HPLC (High Performance Liquid Chromatography) characteristic chromatogram of Xiaoyin capsules / granules as well as construction and

Abstract

The invention discloses an HPLC (High Performance Liquid Chromatography) specific chromatogram of Xiaoyin capsules / granules as well as construction and application thereof, and belongs to the technical field of quality control of traditional Chinese medicine preparations. According to the method, high performance liquid chromatography and mass spectrometry are combined, and chlorogenic acid, gallic acid, hydroxysafflor yellow A, albiflorin, rutin, isovitexin, 5-O-methylvisamidoside, 3, 5-di-O-caffeoylquinic acid, 4, 5-di-O-caffeoylquinic acid, benzoic acid, quercetin, paeonol, kushenone and obakunone are taken as reference substances; the characteristic chromatogram is successfully constructed for detecting different batches of Xiaoyin capsules / particles. The method is simple and easy to implement, high in precision, good in reproducibility, accurate and reliable, at least 14 components can be confirmed at a time under the same chromatographic condition, the detection efficiency is greatly improved, and time and cost are saved; quality monitoring can be accurately, rapidly and simultaneously performed on a plurality of characteristic components of the Xiaoyin capsules / particles, and a powerful guarantee is provided for controlling the quality and ensuring the clinical curative effect of the Xiaoyin capsules / particles.

Description

Technical Field

[0001] The present invention relates to the technical field of quality control of traditional Chinese medicine preparations, and particularly to an HPLC characteristic fingerprint of Xiaoyin capsules / granules and its construction and application. Background Art

[0002] Xiaoyin capsules (National Drug Approval Number: Z20000110) is a traditional Chinese medicine preparation and has been included in the first part of the Chinese Pharmacopoeia (2020 Edition). Xiaoyin granules (National Drug Approval Number: Z20000019) is also a traditional Chinese medicine preparation with the same efficacy as Xiaoyin capsules. It has the effects of clearing heat and cooling blood, nourishing blood and moistening dryness, and dispelling wind and relieving itching. It is mainly used for the treatment of psoriasis of blood-heat and wind-dryness type and blood-deficiency and wind-dryness type. Its prescription consists of 13 Chinese medicinal materials, namely Rehmannia glutinosa, Paeonia suffruticosa, Paeonia lactiflora, Angelica sinensis, Sophora flavescens, Lonicera japonica, Scrophularia ningpoensis, Arctium lappa, Cryptotympana pustulata, Dictamnus dasycarpus, Saposhnikovia divaricata, Isatis indigotica, and Carthamus tinctorius. Among them, Arctium lappa has the effects of dispersing wind-heat and promoting eruption and relieving swelling, and Sophora flavescens can clear heat and dry dampness, kill insects and relieve itching. Combined with other drugs, they jointly play a comprehensive therapeutic effect of clearing heat and cooling blood, nourishing blood and moistening dryness, and dispelling wind and relieving itching.

[0003] Psoriasis, commonly known as psoriasis vulgaris, is a common chronic, recurrent, inflammatory skin disease characterized by abnormal proliferation of epidermal cells in the skin, resulting in symptoms such as erythema, scales, and itching. According to epidemiological surveys, the incidence of psoriasis in China is approximately 0.47% to 0.5%. This data indicates that approximately 1 in every 200 to 250 people in China suffers from psoriasis. There are various treatment methods for psoriasis, including topical treatment, systemic drug treatment, phototherapy, and biological agents. Although existing treatment methods can effectively relieve the symptoms of psoriasis, most therapies have problems such as large side effects, drug resistance, and recurrence. Therefore, finding safe, effective, and long-lasting treatment methods remains the focus of clinical research. The research and development of Xiaoyin capsules and granules are based on traditional Chinese medicine theory, aiming at the causes of psoriasis such as damp-heat, blood stasis, and toxins, and emphasizing the treatment principles of clearing heat and detoxifying, promoting blood circulation to remove blood stasis, and dispelling dampness and relieving itching. By selecting a variety of Chinese medicinal materials and combining modern pharmaceutical technologies, it aims to fundamentally relieve the symptoms of psoriasis and reduce the possibility of recurrence. Compared with the serious side effects that some western medicines and biological agents may bring, Xiaoyin capsules and granules have significant advantages in terms of safety. As traditional Chinese medicine preparations, their prescription ingredients are natural and have small toxic and side effects, especially suitable for psoriasis patients who need long-term management. In addition, the capsule or granule dosage form is not only convenient to take, has good drug stability, but also helps patients adhere to treatment and improve treatment compliance.

[0004] In the quality standard of the current 2020 edition of the Chinese Pharmacopoeia, only paeoniflorin and matrine are used as the indicators for content determination in Xiaoyin Capsules. However, the prescription of Xiaoyin Capsules contains multiple medicinal materials and has complex chemical compositions. Relying solely on the identification or content determination of a single compound makes it difficult to comprehensively reflect its material basis and chemical composition information, and thus it is impossible to achieve comprehensive control of its internal quality. In addition, during the preparation process of the granule dosage form, the extraction and concentration degree of active ingredients are relatively low, while the capsule dosage form is usually designed to enclose and protect the active ingredients, and may more effectively maintain its active ingredients during processing and storage, resulting in a relatively high content. Therefore, when preparing the test solution of Xiaoyin Granules, after ultrasonic treatment, rotary evaporation is carried out, and then an appropriate amount of methanol is added for re-dissolution to ensure the stability of the solution and promote the full dissolution of the medicinal effect components. As a method with characteristics such as integrity, macroscopicity, and fuzzy analysis, the traditional Chinese medicine fingerprint can effectively control the overall quality of traditional Chinese medicine by describing the overall characteristics of traditional Chinese medicine, thus making up for the deficiencies of traditional single-component detection methods. Therefore, the traditional Chinese medicine fingerprint technology provides new ideas and solutions for the quality control of Xiaoyin Capsules / Granules. However, there are currently no literature and patent reports on the construction of HPLC characteristic fingerprints of Xiaoyin Capsules / Granules. Summary of the Invention

[0005] In view of the complex components of Xiaoyin Capsules / Granules, aiming at the existing quality evaluation methods of Xiaoyin Capsules / Granules that only rely on the identification or content determination of a single compound, ignoring the interaction and overall effect between components, resulting in problems such as insufficient systematic evaluation, stability evaluation, and inability to comprehensively reflect the medicinal effect material basis in the existing quality evaluation methods. The present invention aims to provide an HPLC characteristic fingerprint of Xiaoyin Capsules / Granules and its construction and application, and the quality of Xiaoyin Capsules / Granules can be comprehensively evaluated and controlled by using this characteristic fingerprint, so as to ensure the stability of product quality and the safety and effectiveness of clinical medication.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a method for constructing an HPLC characteristic fingerprint of Xiaoyin Capsules / Granules, including:

