Detection method of children exogenous wind-heat composition

Abstract

The invention relates to the technical field of medicine detection, in particular to a detection method of an infant exogenous wind-heat composition. The detection method comprises the following steps: detecting a test solution by adopting liquid chromatography, wherein the test solution comprises the children exogenous wind-heat composition. Wherein in liquid chromatography detection, a mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A is acetonitrile, the mobile phase B is a 0.05%-0.15% formic acid solution, and elution is carried out in the following gradient elution mode. According to the detection method, characteristic components in the children exogenous wind-heat composition are well separated through a specific gradient elution procedure, the separation degree of characteristic peaks is good, the detection of the children exogenous wind-heat composition is effectively realized, and the detection precision is high, the repeatability is good, and the sensitivity is high.

Description

Technical Field

[0001] The present application belongs to the field of drug detection technology, and in particular, relates to a method for detecting a composition for treating exogenous wind-heat in children. Background Art

[0002] Wind-heat cold in children is a common disease in children, with fever as the main symptom. Currently, a variety of Chinese medicine compositions can be used in the clinic to treat wind-heat cold in children, among which the wind-heat cold composition for children with exogenous wind is a Chinese medicine composition with good curative effect on wind-heat cold in children.

[0003] However, since the pediatric exogenous wind-heat composition contains extracts of various Chinese medicines such as mint, Bombyx Batryticatus, Gypsum, Rhizoma Anemarrhenae, Radix Scutellariae, Artemisia Annua, Radix Scrophulariae and Radix Lycii, the ingredients in the pediatric exogenous wind-heat composition are relatively complex, which greatly increases the difficulty of detecting and identifying the pediatric exogenous wind-heat composition. Although the Chinese Pharmacopoeia contains the content determination methods of some raw materials or extracts of the pediatric exogenous wind-heat composition, such as the content determination method of baicalin in Radix Scutellariae extract and the content determination method of mangiferin in Rhizoma Anemarrhenae, the ingredients of the pediatric exogenous wind-heat composition are more complex, resulting in these methods not being suitable for the detection of the pediatric exogenous wind-heat composition, and therefore it is urgent to provide a detection method for the pediatric exogenous wind-heat composition. Summary of the invention

[0004] The purpose of the present application is to provide a method for detecting a composition for exogenous wind-heat in children, so as to solve the technical problem that the detection method for the raw materials of the composition for exogenous wind-heat in children in the prior art cannot realize the detection of the composition for exogenous wind-heat in children.

[0005] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0006] In the first aspect, the present application embodiment provides a method for detecting a composition for exogenous wind-heat in children. The detection method of the present application embodiment comprises the following steps:

[0007] The test solution is detected by liquid chromatography, and the test solution includes a composition for treating exogenous wind-heat in children;

[0008] Wherein, in the liquid chromatography detection, the mobile phase includes mobile phase A and mobile phase B, mobile phase A is acetonitrile, mobile phase B is 0.05% to 0.15% formic acid solution, and the elution is performed by the following gradient elution method:

[0009]

[0010] The detection method of the embodiment of the present application achieves 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 peak in the chromatogram is good, and the separation between adjacent characteristic peaks is good, which effectively realizes 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 be used not only for qualitative detection of the pediatric exogenous wind-heat composition, but also for quantitative detection of characteristic components in the pediatric exogenous wind-heat combination. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained like these drawings without paying any creative work.

[0012] Figure 1 The chromatogram of the test solution of the granules of the composition for treating exogenous wind-heat in children provided in Example 1;

[0013] Figure 2 The standard characteristic spectrum of the granules of the composition for treating exogenous wind-heat in children provided in Example 2;

[0014] Figure 3 The standard characteristic spectrum of the granules for treating exogenous wind-heat in children and the chromatograms of 3 batches of granules for treating exogenous wind-heat in children;

[0015] Figure 4 Chromatogram of the test solution of the granules of the composition for treating exogenous wind-heat in children provided in Comparative Example 1;

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

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

[0018] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0019] In the present application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, a~b (i.e. a and b), a~c, b~c, or a~b~c, where a, b, c can be single or multiple, respectively.

