Comparative Example 1. The chromatographic conditions of the detection method in this comparative example are basically the same as those in Comparative Example 1, the difference being the mobile phase and elution gradient. In this comparative example, mobile phase A is acetonitrile, and mobile phase B is a 0.2% formic acid aqueous solution. The elution gradient is shown in Table 15.

[0159] Table 15

[0160] Time / min Acetonitrile / % 0.2% formic acid in water / % 0 5 95 12 5 95 25 7 93 40 18 82 45 23 77 55 23 77 60 25 75 65 25 75 70 28 72 80 28 72 85 30 70 90 30 70

[0161] like Figure 5 As shown, in the chromatogram of the test solution in this embodiment, peaks before 40 minutes are not easily retained, while there are nine chromatographic peaks after 40 minutes. Because these nine peaks are concentrated after 40 minutes, the detection efficiency is relatively low, and the separation between the peaks is poor, making them difficult to separate.

[0162] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for detecting a pediatric exogenous wind-heat composition, characterized in that, The method comprises the following steps: The test sample solution is detected by liquid chromatography, and the test sample solution comprises the pediatric exogenous wind-heat composition; In the liquid chromatography detection, the mobile phase comprises mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is a 0.05%-0.15% formic acid solution, and the elution is performed in the following gradient elution manner: The conditions of the liquid chromatography detection are as follows: The chromatographic column is a chromatographic column with octadecylsilane-bonded silica gel as the filler; The flow rate is 0.8-1.2 mL / min; The column temperature is 25-30 DEG C; The detection wavelength is 255-261 nm; The pediatric exogenous wind-heat composition comprises a traditional Chinese medicine extract and excipients, the content of the traditional Chinese medicine extract is 20%-30%, the content of the excipients is 80%-70%, and the extraction raw materials of the traditional Chinese medicine extract comprise 3-11 parts of Mentha haplocalyx, 3-11 parts of Scolia sinensis, 15-25 parts of gypsum fibrosum, 7-17 parts of Anemarrhena asphodeloides, 10-14 parts of Scutellaria baicalensis, 10-14 parts of Artemisia annua, 10-14 parts of Scrophularia ningpoensis, and 10-14 parts of Cynanchum paniculatum; The preparation solvent of the test sample solution is water, and the concentration of the test sample solution is 8-12 mg / mL based on the pediatric exogenous wind-heat composition.

2. The detection method of claim 1, wherein, The injection amount is 8-12 muL.

3. The detection method of claim 2, wherein, The chromatographic column is a YMC-Triart C18 chromatographic column, the specification of the chromatographic column is 4.6*250 mm, 5 mu m; and / or The flow rate is 1.0 mL / min; and / or The column temperature is 30 DEG C; and / or The detection wavelength is 258 nm; and / or The injection amount is 10 muL.

4. The detection method according to any one of claims 1 to 3, characterized in that, The test sample solution chromatogram contains at least 9 characteristic peaks, and the 9 characteristic peaks are peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, and peak 9 in order of increasing relative retention time, wherein the peak 9 is a reference peak, and the relative retention times of the peak 1 to the peak 8 are as follows: The relative retention time of the peak 1 is 0.126+ / -5%; The relative retention time of the peak 2 is 0.293+ / -5%; The relative retention time of the peak 3 is 0.313+ / -5%; The relative retention time of the peak 4 is 0.407+ / -5%; The relative retention time of the peak 5 is 0.444+ / -5%; The relative retention time of the peak 6 is 0.793+ / -5%; The relative retention time of the peak 7 is 0.877+ / -5%; The relative retention time of the peak 8 is 0.929+ / -5%.

5. The detection method of claim 4, wherein, The peak 2 is a characteristic peak of mangiferin.

6. The detection method as described in claim 4, characterized in that, The peak 6 is a characteristic peak of baicalin.

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