[0008] S1. Using different batches of Xiaoyin Capsules / Granules as test samples to prepare test solutions; using chlorogenic acid, gallic acid, hydroxysafflor yellow A, albiflorin, rutin, isovitexin, 5-O-methylvisammioside, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, benzoic acid, quercetin, paeonol, kushenone, obakunone as reference substances to prepare single reference substance solutions;

[0009] S2. Performing high performance liquid chromatography analysis on the test solution of Xiaoyin Capsules / Granules and the reference substance solution under the same conditions, and recording the corresponding chromatograms;

[0010] S3. Import the chromatogram of the test sample solution obtained in S2 into the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints and conduct similarity analysis to confirm the reliability of the results;

[0011] S4. Conduct high-resolution mass spectrometry analysis on the chromatogram of the test sample solution obtained in S2 to obtain the total ion current chromatogram; conduct data analysis based on the total ion current chromatogram and the peak emergence situation of the chromatogram of the test sample solution obtained in S2 to obtain the mass spectrometry result chromatogram of each chemical component;

[0012] S5. Compare the chromatogram of the test sample solution obtained in S2, the chromatogram of the reference substance solution, the total ion current chromatogram obtained in S4, and the mass spectrometry chromatograms of each chemical component to obtain the HPLC characteristic chromatogram of Xiaoyin Capsules / Granules composed of common characteristic peaks.

[0013] In S1, take the contents of different batches of Xiaoyin Capsules / Granules, add methanol solution, extract by ultrasonic wave, filter, and filter through a microporous filter membrane to obtain the test sample solution of Xiaoyin Capsules / Granules.

[0014] Furthermore, in every 25 mL of methanol solution, there is 1.0 g of the contents of Xiaoyin Capsules, extract by ultrasonic wave, filter, and filter through a microporous filter membrane to obtain the test sample solution of Xiaoyin Capsules.

[0015] Furthermore, in every 25 mL of methanol solution, there is 2.0 g of the contents of Xiaoyin Granules, extract by ultrasonic wave, evaporate the filtrate to dryness, dissolve the residue in methanol, and filter through a microporous filter membrane to obtain the test sample solution of Xiaoyin Granules.

[0016] Even further, the methanol solution is a pure methanol solution.

[0017] Even further, extract by ultrasonic wave for 30 min.

[0018] Even further, filter through a 0.45 μm microporous filter membrane.

[0019] In S1, use chlorogenic acid, gallic acid, hydroxysafflor yellow A, albiflorin, rutin, isovitexin, 5-O-methylvisammioside, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, benzoic acid, quercetin, paeonol, kurarinone, obacunone as reference substances, dissolve them in methanol solution to prepare a single reference substance solution.

[0020] Further, in S1, in the chlorogenic acid single reference solution, there is 88 μg of chlorogenic acid in every 1 mL of methanol solution; in the gallic acid single reference solution, there is 75 μg of gallic acid in every 1 mL of methanol solution; in the hydroxysafflor yellow A single reference solution, there is 96 μg of hydroxysafflor yellow A in every 1 mL of methanol solution; in the albiflorin single reference solution, there is 83 μg of albiflorin in every 1 mL of methanol solution; in the rutin single reference solution, there is 68 μg of rutin in every 1 mL of methanol solution; in the isovitexin single reference solution, there is 95 μg of isovitexin in every 1 mL of methanol solution; in the 5-O-methylvisammioside single reference solution, there is 123 μg of 5-O-methylvisammioside in every 1 mL of methanol solution; in the 3,5-di-O-caffeoylquinic acid single reference solution, there is 107 μg of 3,5-di-O-caffeoylquinic acid in every 1 mL of methanol solution; in the 4,5-di-O-caffeoylquinic acid single reference solution, there is 99 μg of 4,5-di-O-caffeoylquinic acid in every 1 mL of methanol solution; in the benzoic acid single reference solution, there is 85 μg of benzoic acid in every 1 mL of methanol solution; in the quercetin single reference solution, there is 79 μg of quercetin in every 1 mL of methanol solution; in the paeonol single reference solution, there is 64 μg of paeonol in every 1 mL of methanol solution; in the kushenone single reference solution, there is 136 μg of kushenone in every 1 mL of methanol solution; in the obacunone single reference solution, there is 142 μg of obacunone in every 1 mL of methanol solution.

[0021] In S2, the detection wavelength of the high performance liquid chromatography is 210 - 290 nm, the column temperature is 25 - 35 °C, and the flow rate is 0.6 - 1.0 mL / min.

[0022] Further, the detection wavelength is 225 nm, the column temperature is 30 °C, the flow rate is 1.0 mL / min, and the injection volume is 10 μL.

[0023] In S2, the determination conditions of the high performance liquid chromatography are as follows: a Shim-pack VP-ODS (250 mm × 4.6 mm, 5 μm) chromatographic column; the mobile phase is acetonitrile - 0.1% phosphoric acid, and the gradient elution program is as follows:

[0024]

[0025]

[0026] Specifically, S3 is as follows: Import the chromatograms of the test solutions of Xiaoyin capsules / granules of different batches obtained in S2 into the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints; select the chromatographic peaks that exist in the chromatograms of the test solutions of Xiaoyin capsules / granules of different batches as common peaks, and generate a reference chromatogram of Xiaoyin capsules / granules by the average value calculation method, and calculate the retention time and peak area of each common peak; perform similarity analysis through data import, multi-point correction and data matching; obtain and export the similarity result table between the chromatograms of the test solutions of Xiaoyin capsules / granules of different batches and the common peak pattern; confirm the reliability of the results according to the similarity result table and the chromatograms of the test solutions of Xiaoyin capsules / granules.

[0027] In S4, the detection conditions of the high-resolution mass spectrometry are as follows: electrospray ionization, spray voltage is 3500 V, sheath gas flow rate is 40 arb, auxiliary gas flow rate is 10 arb, capillary temperature is 300 °C, auxiliary gas temperature is 300 °C, the scanning mode is full-scan mode, and the mass-to-charge ratio scanning range m / z is 100 - 1500.

[0028] Specifically, S5 is as follows: Compare the chromatogram of the test solution and the chromatogram of the reference solution obtained in S2, combined with the total ion current chromatogram and the mass spectrometry result chromatogram of chemical components obtained by high-resolution mass spectrometry in S4. Using chlorogenic acid at peak 4 as the reference peak, identify that peak 1 in the chromatogram is gallic acid, peak 5 is hydroxysafflor yellow A, peak 6 is albiflorin, peak 8 is rutin, peak 9 is isovitexin, peak 10 is 5-O-methylvisammioside, peak 11 is 3,5-di-O-caffeoylquinic acid, peak 12 is 4,5-di-O-caffeoylquinic acid, peak 13 is benzoic acid, peak 15 is quercetin, peak 19 is paeonol, peak 21 is kushenone, and peak 22 is obacunone to obtain the characteristic chromatogram of Xiaoyin capsules / granules.

