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

Abstract

The invention discloses a construction method and application of a Xiaoyin capsule / particle HPLC (High Performance Liquid Chromatography) standard fingerprint spectrum, 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; different batches of Xiaoyin capsules / particles are detected, and the characteristic fingerprint spectrum is successfully constructed. The method is simple and easy to implement, high in precision, good in reproducibility, accurate and reliable, at least 20 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, but is not limited to, the technical field of quality control of traditional Chinese medicine preparations, and specifically to a method for establishing an HPLC characteristic spectrum of Xiaoyin capsules / granules and the characteristic spectrum thereof. Background Art

[0002] Xiaoyin Capsule (National Medicine Standard: Z20000110) is a Chinese patent medicine, which has been included in the first volume of the 2020 edition of the Chinese Pharmacopoeia. Xiaoyin Granule (National Medicine Standard: Z20000019) is also a Chinese patent medicine, and its efficacy is the same as that of Xiaoyin Capsule. 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 to treat white ulcers of blood heat and wind dryness type and blood deficiency and wind dryness type. The prescription of this medicine is composed of 13 Chinese medicinal materials, including Rehmannia root, Peony bark, Red Peony Root, Angelica Sinensis, Sophora flavescens, Honeysuckle, Scrophularia ningpoensis, Burdock Fructus, Periostracum Cicadae, Dictamni, Saposhnikovia divaricata, Folium Isatidis, and Safflower. Among them, Burdock Fructus has the effect of dispersing wind-heat, clearing rash and reducing swelling, while Sophora flavescens can clear heat and dry dampness, kill insects and relieve itching. Together with other medicines, it can 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, is a common chronic, recurrent, inflammatory skin disease characterized by abnormal proliferation of epidermal cells, leading to symptoms such as erythema, scaling, and itching. According to epidemiological surveys, the incidence of psoriasis in my country is about 0.47% to 0.5%. This data shows that about 1 in every 200 to 250 people in China suffers from psoriasis. There are various treatments for psoriasis, including local treatment, systemic drug treatment, phototherapy, and biological agents. Although existing treatments 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 lasting treatments remains the focus of clinical research. The research and development of Xiaoyin Capsules and Xiaoyin Granules is based on the theory of traditional Chinese medicine. It focuses on the treatment principles of clearing away heat and detoxification, promoting blood circulation and removing blood stasis, and removing dampness and relieving itching for the causes of psoriasis such as damp heat, blood stasis, and toxins. By selecting a variety of Chinese medicinal materials and combining modern pharmaceutical technology, it aims to fundamentally relieve the symptoms of psoriasis and reduce the possibility of recurrence. Compared with the serious side effects that may be caused by some Western medicines and biological preparations, Xiaoyin capsules and granules have significant advantages in terms of safety. As traditional Chinese medicine preparations, their prescription ingredients are natural and have few toxic side effects, making them particularly suitable for psoriasis patients who require long-term management. In addition, capsules or granules are not only easy to take and have good drug stability, but also help patients adhere to treatment and improve treatment compliance.

[0004] In the quality standards of the current 2020 edition of the Chinese Pharmacopoeia, Xiaoyin Capsules only use paeoniflorin and matrine as indicators for content determination. However, the prescription of Xiaoyin Capsules contains a variety of medicinal materials with complex chemical compositions. It is difficult to fully reflect its material basis and chemical composition information by relying solely on the identification or content determination of a single compound, and thus it is impossible to achieve comprehensive control of its intrinsic quality. In addition, because the extraction and concentration of active ingredients are low during the preparation of granule dosage forms, and capsule dosage forms are usually designed to seal and protect active ingredients, they may be more effective in maintaining their active ingredients during processing and storage, resulting in higher content. Therefore, when preparing the Xiaoyin granule test solution, rotary evaporation is performed after ultrasound, and an appropriate amount of methanol is added for redissolution to ensure the stability of the solution and promote the full dissolution of the active ingredients. As a method with the characteristics of integrity, macroscopicity, and fuzzy analysis, the characteristic spectrum of traditional Chinese medicine can effectively control the overall quality of traditional Chinese medicine by describing the overall characteristics of traditional Chinese medicine, thereby making up for the shortcomings of traditional single component detection methods. Therefore, the traditional Chinese medicine fingerprint technology provides a new idea and solution for the quality control of Xiaoyin capsules / granules. However, there are currently no literature or patent reports on the construction of HPLC characteristic fingerprints of Xiaoyin capsules / granules. Summary of the invention