[0020] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function 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", "an" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0022] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass in the embodiment description of the present application can be μg, mg, g, kg and other mass units known in the chemical industry.

[0023] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0024] In order 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 solution.

[0025] In the first aspect, the present application embodiment provides a method for detecting a composition for exogenous wind-heat in children. The detection method of the present application embodiment comprises the following steps:

[0026] The test solution is detected by liquid chromatography, and the test solution includes a composition for treating exogenous wind-heat in children;

[0027] Wherein, in the liquid chromatography detection, the mobile phase includes mobile phase A and mobile phase B, mobile phase A is acetonitrile, mobile phase B is 0.05% to 0.15% formic acid solution, and the elution is performed by the following gradient elution method:

[0028]

[0029] The detection method of the embodiment of the present application achieves good separation between the characteristic components in the pediatric exogenous wind-heat composition through a specific gradient elution program, and the separation between the characteristic peaks is good, which effectively realizes the detection of the pediatric exogenous wind-heat composition, and the detection has high precision, good repeatability and high sensitivity.

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

[0031] In some embodiments, the test sample of the pediatric exogenous wind-heat composition detected by the detection method of the embodiment of the present application contains a Chinese medicine extract, and the extraction raw materials of the Chinese medicine extract include mint, silkworm, gypsum, rhizoma anemarrhenae, scutellaria, artemisia annua, Scrophularia ningpoensis and radix lycii. In a further embodiment, the extraction raw materials of the Chinese medicine extract include 3 to 11 parts of mint, 3 to 11 parts of silkworm, 15 to 25 parts of gypsum, 7 to 17 parts of rhizoma anemarrhenae, 10 to 14 parts of scutellaria, 10 to 14 parts of artemisia annua, 10 to 14 parts of Scrophularia ningpoensis and 10 to 14 parts of radix lycii.

[0032] In some embodiments, the Chinese medicine extract can be prepared by decoction, extraction and concentration of the extraction raw materials. In the exemplary embodiment, the extraction method of the Chinese medicine extract may include the following steps: mixing the Chinese medicinal materials, immersing the mixed Chinese medicinal materials in water, soaking and decocting, filtering out the primary filtrate and the filter residue; mixing the filter residue with water and decocting, filtering out the secondary filtrate, combining the primary filtrate and the secondary filtrate to obtain an extract, concentrating the extract to obtain an extract containing the Chinese medicinal material extract. The relative density of the extract may be 1.2-1.3.

[0033] In some embodiments, the composition for treating wind-heat in children may further include auxiliary materials. In further embodiments, the content of the Chinese medicine extract may be 20% to 30%, and the content of the auxiliary materials may be 80% to 70%. In a demonstration example, the composition for treating wind-heat in children may be a granule, which includes the extract and auxiliary material dextrin as described above, and the mass ratio of the extract to the auxiliary material dextrin may be 1:3.

[0034] In some embodiments, the detection method of the embodiment of the present application can be used for qualitative detection of the exogenous wind-heat composition for children, and can also be used for quantitative detection of the exogenous wind-heat composition for children. In the exemplary example, the detection method of the embodiment of the present application can be used for identification of the exogenous wind-heat composition for children, can also be used to establish the fingerprint of the exogenous wind-heat composition for children, and can also be used for content detection of characteristic components in the exogenous wind-heat composition for children, without specific limitation.

[0035] In some embodiments, the volume concentration of formic acid in mobile phase B can be 0.06% to 0.14%, optionally 0.07% to 0.13%, 0.08% to 0.12%, 0.09% to 0.11%. In the exemplary embodiment, the volume concentration of formic acid in 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%, etc., which are typical but non-limiting values, or any value between any two numerical ranges. The concentration of formic acid in mobile phase B is controlled within this range, combined with chromatographic conditions such as elution program and column temperature, to further improve the separation degree of characteristic components in the composition for treating exogenous wind-heat in children.