[0029] Further, after obtaining the characteristic chromatogram of Xiaoyin capsules / granules, perform an attribution analysis on the results of the characteristic chromatogram of Xiaoyin capsules / granules.

[0030] Furthermore, the chemical components corresponding to the 14 characteristic peaks in the characteristic chromatogram of Xiaoyin Capsules / Granules were assigned. Among them, the 1st peak, gallic acid, is a common peak of Paeonia lactiflora Pall., Angelica sinensis (Oliv.) Diels, and Carthamus tinctorius L.; the 4th peak, chlorogenic acid, is a common peak of Angelica sinensis (Oliv.) Diels and Arctium lappa L.; the 8th peak, rutin, is a common peak of Lonicera japonica Thunb. and Carthamus tinctorius L.; the 6th peak, albiflorin, is derived from Paeonia lactiflora Pall.; the 13th peak, benzoic acid, and the 19th peak, paeonol, are derived from Cortex Moutan; the 21st peak, kushenone, is derived from Sophora flavescens Ait.; the 11th peak, 3,5-di-O-caffeoylquinic acid, and the 12th peak, 4,5-di-O-caffeoylquinic acid, are derived from Lonicera japonica Thunb.; the 22nd peak, obakunone, is derived from Dictamnus dasycarpus Turcz.; the 10th peak, 5-O-methylvisammioside, is derived from Saposhnikovia divaricata (Turcz.) Schischk.; the 9th peak, isovitexin, is derived from Isatis indigotica Fort.; the 5th peak, hydroxysafflor yellow A, and the 15th peak, quercetin, are derived from Carthamus tinctorius L.

[0031] The present invention provides the HPLC characteristic chromatogram of Xiaoyin Capsules / Granules obtained by the above construction method.

[0032] The present invention provides a method for detecting the quality of Xiaoyin Capsules / Granules. The quality of the Xiaoyin Capsules / Granules sample is detected by using the above HPLC characteristic chromatogram of Xiaoyin Capsules / Granules, and the quality markers are one or more of chlorogenic acid, gallic acid, hydroxysafflor yellow A, albiflorin, rutin, isovitexin, 5-O-methylvisammioside, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, benzoic acid, quercetin, paeonol, kushenone, and obakunone;

[0033] The detected components include one or more of chlorogenic acid, gallic acid, hydroxysafflor yellow A, albiflorin, rutin, isovitexin, 5-O-methylvisammioside, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, benzoic acid, quercetin, paeonol, kushenone, and obakunone.

[0034] Application of the method for detecting the quality of Xiaoyin Capsules / Granules in constructing the quality standard of Xiaoyin Capsules / Granules.

[0035] Application of the method for detecting the quality of Xiaoyin Capsules / Granules in detecting drugs with the same name and the same formula as Xiaoyin Capsules / Granules.

[0036] Compared with the prior art, the technical solution of the present invention has obtained the following beneficial technical effects:

[0037] The method for constructing the HPLC characteristic chromatogram of Xiaoyin capsules / granules provided by the present invention can accurately separate and detect various active ingredients in Xiaoyin capsules / granules through high-performance liquid chromatography technology, combined with gradient elution and specific chromatographic conditions. It has high precision, good reproducibility, good resolution, and high stability. The characteristic chromatogram constructed by this method can ensure the consistency and stability between different batches of Xiaoyin capsules / granules, providing a reliable reference standard for the quality control of Xiaoyin capsules / granules. Through the similarity evaluation with the characteristic chromatogram, it can quickly and accurately judge whether the quality of the sample meets the requirements, ensure the consistency between different batches of products, and contribute to ensuring the stability and reliability of product quality. Compared with traditional quality detection methods, the HPLC characteristic chromatogram technology has higher detection efficiency, can detect a large number of samples in a short time, and automatically generate a detection result report, which can comprehensively, objectively, and accurately detect and evaluate the quality of Xiaoyin capsules (granules), and is of great significance for ensuring clinical efficacy and providing effective guarantee for clinical application.

[0038] The characteristic chromatogram of Xiaoyin capsules / granules constructed by the present invention realizes the characterization of 26 common chromatographic peaks, and clearly identifies the chemical structures of 14 main chromatographic peaks, covering 10 prescription drug flavors including Cortex Moutan, Radix Paeoniae Rubra, Radix Angelicae Sinensis, Radix Sophorae Flavescentis, Flos Lonicerae, Fructus Arctii, Cortex Dictamni, Radix Saposhnikoviae, Folium Isatidis, and Flos Carthami, controlling the quality and components of Xiaoyin capsules (granules) as a whole; the present invention makes up for the deficiencies in the content determination of Xiaoyin capsules / granules in the implementation standards, as well as the one-sidedness that the existing detection methods can only reflect the quality of very few drug components. The established characteristic chromatogram determination method is an effective supplement to the quality control method of Xiaoyin capsules / granules, can reflect the types and quantities of the contained chemical components, can effectively characterize its quality, and is conducive to comprehensively controlling the quality of products.

[0039] The quality detection method of Xiaoyin capsules / granules of the present invention can quickly and accurately identify products of different batches and different manufacturers. By comparing the presence or absence of common peaks in the obtained characteristic chromatograms, a more comprehensive evaluation of the quality of the preparation can be carried out, more effectively ensuring the quality of the products. It has the advantages of simplicity, high efficiency, good repeatability and stability, and is not only applicable to the quality control of Xiaoyin capsules / granules, but also can provide reference for the quality control of other traditional Chinese medicine preparations. Description of the Drawings

[0040] Figure 1 It is the chromatogram obtained by optimizing the extraction method in the preparation process of the test solution of Xiaoyin capsules of the present invention;

[0041] Figure 2 It is the chromatogram obtained by optimizing the extraction solvent in the preparation process of the test solution of Xiaoyin capsules of the present invention;

[0042] Figure 3This is the chromatogram obtained by optimizing the extraction time during the preparation of the test solution for the anti-silver capsules of the present invention;

[0043] Figure 4 This is the chromatogram obtained during the optimization of the preparation of the test solution for the anti-silver granules of the present invention;

[0044] Figure 5 This is the chromatogram (A), full-wavelength scan (190 - 400nm) (B) obtained by optimizing the detection wavelength under the chromatographic conditions of the present invention;

[0045] Figure 6 This is the chromatogram obtained by optimizing the column temperature under the chromatographic conditions of the present invention;

[0046] Figure 7 This is the chromatogram obtained by optimizing the flow rate under the chromatographic conditions of the present invention

[0047] Figure 8 This is the chromatogram obtained by optimizing the mobile phase composition under the chromatographic conditions of the present invention;