[0005] In view of the complex ingredients of Xiaoyin capsules / granules, the existing Xiaoyin capsule / granule quality evaluation method only relies on the identification or content determination of a single compound, ignoring the interaction and overall effect between the ingredients. As a result, the existing quality evaluation method has the problems of lack of systematic evaluation, insufficient stability evaluation, and inability to fully reflect the material basis of drug efficacy. The present invention aims to provide an HPLC characteristic fingerprint of Xiaoyin capsules / granules and its construction and application.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: The present invention provides a method for constructing an HPLC standard fingerprint of Xiaoyin capsules / granules, comprising: S1. Prepare the test sample Xiaoyin capsule / granule solution using different batches of Xiaoyin capsule / granule as test samples; prepare the single reference solution using chlorogenic acid, gallic acid, hydroxysafflor yellow A, paeoniflorin, rutin, isovitexin, 5-O-methylvisaminol glycosides, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, benzoic acid, quercetin, paeonol, matrine, and phellodendron as reference substances; S2, performing HPLC analysis on the test Xiaoyin capsule / granule solution and the reference solution under the same conditions, and recording the corresponding chromatograms; S3, importing the chromatograms of the test sample and the reference sample obtained in S2 into the similarity evaluation system for the chromatographic characteristic spectra of traditional Chinese medicine and performing similarity analysis to confirm the reliability of the results; S4, performing high-resolution mass spectrometry analysis based on the chromatogram of the test sample obtained in S2 to obtain a total ion current diagram; performing data analysis based on the total ion current diagram and the peak conditions of the chromatogram of the test sample obtained in S2 to obtain a mass spectrometry result diagram of each chemical component; S5, comparing the chromatograms of the test sample and the reference sample obtained in S2, the total ion flow diagram obtained in S4, and the mass spectra of each chemical component, to obtain an HPLC characteristic fingerprint of the Xiaoyin capsule / granules consisting of common characteristic peaks.

[0007] In S1, the contents of different batches of Xiaoyin capsules / granules were taken, methanol solution was added, ultrasonic extraction was performed, filtration was performed, and microporous membrane filtration was performed to obtain the test sample Xiaoyin capsule / granule solution.

[0008] Furthermore, every 25 mL of methanol solution contained 1.0 g of the contents of Xiaoyin Capsules, which were extracted by ultrasonic, filtered, and filtered through a microporous membrane to obtain the test sample Xiaoyin Capsule solution; every 25 mL of methanol solution contained 2.0 g of the contents of Xiaoyin Granules, which were extracted by ultrasonic, the filtrate was evaporated to dryness, methanol was added to the residue to dissolve it, and filtered through a microporous membrane to obtain the test sample Xiaoyin Granule solution.

[0009] Furthermore, the methanol solution is a pure methanol solution; ultrasonic extraction is performed for 30 min; and filtration is performed with a 0.45 µm microporous membrane.

[0010] In S1, chlorogenic acid, gallic acid, hydroxysafflor yellow A, paeoniflorin, rutin, isovitexin, 5-O-methylvisaminol glycosides, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, benzoic acid, quercetin, paeonol, matrine, and phellodendron were used as reference substances and dissolved in methanol solution to prepare a single reference substance solution.