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

[0037]

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

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

[0040] In some embodiments, the concentration of the test solution can be 8-12 mg / mL, optionally 9 mg / mL 10 mg / mL, according to the composition for exogenous wind-heat in children. In the demonstration example, the concentration of the test solution can be 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL and other typical but non-limiting sizes according to the composition for exogenous wind-heat in children. In one demonstration example, 0.4-0.6 g of the test sample can be dissolved in water and diluted to 50 mL. Controlling the concentration of the test solution within this range further improves the detection sensitivity while improving the peak shape of the characteristic peak and improving the separation 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 a filler. In a further embodiment, the chromatographic column can be a YMC-Triart C18 chromatographic column, and the specifications of the chromatographic column can be 4.6×250mm, 5μm. The chromatographic column is used for liquid chromatography detection to further improve the separation and peak shape of the characteristic peaks in the chromatogram of the test 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, and can be 0.9-1.1 mL / min. In the exemplary embodiment, 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, etc., which are typical but non-limiting values, or any flow rate value between any two numerical ranges.

[0043] In some embodiments, the column temperature of the liquid chromatography detection can be 25-30° C., and can be 28-32° C. In an exemplary embodiment, 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., etc., which are typical but non-limiting values, or any temperature value between any two numerical ranges.

[0044] By controlling the flow rate and column temperature within this range and combining the chromatographic conditions such as the gradient elution program and the mobile phase, the separation effect of the index components in the test solution can be further improved, the separation degree between the characteristic peaks of the chromatogram of the test solution can be improved, the accuracy of the detection can be improved, and the repeatability of the detection results can be improved.

[0045] In some embodiments, the injection volume for liquid chromatography detection can be 8 to 12 μL, optionally 9 to 11 μL. In the exemplary embodiment, the injection volume can be 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, and other typical but non-limiting sizes, or any size between any two numerical ranges. By controlling the injection volume within this range, combined with conditions such as the concentration of the test solution, the gradient elution program, and the detection wavelength, the peak shape of the characteristic peak of the test solution can be further improved, the situation of flat peaks due to overloading can be reduced, and the sensitivity of the detection can be improved.

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

[0047] In some embodiments, the chromatogram of the test solution may contain at least 9 characteristic peaks, which are sorted from small to large according to the 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 a further embodiment, 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 was 0.126 ± 5%;

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

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

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

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

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

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

[0055] The relative retention time of peak 8 was 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%, etc. Typical but non-limiting sizes. 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%, etc. Typical but non-limiting sizes. 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%, etc. Typical but non-limiting sizes. 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%, etc. Typical but non-limiting sizes. 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%, etc. Typical but non-limiting sizes. 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%, etc. Typical but non-limiting sizes. 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%, etc. Typical but non-limiting sizes. 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%, etc. Typical but non-limiting sizes.

[0057] The nine characteristic peaks are used to identify and detect the exogenous wind-heat composition for children, with good specificity and fast and convenient detection. The identification of the exogenous wind-heat composition for children containing a variety of Chinese herbal medicine extracts can be completed at one time. In some embodiments, peaks 1 to 3 belong to Rhizoma Anemarrhenae, and peaks 4 to 9 belong to Radix Scutellariae extract. The detection method of the embodiment of the present application is controlled by liquid chromatogram conditions, and the characteristic component detection of Rhizoma Anemarrhenae and Radix Scutellariae in the exogenous wind-heat composition for children can be completed at one time, and the detection efficiency is high.