[0048] Figure 9 This is the chromatogram obtained by optimizing the elution program under the chromatographic conditions of the present invention;

[0049] Figure 10 This is the chromatogram (A), 3D map (B) and UV map (C) of chlorogenic acid of the present invention;

[0050] Figure 11 This is the chromatogram (A), 3D map (B) and UV map (C) of gallic acid of the present invention;

[0051] Figure 12 This is the chromatogram (A), 3D map (B) and UV map (C) of hydroxysafflor yellow A of the present invention;

[0052] Figure 13 This is the chromatogram (A), 3D map (B) and UV map (C) of albiflorin of the present invention;

[0053] Figure 14 This is the chromatogram (A), 3D map (B) and UV map (C) of rutin of the present invention;

[0054] Figure 15 This is the chromatogram (A), 3D map (B) and UV map (C) of isovitexin of the present invention;

[0055] Figure 16 This is the chromatogram (A), 3D map (B) and UV map (C) of 5-O-methylvisammioside of the present invention;

[0056] Figure 17 This is the chromatogram (A), 3D map (B) and UV map (C) of 3,5-di-O-caffeoylquinic acid of the present invention;

[0057] Figure 18 These are the chromatogram (A), 3D diagram (B), and UV diagram (C) of 4,5-di-O-caffeoylquinic acid of the present invention;

[0058] Figure 19 These are the chromatogram (A), 3D diagram (B), and UV diagram (C) of benzoic acid of the present invention;

[0059] Figure 20 These are the chromatogram (A), 3D diagram (B), and UV diagram (C) of quercetin of the present invention;

[0060] Figure 21 These are the chromatogram (A), 3D diagram (B), and UV diagram (C) of paeonol of the present invention;

[0061] Figure 22 These are the chromatogram (A), 3D diagram (B), and UV diagram (C) of kushenone of the present invention;

[0062] Figure 23 These are the chromatogram (A), 3D diagram (B), and UV diagram (C) of obacunone of the present invention;

[0063] Figure 24 This is the total ion current diagram of Xiaoyin Capsule of the present invention in positive ion mode;

[0064] Figure 25 This is the total ion current diagram of Xiaoyin Capsule of the present invention in negative ion mode.

[0065] Figure 26 This is the mass spectrum diagram of oxypaeoniflorin of the present invention;

[0066] Figure 27 This is the mass spectrum diagram of paeoniflorin of the present invention;

[0067] Figure 28 This is the mass spectrum diagram of rutin of the present invention;

[0068] Figure 29 This is the mass spectrum diagram of isovitexin of the present invention;

[0069] Figure 30 This is the mass spectrum diagram of hyperoside of the present invention;

[0070] Figure 31 This is the mass spectrum diagram of acteoside of the present invention;

[0071] Figure 32 This is the mass spectrum diagram of chlorogenic acid / neochlorogenic acid / cryptochlorogenic acid of the present invention;

[0072] Figure 33 This is the mass spectrum diagram of kushenone of the present invention;

[0073] Figure 34 This is the mass spectrum diagram of obacunone of the present invention;

[0074] Figure 35 This is the mass spectrum of hydroxysafflor yellow A of the present invention;

[0075] Figure 36 This is the mass spectrum of 3,5-di-O-caffeoylquinic acid / 4,5-di-O-caffeoylquinic acid / isochlorogenic acid C / isochlorogenic acid A of the present invention;

[0076] Figure 37 This is the mass spectrum of isoquercitrin of the present invention;

[0077] Figure 38 This is the mass spectrum of gallic acid of the present invention;

[0078] Figure 39 This is the mass spectrum of norsophoranol of the present invention;

[0079] Figure 40 This is the mass spectrum of angoroside C of the present invention;

[0080] Figure 41 This is the characteristic chromatogram of the test samples of Xiaoyin capsules of different batches of the present invention;

[0081] Figure 42 This is the characteristic chromatogram of the test samples of Xiaoyin granules of different batches of the present invention;

[0082] Figure 43 This is the HPLC characteristic chromatogram of Xiaoyin capsules / granules of the present invention. Detailed implementation manners

[0083] The following examples are used to further illustrate the present invention, but these examples are only for better understanding of the invention and are not used to limit the scope or implementation principle of the present invention. The implementation manners of the present invention are not limited to the following content. Unless otherwise specified, the test methods used are conventional methods, and the raw materials used are commercially available products.

[0084] The instruments used in the present invention are shown in the following table.

[0085]

[0086] The samples of Xiaoyin capsules and Xiaoyin granules of different batches used in the present invention are all purchased from the market. The Xiaoyin capsules are produced by Shaanxi Momeide Qixuehe Pharmaceutical Co., Ltd., and the Xiaoyin granules are produced by Shaanxi Kanghui Pharmaceutical Co., Ltd. The reagents used are shown in the following table.

[0087]

[0088] Reference substances: Chlorogenic acid reference substance (batch number: 110753-202018, purity: 98%), Gallic acid reference substance (batch number: 110831-20190, purity: 91.5%), Hydroxysafflor yellow A reference substance (batch number: 111637-202111, purity: 96.8%), Rutin (batch number: 100080-201811, purity: 92.4%), Paeonol (batch number: 110708-200505, purity: 99.8%), all purchased from the National Institutes for Food and Drug Control; Albiflorin (batch number: 22040205, purity: 99.7%), Quercetin (batch number: 21122007, purity: 98.96%), all purchased from Chengdu Profide Biotechnology Co., Ltd.; Isovitexin (batch number: PS001065, purity: >98%), Benzoic acid (batch number: PS012647, purity: 98%), Kushenone (batch number: PS000539, purity: >95%), all purchased from Chengdu Pusi Biotechnology Co., Ltd.; 5-O-Methylvisammioside (batch number: 22040205, purity: >99.7%), Obacunone (batch number: AZBG0405, purity: 98%), all purchased from Chengdu Efa Biotechnology Co., Ltd.; 3,5-Di-O-caffeoylquinic acid (batch number: 103963, purity: >98%), 4,5-Di-O-caffeoylquinic acid (batch number: 104311-230601, purity: >99%), all purchased from Jiangsu Yongjian Medical Technology Co., Ltd.