[0011] Furthermore, in S1, the chlorogenic acid single reference solution contained 88 μg of chlorogenic acid per 1 mL of methanol solution; the gallic acid single reference solution contained 75 μg of gallic acid per 1 mL of methanol solution; the hydroxysafflor yellow A single reference solution contained 96 μg of hydroxysafflor yellow A per 1 mL of methanol solution; the paeoniflorin single reference solution contained 83 μg of paeoniflorin per 1 mL of methanol solution; the rutin single reference solution contained 68 μg of rutin per 1 mL of methanol solution; the isovitexin single reference solution contained 95 μg of isovitexin per 1 mL of methanol solution; the 5-O-methylvisaminol single reference solution contained 123 μg of 5-O-methylvisaminol per 1 mL of methanol solution; the 3,5-di-O-caffeoylquinic acid single reference solution contained 1 mL of methanol solution contains 107 μg of 3,5-di-O-caffeoylquinic acid; 1 mL of methanol solution contains 99 μg of 4,5-di-O-caffeoylquinic acid in the single reference solution of 4,5-di-O-caffeoylquinic acid; 1 mL of methanol solution contains 85 μg of benzoic acid in the single reference solution of benzoic acid; 1 mL of methanol solution contains 79 μg of quercetin in the single reference solution of quercetin; 1 mL of methanol solution contains 64 μg of paeonol in the single reference solution of paeonol; 1 mL of methanol solution contains 136 μg of quercetin in the single reference solution of matrine; 1 mL of methanol solution contains 142 μg of quercetin in the single reference solution of quercetin.

[0012] In S2, the HPLC detection wavelength is 210-290 nm, the column temperature is 25-35° C., and the flow rate is 0.6-1.0 mL / min.

[0013] Furthermore, the detection wavelength was 225 nm, the column temperature was 30 °C, the flow rate was 1.0 mL / min, and the injection volume was 10 μL.

[0014] In S2, the HPLC determination conditions are: 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: .

[0016] S3 specifically includes: importing the chromatograms of the test solutions of different batches of Xiaoyin capsules / granules and the chromatograms of the reference substance obtained in S2 into the similarity evaluation system of the chromatographic characteristic spectra of traditional Chinese medicine; selecting the chromatographic peaks existing in the chromatograms of the test solutions of different batches of Xiaoyin capsules / granules as common peaks, generating the reference spectra of Xiaoyin capsules / granules by the average value calculation method, and calculating the relative retention time and relative peak area of ​​each common peak; performing similarity analysis after data import, multi-point correction and data matching; obtaining and exporting the similarity result table between the chromatograms of the test solutions of Xiaoyin capsules from different batches and the common peak patterns; confirming the reliability of the results based on the similarity result table and the chromatograms of the test solutions of Xiaoyin capsules / granules.

[0017] In S4, the detection conditions of the high-resolution mass spectrometry are: electrospray ionization, spray voltage of 3500 V, sheath gas flow rate of 40 arb, auxiliary gas flow rate of 10 arb, capillary temperature of 300°C, auxiliary gas temperature of 300°C, scanning mode of full scan mode, and mass-to-charge ratio scanning range of m / z 100-1500.

[0018] S5 is specifically as follows: according to the chromatograms of the test sample and the reference sample obtained in S2, combined with the total ion flow diagram obtained by high-resolution mass spectrometry in S4 and the mass spectrum results of the chemical components, comparison is performed, and peak 4 chlorogenic acid is used as the reference peak. Peak 1 in the chromatogram is identified as gallic acid, peak 5 as hydroxysafflor yellow A, peak 6 as paeoniflorin, peak 8 as rutin, peak 9 as isovitexin, peak 10 as 5-O-methylvisaminol glycoside, peak 11 as 3,5-di-O-caffeoylquinic acid, peak 12 as 4,5-di-O-caffeoylquinic acid, peak 13 as benzoic acid, peak 15 as quercetin, peak 19 as paeonol, peak 21 as matrine, and peak 22 as phellodendron to obtain the characteristic spectrum of Xiaoyin capsules / granules.

[0019] Furthermore, after obtaining the characteristic spectrum of the Xiaoyin capsules / granules, attribution analysis is performed on the characteristic spectrum of the Xiaoyin capsules / granules.