[0058] In some embodiments, the detection method of the embodiment of the present application further comprises using liquid chromatography to detect a reference solution, and the reference solution contains a baicalin reference and a mangiferin reference. The Chinese medicinal material Anemarrhena asphodeloides for preparing a pediatric exogenous wind-heat composition contains mangiferin, and Scutellaria baicalensis contains baicalin. The characteristic peaks of mangiferin and baicalin in the chromatogram of the test solution are located by detecting the reference solution. It can be seen that with peak 9 in the chromatogram of the test solution as a reference, the relative retention time of the characteristic peak of mangiferin matches the relative retention time of peak 2, and the relative retention time of the characteristic peak of baicalin matches the relative retention time of peak 6. That is, peak 2 in the chromatogram of the test solution is the characteristic peak of mangiferin, and peak 6 is the characteristic peak of baicalin.

[0059] In some embodiments, the detection method of the embodiment of the present application can be used to construct the fingerprint of the composition for exogenous wind-heat in children. When the detection method of the embodiment of the present application is used to construct the fingerprint of the composition for exogenous wind-heat in children, the following steps can be included:

[0060] The liquid chromatography method described above is used to test the qualified test solution and reference solution of the composition for exogenous wind-heat in children, and the chromatogram is recorded to obtain the chromatogram of the test solution and the reference solution;

[0061] The chromatograms of the test solution and the reference solution were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" to develop a fingerprint of the pediatric exogenous wind-heat composition.

[0062] Through the fingerprint spectrum of the pediatric exogenous wind-heat composition, a quick comparison can be made when performing identification and other tests on the pediatric exogenous wind-heat composition to confirm whether the chromatogram of the sample to be tested matches the fingerprint spectrum of the pediatric exogenous wind-heat composition, and whether the identification test items of the pediatric exogenous wind-heat composition are qualified.

[0063] In some embodiments, the detection method of the embodiment of the present application can also be used to identify the composition for exogenous wind-heat in children. When the detection method of the embodiment of the present application is used to identify the composition for exogenous wind-heat in children, the following steps can be included:

[0064] Use liquid chromatography to detect the test solution and record the chromatogram of the test solution;

[0065] Compare the relative retention time of the characteristic peak in the chromatogram of the test solution with the relative retention time of peaks 1 to 9 mentioned above. If the chromatogram of the test solution contains characteristic peaks corresponding to the relative retention times of peaks 1 to 9 mentioned above, the identification result is qualified.

[0066] In other embodiments, when the detection method of the embodiment of the present application is used to identify and check the exogenous wind-heat composition in children, the following steps may be included:

[0067] Use liquid chromatography to detect the test solution and record the chromatogram of the test solution;

[0068] The chromatogram of the test sample solution is imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" and compared with the standard fingerprint, the similarity is calculated, and the qualification of the identification result of the test sample is confirmed based on the similarity.

[0069] In some embodiments, the detection method of the embodiment of the present application can also be used to quantitatively detect the composition for exogenous wind-heat in children. When the detection method of the embodiment of the present application is used to quantitatively detect the composition for exogenous wind-heat in children, the following steps can be included:

[0070] Using liquid chromatography to detect reference solutions of different concentrations, obtaining a regression equation based on the measured peak area and the concentration of the corresponding reference solution, and obtaining a standard curve, wherein the peak area or peak height is used as the ordinate and the concentration of the reference solution is used as the abscissa, and the reference solution may contain a mangiferin reference substance and / or a baicalin reference substance;

[0071] The test solution is detected by liquid chromatography, and the peak area or peak height of the characteristic peak corresponding to the reference solution in the chromatogram of the test solution is substituted into the regression equation to calculate the concentration of the target component in the test solution, thereby detecting the content of baicalin and / or mangiferin in the composition for exogenous wind-heat in children.

[0072] The detection method of the embodiment of the present application has a good effect on separating the characteristic components in the exogenous wind-heat composition for children. Therefore, the detection method of the embodiment of the present application is used to perform quantitative detection of the exogenous wind-heat composition for children, which 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, as well as to demonstrate the significant improvement in the performance of the detection method for the pediatric exogenous wind-heat composition in the embodiment of the present application, the above-mentioned technical scheme is illustrated by means of multiple embodiments below.