[0089] The present invention provides an HPLC characteristic fingerprint of Xiaoyin capsules / granules, and the specific construction method is as follows:

[0090] S1. Using different batches of Xiaoyin capsules / granules as test samples, prepare test sample solutions; using Chlorogenic acid, Gallic acid, Hydroxysafflor yellow A, Albiflorin, Rutin, Isovitexin, 5-O-Methylvisammioside, 3,5-Di-O-caffeoylquinic acid, 4,5-Di-O-caffeoylquinic acid, Benzoic acid, Quercetin, Paeonol, Kushenone, Obacunone as reference substances, prepare single reference substance solutions;

[0091] S2. Under the same conditions, perform high-performance liquid chromatography analysis on the test sample solutions and reference substance solutions of Xiaoyin capsules / granules, and record the corresponding chromatograms;

[0092] S3. Import the chromatograms of the test sample solutions obtained in S2 into the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints and perform similarity analysis to confirm the reliability of the results;

[0093] S4. Perform high-resolution mass spectrometry analysis on the chromatogram of the test sample solution obtained in S2 to obtain the total ion current chromatogram; perform data analysis based on the total ion current chromatogram and the peak emergence conditions of the chromatogram of the test sample solution obtained in S2 to obtain the mass spectrometry result chromatograms of each chemical component.

[0094] S5. Compare the chromatogram of the test sample solution obtained in S2, the chromatogram of the reference substance solution, the total ion current chromatogram obtained in S4, and the mass spectrometry chromatograms of each chemical component to obtain the HPLC characteristic chromatogram of Xiaoyin Capsules / Granules composed of common characteristic peaks.

[0095] Among them, the preparation method of the test sample solution of Xiaoyin Capsules in S1 is preferably: Weigh 1.0 g of the contents of Xiaoyin Capsules in different batches respectively, place them in a stoppered conical flask, add 25 mL of pure methanol solution, ultrasonically extract for 30 min, filter, and filter the filtrate through a 0.45 μm microporous filter membrane to obtain the test sample solution of Xiaoyin Capsules.

[0096] The preparation method of the test sample solution of Xiaoyin Granules is: Weigh 2.0 g of Xiaoyin Granules in different batches respectively, place them in a stoppered conical flask, add 25 mL of pure methanol solution, ultrasonically extract for 30 min, filter, evaporate the filtrate to dryness, dissolve the residue in 5 mL of methanol, filter, and then filter through a 0.45 μm microporous filter membrane to obtain the test sample solution of Xiaoyin Granules.

[0097] The preparation method of the standard substance solution in S2 is preferably: Accurately weigh chlorogenic acid, gallic acid, hydroxysafflor yellow A, albiflorin, rutin, isovitexin, 5-O-methylvisammioside, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, benzoic acid, quercetin, paeonol, kushenone, and phellodendrone respectively, and dissolve them in pure methanol solution to form a single reference substance solution containing 88 μg of chlorogenic acid, 75 μg of gallic acid, 96 μg of hydroxysafflor yellow A, 83 μg of albiflorin, 68 μg of rutin, 95 μg of isovitexin, 123 μg of 5-O-methylvisammioside, 107 μg of 3,5-di-O-caffeoylquinic acid, 99 μg of 4,5-di-O-caffeoylquinic acid, 85 μg of benzoic acid, 79 μg of quercetin, 64 μg of paeonol, 136 μg of kushenone, and 142 μg of phellodendrone per 1 mL.

[0098] The liquid chromatography conditions in S3 are: Chromatographic column: Shim-pack VP-ODS (250 mm × 4.6 mm, 5 μm) chromatographic column; Detector: Diode array detector; Detection wavelength: 225 nm; Flow rate: 1.0 mL / min; Injection volume: 10 μL; Column temperature: 30 °C; Mobile phase: Acetonitrile (A) - 0.1% phosphoric acid aqueous solution (B), gradient elution, and the elution program is shown in Table 1:

[0099] Table 1: Liquid Chromatography Elution Program

[0100]

[0101]

[0102] The present invention provides an optimization process for the HPLC characteristic fingerprint detection of the above-mentioned Xiaoyin capsules / granules:

[0103] (1) Optimization of the preparation of the test solution

[0104] In this example, different extraction methods (ultrasonic extraction, reflux extraction, maceration) were experimentally investigated. Specifically, see the appendix Figure 1 As shown, from the appendix Figure 1 data, it can be seen that the chromatogram components obtained by ultrasonic extraction are relatively comprehensive and the resolution is good. Therefore, the ultrasonic extraction method is adopted;

[0105] In this example, the extraction effects of different extraction solvents (pure ethanol solution, 70% methanol solution, 50% methanol solution, pure methanol solution) were compared. Specifically, see the appendix Figure 2 As shown, from the appendix Figure 2 data, it was found that when the pure methanol solution was used as the extraction solvent, the chromatogram of the extract had the most information and the highest component content. Therefore, the pure methanol solution was selected for extraction;

[0106] In this example, the extraction effects of different extraction times (30 min, 45 min, 1 h) were compared. Specifically, see the appendix Figure 3 As shown, from the appendix Figure 3 data, it was found that when ultrasonic extraction was carried out for 30 min, the chromatogram components had the highest content, good peak shape and high resolution. Therefore, 30 min was selected as the ultrasonic extraction time.

[0107] Based on the optimization results of the test solution of Xiaoyin capsules, this example conducted an experimental comparison of different extraction methods. Specifically, see the appendix Figure 4 As shown, from the appendix Figure 4 data, it can be seen that for the test solution of Xiaoyin granules obtained by ultrasonic extraction, filtration, evaporation to dryness of the filtrate, re-dissolution with methanol and then filtration, the components in the chromatogram are relatively comprehensive and the resolution is good;

[0108] (2) Optimization of chromatographic conditions

[0109] In this example, a diode array detector was used to investigate the detection wavelength, and chromatograms at 220 nm, 225 nm, 230 nm, 275 nm, and 290 nm were extracted. Specifically, see the appendix Figure 5 As shown, from the appendix Figure 5As can be seen from the data, when the detection wavelength condition is 225 nm, the chromatogram contains the most comprehensive information and the baseline is stable. Therefore, this method is selected as the detection wavelength condition;

[0110] In this embodiment, the column temperature (25 °C, 30 °C, 35 °C) is screened. For details, see the appendix Figure 6 As shown. From the appendix Figure 6 As can be seen from the data, when the column temperature is maintained at 30 °C, the peak emergence situation is the best and the separation effect of each component is good. Therefore, the column temperature of 30 °C is finally selected;

[0111] In this embodiment, the flow rate (0.6 mL / min, 0.8 mL / min, 1.0 mL / min) is screened. For details, see the appendix Figure 7 As shown. From the appendix Figure 7 As can be seen from the data, when the flow rate is 1.0 mL / min, the peak emergence situation is the best and the separation effect of each component is good. Therefore, the flow rate of 1.0 mL / min is finally selected;

[0112] After determining the optimal detection wavelength, column temperature and flow rate, the present invention screens the mobile phase through a large number of experiments. For details, see the appendix Figure 8 As shown. From the appendix Figure 8 As can be seen from the data, finally, acetonitrile - 0.1% phosphoric acid is selected as the mobile phase, and the elution program is optimized. Some elution programs are as follows:

[0113] Table 2: Elution Program 1

[0114]

[0115] Table 3: Elution Program 2

[0116]

[0117]

[0118] Table 4: Elution Program 3

[0119]

[0120] Table 5: Elution Program 4

[0121]

[0122] Table 6: Elution Program 5

[0123]

[0124] Table 7: Elution Program 6

[0125]

[0126] The detection results are as Figure 9 shown. ThroughFigure 9 It can be seen that the elution program 6 has good resolution, high peak height, stable baseline, and complete chromatographic information. Therefore, the elution program 6 is selected as the optimal elution program.