[0020] Furthermore, the chemical components corresponding to the 14 characteristic peaks in the characteristic fingerprint of Xiaoyin Capsules / Granules were attributed and analyzed, among which, peak 1 gallic acid is a common peak of red peony root, Chinese angelica root and safflower; peak 4 chlorogenic acid is a common peak of Chinese angelica root and arctium fruit; peak 8 rutin is a common peak of honeysuckle and safflower; peak 6 paeoniflorin is derived from red peony root; peak 19 paeonol is derived from peony bark; peak 21 matrine is derived from sophora flavescens; peak 11 3,5-di-O-caffeoylquinic acid and peak 12 4,5-di-O-caffeoylquinic acid are derived from honeysuckle; peak 22 phellodendron is derived from Dictamni cortex; peak 10 5-O-methylvisamin glycoside is derived from Saposhnikovia divaricata; peak 9 isovitexin is derived from Isatis indigotica; peak 5 hydroxysafflor yellow A and peak 15 quercetin are derived from safflower.

[0021] The present invention provides an HPLC characteristic fingerprint of Xiaoyin capsules / granules obtained by the above-mentioned construction method.

[0022] The present invention provides a method for detecting the quality of Xiaoyin capsules / granules, and utilizes the HPLC characteristic fingerprint spectrum of Xiaoyin capsules / granules obtained by the above-mentioned construction method to detect the quality of Xiaoyin capsules / granules samples.

[0023] Compared with the prior art, the technical solution of the present invention has achieved the following beneficial technical effects: The method for constructing the HPLC standard fingerprint of Xiaoyin capsule / granules provided by the present invention can accurately separate and detect multiple active ingredients in Xiaoyin capsule / granules through high-performance liquid chromatography technology, combined with gradient elution and specific chromatographic conditions, with high precision, good reproducibility, good separation and high stability. The characteristic fingerprint constructed by this method can ensure the consistency and stability between different batches of Xiaoyin capsule / granules, and provide a reliable reference standard for the quality control of Xiaoyin capsule / granules. Through the similarity evaluation with the characteristic fingerprint, the quality of the sample can be quickly and accurately judged, and the consistency between different batches of products can be ensured, which helps to ensure the stability and reliability of product quality. Compared with traditional quality detection methods, HPLC characteristic fingerprint technology has higher detection efficiency, can detect a large number of samples in a short time, and automatically generate a test report, which can comprehensively, objectively and accurately detect and evaluate the quality of Xiaoyin capsule (granules), which is of great significance for ensuring clinical efficacy.

[0024] The characteristic fingerprint of Xiaoyin capsule / granule constructed by the present invention realizes the characterization of 26 common chromatographic peaks, and clearly chemically identifies 14 main chromatographic peaks, covering 10 prescription medicinal flavors of Paeonia suffruticosa, Paeonia lactiflora, Angelica sinensis, Sophora flavescens, Lonicera japonica, Scrophularia ningpoensis, Arctium lappa, Saposhnikovia divaricata, Folium Isatidis, and Carthamus tinctorius, so as to control the quality and composition of Xiaoyin capsule (granule) as a whole; the present invention makes up for the deficiency of Xiaoyin capsule / granule in content determination in the implementation standard, and the one-sidedness that the detection method in the prior art can only reflect the quality of very few drug components. The established characteristic spectrum determination method is an effective supplement to the quality control method of Xiaoyin capsule / granule, can reflect the types and quantities of the chemical components contained, can effectively characterize its quality, and is conducive to comprehensive control of product quality.