[0074] Example 1

[0075] This embodiment provides a method for detecting a composition for exogenous wind-heat in children. The detection method of this embodiment is to detect the test solution using a high performance liquid chromatograph.

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

[0077] Table 1

[0078] Time / min Acetonitrile / % 0.1% formic acid aqueous solution / % 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 this embodiment includes the following steps:

[0080] Step S1: Preparation of test solution. Accurately weigh 0.5 g of the pediatric exogenous wind-heat composition granules, put it in a 50 mL volumetric flask, add 25 mL of water, dissolve it by ultrasonic for 30 min, add water to the scale line after cooling, shake well, filter, and obtain the test solution. The preparation method of the pediatric exogenous wind-heat composition granules used in this example is the same as the preparation method of the pediatric exogenous wind-heat composition granules in Example 2.

[0081] Step S2: Inject the test solution into the high performance liquid chromatograph and record the chromatogram. Figure 1 shown.

[0082] like Figure 1 As shown, there are 9 chromatographic peaks in the retention time range of about 8min to 64min in the chromatogram of the test solution of the detection method in this embodiment, and the separation of the 9 chromatographic peaks is good. The test solution is continuously injected multiple times, and the retention time and peak area of ​​the chromatographic peaks in each spectrum obtained are relatively stable.

[0083] Example 2

[0084] This embodiment provides a method for detecting a composition for exogenous wind-heat in children. The detection method of this embodiment is to detect the test solution using a high performance liquid chromatograph.

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

[0086] Table 2

[0087] Time / min Acetonitrile / % 0.1% formic acid aqueous solution / % 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] 1. Labeling and attribution of characteristic peaks

[0089] (1) Preparation of experimental samples

[0090] 1) Preparation of test solution:

[0091] Accurately weigh 0.5 g of the granules of the pediatric exogenous wind-heat combination into a 50 mL volumetric flask, add 25 mL of water, and dissolve by ultrasonic for 30 min. After cooling, add water to the scale line, shake well, and filter to obtain the test solution.

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

[0093] Step G1: Preparation of Chinese herbal medicine extract. Mix 9 parts of mint, 9 parts of Bombyx Batryticatus, 18 parts of Gypsum, 10 parts of Anemarrhena, 12 parts of Scutellaria, 12 parts of Artemisia annua, 12 parts of Scrophularia and 10 parts of Lycium bark, and immerse the mixed Chinese herbal medicines in water for 15 minutes. After boiling for 30 minutes, filter out the primary filtrate and the residue, mix the residue with water at a mass ratio of 1:6, boil for 30 minutes, filter out the secondary filtrate, combine the primary filtrate and the secondary filtrate to obtain an extract. Concentrate the extract at a temperature not exceeding 70°C and a vacuum degree of -0.07MPa to obtain a Chinese herbal medicine extract with a relative density of 1.2.

[0094] Step G2: Preparation of fine powder of Chinese medicine composition. After the air is dried by the rotary dehumidifier, the dry air is heated to 60°C through the primary filter and the heater, and then enters the fluidized bed from the bottom of the fluidized bed for preheating for 30 minutes. The extract is then dried and passed through a 100-mesh sieve and mixed with the auxiliary material (dextrin) at a mass ratio of 1:3 to obtain fine powder of Chinese medicine composition;

[0095] Step G3: Preparation of granules. The hot air drives the fine powder of the Chinese medicine composition to be fluidized, and water is added in a mass ratio of 1:3 to the fine powder of the Chinese medicine composition. The water is transported to the spray gun tube by a peristaltic pump, and the water is atomized into small droplets by 1 bar compressed air. The water is dispersed in the bed layer of the fluidized bed at a speed of 5.4 g / min / kg and collides with the fine powder of the Chinese medicine composition to form particles. After continuing to let in air for 5 minutes, the particles are made and passed through a 14-mesh sieve to obtain pediatric exogenous wind-heat composition particles for treating exogenous wind-heat in children.