[0127] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0128] Example 1

[0129] Based on the above optimization conditions, this example provides a method for constructing the HPLC characteristic fingerprint of Xiaoyin capsules / granules.

[0130] S1. Preparation of test solution:

[0131] Weigh 1.0 g of the contents of Xiaoyin capsules from different batches respectively, place them in a stoppered conical flask, add 25 mL of pure methanol solution, extract by ultrasonic for 30 min, filter, and filter the filtrate through a 0.45 μm microporous filter membrane to obtain the test solution of Xiaoyin capsules.

[0132] Weigh 2.0 g of Xiaoyin granules from different batches respectively, place them in a stoppered conical flask, add 25 mL of pure methanol solution, extract by ultrasonic for 30 min, filter, evaporate the filtrate to dryness, dissolve the residue in 5 mL of methanol, filter, and then filter through a 0.45 μm microporous filter membrane to obtain the test solution of Xiaoyin granules.

[0133] S2. Preparation of reference solution:

[0134] Accurately weigh chlorogenic acid, gallic acid, hydroxysafflor yellow A, albiflorin, rutin, isovitexin, 5-O-methylvisamminol, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, benzoic acid, quercetin, paeonol, kurarinone, obakunone respectively, dissolve them in pure methanol solution, and prepare a single reference solution containing 88 μg of chlorogenic acid, 75 μg of gallic acid, 96 μg of hydroxysafflor yellow A, 83 μg of albiflorin, 68 μg of rutin, 95 μg of isovitexin, 123 μg of 5-O-methylvisamminol, 107 μg of 3,5-di-O-caffeoylquinic acid, 99 μg of 4,5-di-O-caffeoylquinic acid, 85 μg of benzoic acid, 79 μg of quercetin, 64 μg of paeonol, 136 μg of kurarinone, and 142 μg of obakunone per 1 mL.

[0135] S3. Chromatographic analysis:

[0136] Inject the test solution in S1 and the reference solution in S2 into a high performance liquid chromatograph for chromatographic analysis respectively, and conduct the detection under the same conditions. Use an UltiMate 3000 chromatograph. The liquid chromatography conditions are as follows: chromatographic column: Shim-pack VP-ODS (250mm×4.6mm, 5μm); detector: diode array detector, detection wavelength is 225nm; volume flow rate: 1.0mL / min; injection volume: 10μL; column temperature: 30°C; mobile phase: acetonitrile (A) - 0.1% phosphoric acid aqueous solution (B), gradient elution, adopt the 6-parameter optimized elution program in Example 1. The chromatograms of different single reference solutions are as Figures 10 to 23 shown.

[0137] S4. High performance liquid chromatography-mass spectrometry (HPLC-MS) analysis

[0138] According to the chromatogram of the test solution of Xiaoyin capsules / granules obtained in S3 and the chromatogram of the single reference solution, conduct high-resolution mass spectrometry analysis on the test solution of Xiaoyin capsules / granules. The high-resolution mass spectrometry detection conditions are as follows: electrospray ionization, spray voltage 3500V, sheath gas flow rate 40arb, auxiliary gas flow rate 10arb, capillary temperature 300°C, auxiliary gas temperature 300°C, scanning mode is full scan mode, mass-to-charge ratio scanning range m / z is 100 - 1500, and the total ion chromatogram ( Figure 24 and Figure 25 ) is obtained. Import the total ion chromatogram into Xcalibur software, and conduct data analysis according to the peak emergence situation of the chromatogram of the test solution obtained in S3 to obtain the mass spectrometry result diagram of each chemical component ( Figures 26 to 40 ).

[0139] Through high-resolution mass spectrometry analysis of the material reference substance of Xiaoyin Capsules, 20 chemical components were resolved. Combining with the HPLC-MS component analysis results of the extracts of 13 herbs including Rehmannia glutinosa, Paeonia suffruticosa, Paeonia lactiflora, Angelica sinensis, Sophora flavescens, Lonicera japonica, Scrophularia ningpoensis, Arctium lappa, Cryptotympana pustulata, Dictamnus dasycarpus, Saposhnikovia divaricata, Isatis indigotica, and Carthamus tinctorius in the preparation prescription in the early stage of the present invention, attribution analysis was carried out on the 20 chemical components, covering 11 herbs. The results showed that paeoniflorin is a common chemical component of Paeonia suffruticosa and Paeonia lactiflora; gallic acid is a common chemical component of Paeonia lactiflora, Angelica sinensis, and Carthamus tinctorius; acteoside is a common chemical component of Rehmannia glutinosa and Scrophularia ningpoensis; chlorogenic acid is a common chemical component of Angelica sinensis, Lonicera japonica, and Arctium lappa; albiflorin is a common chemical component of Lonicera japonica and Isatis indigotica; rutin is a common chemical component of Lonicera japonica and Carthamus tinctorius; oxypaeoniflorin is derived from Paeonia suffruticosa; kushenone and nor-kushenone are derived from Sophora flavescens; 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, neochlorogenic acid, and isochlorogenic acid C are derived from Lonicera japonica; angoroside C is derived from Scrophularia ningpoensis; isochlorogenic acid A and cryptochlorogenic acid are derived from Arctium lappa; obacunone is derived from Dictamnus dasycarpus; isoswertisin and isovitexin are derived from Isatis indigotica; hydroxysafflor yellow A is derived from Carthamus tinctorius.

[0140] S5. Reliability confirmation

[0141] Import the chromatograms of the test sample solutions of different batches of Xiaoyin Capsules obtained in S3 into the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (Version 2012A); select the chromatographic peaks that exist in the chromatograms ( Figures 41 - 42 ) of the test sample solutions of different batches of Xiaoyin Capsules / Granules as common peaks, and generate the reference chromatogram of Xiaoyin Capsules by the average value calculation method, and calculate the retention time and peak area of each common peak; through data import, multi-point correction, and data matching, similarity analysis is carried out; obtain the similarity result table between the chromatograms of the test sample solutions of different batches of Xiaoyin Capsules and the common peak pattern and export it; according to the similarity result table and the chromatograms of the test sample solutions of Xiaoyin Capsules, confirm the reliability of the results.

[0142] S6. Characteristic chromatogram of Xiaoyin Capsules / Granules

[0143] Based on the chromatograms of the test solution of Xiaoyin Capsules / Granules obtained in S3 and the chromatograms of single reference substance solutions, and by comparing with the total ion current chromatogram obtained by high-resolution mass spectrometry and the mass spectrometry result chromatogram of chemical components, taking chlorogenic acid (peak No. 4) as the reference peak, it was identified that peak No. 1 in the chromatogram was gallic acid, peak No. 5 was hydroxysafflor yellow A, peak No. 6 was albiflorin, peak No. 8 was rutin, peak No. 9 was isovitexin, peak No. 10 was 5-O-methylvisammioside, peak No. 11 was 3,5-di-O-caffeoylquinic acid, peak No. 12 was 4,5-di-O-caffeoylquinic acid, peak No. 13 was benzoic acid, peak No. 15 was quercetin, peak No. 19 was paeonol, peak No. 21 was kushenone, and peak No. 22 was obacunone, thus obtaining the characteristic chromatogram of Xiaoyin Capsules / Granules. See Appendix Figure 43 as shown.

[0144] By comparing with the high-performance liquid chromatograms of 14 reference substances such as chlorogenic acid, and combining with the HPLC-MS component analysis results of the extracts of 13 herbs including Rehmannia glutinosa, Cortex Moutan, Radix Paeoniae Rubra, Angelica sinensis, Sophora flavescens, Lonicera japonica, Scrophularia ningpoensis, Arctium lappa, Cryptotympana pustulata, Dictamnus dasycarpus, Saposhnikovia divaricata, Isatis indigotica, and Carthamus tinctorius in the preparation formula in the early stage of the present invention, the chemical components corresponding to the 14 characteristic peaks in the characteristic chromatogram of Xiaoyin Capsules (Granules) were attributed and analyzed. Among them, gallic acid (peak No. 1) was a common peak of Radix Paeoniae Rubra, Angelica sinensis, and Carthamus tinctorius; chlorogenic acid (peak No. 4) was a common peak of Angelica sinensis and Arctium lappa; rutin (peak No. 8) was a common peak of Lonicera japonica and Carthamus tinctorius; albiflorin (peak No. 6) was derived from Radix Paeoniae Rubra; benzoic acid (peak No. 13) and paeonol (peak No. 19) were derived from Cortex Moutan; kushenone (peak No. 21) was derived from Sophora flavescens; 3,5-di-O-caffeoylquinic acid (peak No. 11) and 4,5-di-O-caffeoylquinic acid (peak No. 12) were derived from Lonicera japonica; obacunone (peak No. 22) was derived from Dictamnus dasycarpus; 5-O-methylvisammioside (peak No. 10) was derived from Saposhnikovia divaricata; isovitexin (peak No. 9) was derived from Isatis indigotica; hydroxysafflor yellow A (peak No. 5) and quercetin (peak No. 15) were derived from Carthamus tinctorius.

[0145] Example 2

[0146] Methodology study on the detection method of characteristic chromatogram

[0147] (1) Similarity study

[0148] Meanwhile, the present invention uses the automatically generated reference chromatogram R to generate the common chromatographic peak pattern. It is analyzed and calculated that there is relatively good similarity among the common chromatographic peaks of the test samples of Xiaoyin Capsules and Xiaoyin Granules in different batches, indicating that the characteristic chromatograms of Xiaoyin Capsules and Xiaoyin Granules established by this method can well detect the quality of Xiaoyin Capsules and Xiaoyin Granules. The results are shown in Table 8-9.

[0149] Table 8: Similarity between each batch of Xiaoyin Capsule samples and the common chromatographic peak pattern

[0150]

[0151] Table 9: Similarity between the samples of Xiaoyin Granules in each batch and the common chromatographic peak pattern

[0152]

[0153]

[0154] (2) Precision study

[0155] Take the test solution prepared by the method of Example 1, analyze it according to the detection method of Example 1, inject samples in parallel 6 times, the injection volume is 10 μL, and take chlorogenic acid, gallic acid, hydroxysafflor yellow A, albiflorin, rutin, isovitexin, 5-O-methylvisammioside, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, benzoic acid, quercetin, paeonol, kushenone, phellodendrone as reference peaks. By analyzing the peak areas and retention times of the common characteristic peaks and calculating the RSD values, see Table 10 for details. It can be seen from the data in Table 10 that the RSDs of the retention times are all less than 0.871%, and the RSDs of the peak areas are all less than 2.727%, indicating that the parallel injection precision of this equipment is good.

[0156] Table 10: Peak areas and retention times of the common characteristic peaks of the samples of Xiaoyin Granules in each batch and calculation of RSD values

[0157]

[0158]

[0159] (3) Stability study

[0160] Take the test solution prepared by the method of Example 1, analyze it according to the detection method of Example 1, inject samples at different times of 0, 2, 6, 12, 18, 24 h for analysis, the injection volume is 10 μL, and take chlorogenic acid, gallic acid, hydroxysafflor yellow A, albiflorin, rutin, isovitexin, 5-O-methylvisammioside, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, benzoic acid, quercetin, paeonol, kushenone, phellodendrone as reference peaks. By analyzing the peak areas and retention times of the common characteristic peaks of the HPLC characteristic chromatogram of the sample and calculating the RSD values, see Table 11 for details. It can be seen from the data in Table 11 that the results show that the RSDs of the retention times are all less than 0.659%, and the RSDs of the peak areas are all less than 1.876%, indicating that the chromatographic peaks of the test solution of Xiaoyin Capsules hardly change within 24 h and have good stability.

[0161] Table 11: Peak areas and retention times of common characteristic peaks of Xiaoyin Granule samples at different times and calculation of RSD values

[0162]

[0163] (4) Reproducibility study

[0164] Prepare 6 batches of sample solutions according to the method of the test solution in Example 1. Refer to the chromatographic conditions in Example 1, with an injection volume of 10 μL. Using chlorogenic acid, gallic acid, hydroxysafflor yellow A, albiflorin, rutin, isovitexin, 5-O-methylvisammioside, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, benzoic acid, quercetin, paeonol, kurarinone, obakunone as reference peaks, analyze the peak areas and retention times of the common characteristic peaks of the HPLC characteristic chromatogram of the sample and calculate the RSD values. See Table 12 for details. From the data in Table 12, it can be seen that the RSD of the retention times are all less than 0.855%, and the RSD of the peak areas are all less than 2.722%, indicating that the chromatographic peaks of the sample have good reproducibility and the repeatability of this method is good.

[0165] Table 12: Peak areas and retention times of common characteristic peaks of each batch of Xiaoyin Granule samples and calculation of RSD values

[0166]

[0167] The above experimental results show that the method for constructing the characteristic chromatogram of the Xiaoyin capsules / granules provided by the present invention has the characteristics of good stability, high precision and good repeatability, can comprehensively and objectively evaluate the quality of the Xiaoyin capsules / granules, and provide quality assurance for clinical efficacy. The above embodiments are only exemplary embodiments of the present invention and are not used to limit the present invention. The protection scope of the present invention is defined by the claims. The above content is only an example and explanation of the concept of the present invention. Those skilled in the art in the technical field to which the present invention pertains can make various modifications or supplements to the specific embodiments described or use similar methods to replace them, as long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all belong to the protection scope of the present invention.