[0096] 2) Preparation of reference solution:

[0097] Take appropriate amount of mangiferin reference substance and baicalin reference substance respectively, add 80% methanol to prepare reference substance solutions with a content of 0.1 mg / mL.

[0098] 3) Preparation of positive sample solution:

[0099] Take each single herbal decoction piece, and prepare the single herbal dry paste powder of each herbal flavor according to the extraction, concentration and drying process conditions. The preparation method of the dry paste is as follows:

[0100] Each Chinese herbal medicine was immersed in water for 15 minutes, and then filtered out the primary filtrate and residue after decocting for 30 minutes. The residue was mixed with water at a mass ratio of 1:6, and then filtered out the secondary filtrate after decocting for 30 minutes. The primary filtrate and the secondary filtrate were combined to obtain an extract. The extract was concentrated at a temperature not exceeding 70°C and a vacuum degree of -0.07MPa to obtain a Chinese herbal medicine extract with a relative density of 1.2. After the air was dried by a rotary dehumidifier, the dry inlet air was heated to 60°C through a primary filter and a heater, and then entered the fluidized bed from the bottom of the fluidized bed for preheating for 30 minutes. The extract was then dried and passed through a 100-mesh sieve and mixed with the auxiliary material (dextrin) at a mass ratio of 1:3 to obtain a single-flavor dry paste powder of each flavor.

[0101] Take 0.4 g of the prepared dry paste powder of each single medicinal flavor, and treat it according to the preparation method of the test sample solution to obtain the positive sample solution of each single medicinal flavor.

[0102] 4) Preparation of negative sample solution:

[0103] According to the prescription ratio of the Chinese medicinal materials in the above-mentioned pediatric exogenous wind-heat composition granules in this example, the prescription pieces of each missing single medicine were weighed respectively, and the negative sample dry paste powder of each medicinal flavor was prepared according to the extraction, concentration and drying process conditions of the above-mentioned single medicine dry paste. 0.4 g of the negative sample dry paste powder of each medicinal flavor was taken and processed according to the preparation method of the test sample solution to obtain the negative sample solution of each single medicine.

[0104] The test solution, reference solution, positive sample solution and negative sample solution were respectively injected into the liquid chromatograph for analysis. Three different batches of pilot-scale pediatric exogenous wind-heat composite granules were introduced into the "Chinese Medicine Fingerprint Similarity Evaluation System 2004A Edition" to form a standard feature map, such as Figure 2 As shown, Figure 2 This is the standard characteristic spectrum of the granules of the combination of wind and heat in children. There are 9 characteristic peaks in the spectrum. Peak 9 is used as the reference peak (S). 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 reference solution was used for calibration, and the positive sample solution and the negative sample solution were compared to confirm that the characteristic peaks 1 to 3 belonged to Anemarrhena asphodeloides; peaks 4 to 9 belonged to Scutellaria baicalensis. Two characteristic peaks were calibrated, of which peak 2 was the characteristic peak of mangiferin and peak 6 was the characteristic peak of baicalin.

[0105] 2. Methodological Investigation

[0106] (1) Precision inspection

[0107] Take an appropriate amount of the granules of the pediatric exogenous wind-heat combination from the same batch number to prepare the test solution. According to the above chromatographic conditions, parallel injection was performed 6 times to determine 9 common peaks. Peak No. 9 was used as the reference peak, and the relative retention time and relative peak area of ​​the remaining characteristic peaks were calculated according to the following formulas (1) and (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 results of relative retention time investigation of precision experiment, and Table 4 is the results of relative retention peak area investigation 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 Table 3 and Table 4, in the precision investigation experiment, the relative retention time RSD value and relative peak area RSD of each chromatographic peak from peak 1 to peak 9 of the chromatogram were less than 2%, indicating that the detection method of this embodiment has good precision.