Claims

1. A method for constructing the HPLC characteristic fingerprint of Xiaoyin capsules / granules, which is characterized in that, The prescription of Xiaoyin Capsules / Granules consists of 13 Chinese medicinal materials, namely Rehmanniae Radix, Moutan Cortex, Paeoniae Radix Rubra, Angelicae Sinensis Radix, Sophorae Flavescentis Radix, Lonicerae Japonicae Flos, Scrophulariae Radix, Arctii Fructus, Cryptotympanae Periostracum, Dictamni Cortex, Saposhnikoviae Radix, Isatidis Folium, and Carthami Flos. The method for constructing its characteristic fingerprint includes: S1. Using different batches of Xiaoyin Capsules / Granules as test samples to prepare test sample solutions of Xiaoyin Capsules / Granules; using chlorogenic acid, gallic acid, hydroxysafflor yellow A, albiflorin, rutin, isovitexin, 5-O-methylvisamminol, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, benzoic acid, quercetin, paeonol, kushenone, and phellodendrone as reference substances to prepare single reference substance solutions. S2. Under the same conditions, performing high-performance liquid chromatography analysis on the test sample solutions and reference substance solutions of Xiaoyin Capsules / Granules, and recording the corresponding chromatograms. S3. Importing the chromatograms of the test sample solutions obtained in S2 into the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints and performing similarity analysis to confirm the reliability of the results. S4. Performing high-resolution mass spectrometry analysis on the chromatograms of the test sample solutions obtained in S2 to obtain the total ion current chromatogram; performing data analysis based on the total ion current chromatogram and the peak elution conditions of the test sample solutions obtained in S2 to obtain the mass spectrometry result chromatograms of each chemical component. S5. Comparing the chromatograms of the test sample solutions and reference substance solutions obtained in S2, the total ion current chromatogram obtained in S4, and the mass spectrometry chromatograms of each chemical component to obtain the HPLC characteristic fingerprint of Xiaoyin Capsules / Granules composed of common characteristic peaks.

2. The method for constructing the HPLC characteristic fingerprint of Xiaoyin capsules / granules according to claim 1, wherein In S1, taking test samples of different batches of Xiaoyin Capsules / Granules, adding methanol solution, ultrasonic extracting, and filtering through a microporous membrane to obtain the test sample solutions of Xiaoyin Capsules / Granules.

3. The method for constructing the HPLC characteristic fingerprint of Xiaoyin Capsule / Granule according to claim 1, wherein In S2, the conditions for high-performance liquid chromatography determination are as follows: Shim-pack VP-ODS (250mm×4.6mm, 5μm) chromatographic column; the mobile phase is acetonitrile - 0.1% phosphoric acid, the detection wavelength is 210 - 290nm, the column temperature is 25 - 35°C, the flow rate is 0.6 - 1.0mL / min, and the gradient elution program is as follows:

4. The method for constructing the HPLC characteristic chromatogram of Xiaoyin capsules / granules according to claim 1, wherein, S3 specifically includes: Importing the chromatograms of the test sample solutions of different batches of Xiaoyin Capsules / Granules obtained in S2 into the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints; selecting the chromatographic peaks that exist in the chromatograms of the test sample solutions of different batches of Xiaoyin Capsules / Granules as common peaks, generating the reference chromatogram of Xiaoyin Capsules / Granules by the average value calculation method, and calculating the retention time and peak area of each common peak; performing similarity analysis through data import, multi-point correction, and data matching; obtaining and exporting the similarity result table between the chromatograms of the test sample solutions of different batches of Xiaoyin Capsules / Granules and the common peak pattern; and confirming the reliability of the results based on the similarity result table and the chromatograms of the test sample solutions of Xiaoyin Capsules / Granules.

5. The method for constructing the HPLC characteristic fingerprint of Xiaoyin capsules / granules according to claim 1, wherein, In S4, the detection conditions for high-resolution mass spectrometry are as follows: electrospray ionization, spray voltage is 3500V, sheath gas flow rate is 40arb, auxiliary gas flow rate is 10arb, capillary temperature is 300°C, auxiliary gas temperature is 300°C, the scanning mode is full-scan mode, and the mass-to-charge ratio scanning range m / z is 100 - 1500.

6. The method for constructing the HPLC characteristic fingerprint of Xiaoyin capsules / granules according to claim 1, wherein Specifically, S5 is as follows: Based on the chromatograms of the test sample solution and the reference substance solution obtained in S2, combined with the total ion current map obtained by high-resolution mass spectrometry in S5 and the mass spectrometry result map of chemical components, with chlorogenic acid at peak 4 as the reference peak, it is identified that peak 1 in the chromatogram is gallic acid, peak 5 is hydroxysafflor yellow A, peak 6 is albiflorin, peak 8 is rutin, peak 9 is isovitexin, peak 10 is 5-O-methylvisammioside, peak 11 is 3,5-di-O-caffeoylquinic acid, peak 12 is 4,5-di-O-caffeoylquinic acid, peak 13 is benzoic acid, peak 15 is quercetin, peak 19 is paeonol, peak 21 is kushenone, and peak 22 is obacunone to obtain the characteristic chromatogram of Xiaoyin capsules / granules.

7. The method for constructing the HPLC characteristic chromatogram of Xiaoyin Capsule / Granule according to claim 1, wherein After obtaining the characteristic chromatogram of Xiaoyin capsules / granules, an attribution analysis is performed on the results of the characteristic chromatogram of Xiaoyin capsules / granules.

8. The HPLC characteristic chromatogram of Xiaoyin capsules / granules obtained by the construction method according to any one of claims 1 to 7.

9. A method for detecting the quality of Xiaoyin capsules / granules, characterized in that, Using the HPLC characteristic chromatogram of Xiaoyin capsules / granules described in claim 8, the quality of Xiaoyin capsules / granules or their drugs with the same name and the same formula is detected, with one or more of chlorogenic acid, gallic acid, hydroxysafflor yellow A, albiflorin, rutin, isovitexin, 5-O-methylvisammioside, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, benzoic acid, quercetin, paeonol, kushenone, and obacunone as quality markers.

10. A method for detecting the quality of Xiaoyin capsules / granules according to claim 9, characterized in that, The detected components are one or more of chlorogenic acid, gallic acid, hydroxysafflor yellow A, albiflorin, rutin, isovitexin, 5-O-methylvisammioside, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, benzoic acid, quercetin, paeonol, kushenone, and obacunone.

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