[0117] (2) Repeatability study

[0118] Take an appropriate amount of the same batch of pediatric exogenous wind-heat composite granules, prepare 6 test sample solutions in parallel, and inject them for determination according to the above chromatographic conditions. Take peak No. 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, where Table 5 is the result of the relative retention time investigation of the repeatability experiment, and Table 6 is the result of the relative retention peak area investigation of the 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 test, the relative retention time RSD value and relative peak area RSD of each chromatographic peak from peak 1 to peak 9 of the chromatogram were less than 2%, indicating that the detection method of this embodiment has good repeatability.

[0125] (3) Stability investigation

[0126] Take an appropriate amount of the same batch of pediatric exogenous wind-heat composite granules to prepare a test solution, and inject the sample at 0, 2, 6, 12, 18, and 24 hours. Take peak 9 as the reference peak, and calculate the relative retention time and relative peak area according to formula (1) and formula (2). The results are shown in Table 7 and Table 8, where Table 7 is the relative retention time investigation result of the stability experiment, and Table 8 is the relative retention peak area investigation result of the stability experiment.

[0127] Table 7

[0128]

[0129]

[0130] Table 8

[0131]

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

[0133] (4) Durability test

[0134] Take an appropriate amount of the same batch number of pediatric exogenous wind-heat composition particles 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 respectively. The durability chromatographic condition change parameters are shown in Table 9, and the experimental results are shown in Tables 10 to 12. Among them, 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] It can be seen from Tables 10 to 12 that for different column temperatures, flow rates, and chromatographic columns, the RSDs of the relative retention times of the characteristic peaks and the S peak are all less than 2.0%, indicating that the method has good durability.

[0144] (5) Characteristic spectrum detection of 3 batches of samples

[0145] Three batches of samples of the pediatric exogenous wind-heat combination granules (batch numbers: 20230901, 20231001, and 20231101) were tested. The chromatograms of the test solutions of the three batches of pediatric exogenous wind-heat combination granules are as follows: Figure 3 shown. Figure 3 In the figure, R is the generated reference spectrum, that is, the standard characteristic spectrum, S1 is the chromatogram of batch 20230901, S2 is the chromatogram of batch 20231001, and S3 is the chromatogram of batch 20231101. The chromatograms of the three batches of test solution use peak 9 as the reference peak, and the relative retention time and relative peak area are calculated according to formula (1) and formula (2). The average relative retention time and relative peak area are shown in Table 13. The "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software developed by the Chinese Pharmacopoeia Committee was used to calculate and compare the similarity between the chromatograms of the three batches of particles and the standard characteristic spectrum.

[0146] Table 13

[0147]

[0148] As shown in Table 13, the average relative retention time of the chromatograms of the three batches of pediatric exogenous wind-heat composition granules test solution was similar to the standard characteristic spectrum, and there was no significant difference in the relative peak area of ​​the chromatograms of the three batches of pediatric exogenous wind-heat composition granules test solution. The similarities between the chromatograms of the three batches of pediatric exogenous wind-heat composition granules test solution and the standard characteristic spectrum were 0.986, 0.992, and 0.989, respectively, with good similarity and good quality uniformity and stability between batches.

[0149] Comparative Example 1

[0150] This comparative example provides a method for detecting a composition for treating exogenous wind-heat in children. The comparative example detection method is to use a high performance liquid chromatograph to detect the test solution. The chromatogram of the test solution is as follows: Figure 4 shown.

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

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

[0153] Table 14

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

[0155] like Figure 4 As shown, in the chromatogram of the test solution of this comparative example, there is no chromatographic peak after the retention time of 44 min, and the separation between the chromatographic peaks is poor.

[0156] Comparative Example 2

[0157] This comparative example provides a method for detecting a composition for treating exogenous wind-heat in children. The comparative example detection method is to use a high performance liquid chromatograph to detect the test solution. The chromatogram of the test solution is as follows: Figure 5 shown.

[0158] The preparation method of the test solution of this comparative example is the same as that of the test solution of comparative example 1. The chromatographic conditions of the detection method of this comparative example are basically the same as those of comparative example 1, except that the mobile phase and elution gradient are different. The mobile phase A of the detection method of this comparative example is acetonitrile, the mobile phase B is 0.2% formic acid aqueous solution, and the elution gradient is shown in Table 15:

[0159] Table 15

[0160] Time / min Acetonitrile / % 0.2% formic acid aqueous solution / % 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, the peaks before the retention time of 40 minutes in the chromatogram of the test solution of the detection method of this embodiment are not easily retained, and there are 9 chromatographic peaks after 40 minutes. Since the 9 chromatographic peaks are concentrated after 40 minutes, the detection efficiency is relatively low, and the separation between the chromatographic peaks is poor and difficult to separate.

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

Claims

1. A method for detecting a composition for exogenous wind-heat in children, characterized in that: The steps include: A liquid chromatography is used to detect a test solution, wherein the test solution includes a composition for treating exogenous wind-heat in children; Wherein, in the liquid chromatography detection, the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, the mobile phase B is 0.05% to 0.15% formic acid solution, and the elution is performed by the following gradient elution method:

2. The detection method according to claim 1, characterized in that The mobile phase B is 0.1% formic acid solution, and the gradient elution procedure is as follows:

3. The detection method according to claim 1, characterized in that The conditions for the liquid chromatography detection include at least one of the following (1) to (5): (1) The chromatographic column is a chromatographic column using octadecylsilane bonded silica gel as a filler; (2) Flow rate: 0.8-1.2 mL / min; (3) Column temperature is 25-30°C; (4) The detection wavelength is 255-261 nm; (5) The injection volume is 8 to 12 μL.

4. The detection method according to claim 3, characterized in that The chromatographic column is a YMC-Triart C18 chromatographic column, and the specification of the chromatographic column is 4.6×250 mm, 5 μm; and / or The flow rate is 1.0 mL / min; and / or The column temperature is 30°C; and / or The detection wavelength is 258 nm; and / or The injection volume was 10 μL.

5. The detection method according to any one of claims 1 to 4, characterized in that: The chromatogram of the test solution contains at least 9 characteristic peaks, which are arranged from small to large according to the relative retention time. The 9 characteristic peaks are peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8 and peak 9. Taking peak 9 as the reference peak, the relative retention times of peak 1 to peak 8 are as follows: The relative retention time of peak 1 is 0.126±5%; The relative retention time of peak 2 is 0.293±5%; The relative retention time of peak 3 is 0.313±5%; The relative retention time of peak 4 is 0.407±5%; The relative retention time of peak 5 is 0.444±5%; The relative retention time of peak 6 is 0.793±5%; The relative retention time of peak 7 is 0.877±5%; The relative retention time of peak 8 is 0.929±5%.

6. The detection method according to claim 5, characterized in that The peak 2 is the characteristic peak of mangiferin.

7. The detection method according to claim 5, characterized in that: The peak 6 is the characteristic peak of baicalin.

8. The detection method according to any one of claims 1 to 4, characterized in that: The solvent of the test solution includes water.

9. The detection method according to any one of claims 1 to 4, characterized in that: According to the composition for treating exogenous wind-heat in children, the concentration of the test solution is 8-12 mg / mL.

10. The detection method according to any one of claims 1 to 4, characterized in that: The composition for treating exogenous wind-heat in children comprises a Chinese herbal extract, wherein the extracting raw materials of the Chinese herbal extract comprise 3-11 parts of mint, 3-11 parts of bombyx batryticatus, 15-25 parts of gypsum, 7-17 parts of rhizoma anemarrhenae, 10-14 parts of scutellaria baicalensis, 10-14 parts of artemisia annua, 10-14 parts of scrophulariae ningpoensis and 10-14 parts of cortex lycii; and / or The pediatric exogenous wind-heat composition also includes auxiliary materials, and the content of the traditional Chinese medicine extract is 20% to 30%, and the content of the auxiliary materials is 80% to 70%.

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