[0025] The quality detection method for Xiaoyin capsules / granules of the present invention can quickly and accurately identify products from different batches and different manufacturers. By comparing the presence or absence of common peaks in the obtained fingerprint spectrum, a more comprehensive evaluation of the quality of the preparation can be performed, and the quality of the product can be more effectively guaranteed. The method has the advantages of simplicity, high efficiency, good repeatability and stability, and is not only suitable for the quality control of Xiaoyin capsules / granules, but also can provide a reference and reference for the quality control of other traditional Chinese medicine preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a chromatogram obtained by optimizing the extraction method during the preparation of the test solution for Xiaoyin Capsule of the present invention; Figure 2 This is a chromatogram obtained by optimizing the extraction solvent during the preparation of the test solution of the Xiaoyin Capsule of the present invention; Figure 3 This is a chromatogram obtained by optimizing the extraction time during the preparation of the test solution for the Xiaoyin Capsule of the present invention; Figure 4 This is a chromatogram obtained during the preparation of the optimized test solution of the Xiaoyin granules of the present invention; Figure 5 The chromatogram obtained by optimizing the detection wavelength under the chromatographic conditions of the present invention; Figure 6 The chromatogram obtained by optimizing the column temperature in the chromatographic conditions of the present invention; Figure 7 Chromatogram obtained by optimizing the flow rate in the chromatographic conditions of the present invention Figure 8 The chromatogram obtained by optimizing the mobile phase composition under the chromatographic conditions of the present invention; Fig. 9 A chromatogram obtained by optimizing the elution procedure in the chromatographic conditions of the present invention; Fig.10 is the chromatogram of chlorogenic acid of the present invention; Fig.11 is the gallic acid chromatogram of the present invention; Fig.12 is the chromatogram of hydroxysafflor yellow A of the present invention; Fig.13 This is the chromatogram of paeoniflorin of the present invention; Fig.14 is the rutin chromatogram of the present invention; Fig.15 is the chromatogram of isovitexin of the present invention; Fig.16 This is the chromatogram of 5-O-methylvisaminol glycoside of the present invention; Fig.17 This is the chromatogram of 3,5-di-O-caffeoylquinic acid of the present invention; Fig.18 This is the chromatogram of 4,5-di-O-caffeoylquinic acid of the present invention; Fig.19 is the benzoic acid chromatogram of the present invention; Fig. 20 is the quercetin chromatogram of the present invention; Fig.21 is the chromatogram of paeonol of the present invention; Fig. 22 is the chromatogram of matrine of the present invention; Fig.23 is the chromatogram of the present invention; Fig.24 This is the total ion current diagram of the Xiaoyin capsule of the present invention under the positive ion mode; Fig.25 This is the total ion current diagram of the Xiaoyin capsule of the present invention under the negative ion mode.

[0027] Fig.26 This is the mass spectrum of oxidized paeoniflorin of the present invention; Fig. 27 This is the mass spectrum of paeoniflorin of the present invention; Fig.28 is the mass spectrum of rutin of the present invention; Fig.29 This is the mass spectrum of Vitex quinata of the present invention; Fig.30 This is the mass spectrum of hyperoside of the present invention; Fig.31 is the mass spectrum of verbascoside of the present invention; Fig.32 The mass spectrum of chlorogenic acid / neochlorogenic acid / cryptochlorogenic acid of the present invention; Fig.33 This is the mass spectrum of matrine of the present invention; Fig.34 is the mass spectrum of the present invention; Fig.35 This is the mass spectrum of hydroxysafflor yellow A of the present invention; Fig.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; Fig.37 This is the mass spectrum of the Isogorgonium japonicum of the present invention; Fig.38 is the mass spectrum of gallic acid of the present invention; Fig.39 This is the mass spectrum of the oxymatrine of the present invention; Fig.40 This is the mass spectrum of Angloside C of the present invention; Fig.41 This is the characteristic spectrum of 15 batches of Xiaoyin Capsules tested in the present invention; Fig.42 The characteristic spectra of 15 batches of Xiaoyin granules tested in the present invention; Fig.43 It is the HPLC characteristic fingerprint of the Xiaoyin capsule / granule of the present invention. DETAILED DESCRIPTION

[0028] 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 intended to limit the scope or implementation principles of the present invention. The implementation methods of the present invention are not limited to the following contents. Unless otherwise specified, the test methods used are conventional methods, and the raw materials used are commercially available products. The instruments used in the present invention include: high performance liquid chromatograph (UltiMate 3000, Dionex China Co., Ltd.), chromatographic column (Shim-pack VP-ODS, Dionex China Co., Ltd.), mass spectrometer (6200 series TOF / 6500, Agilent), and the rest are conventional equipment; different batches of Xiaoyin capsule samples and Xiaoyin granule samples used in the present invention are purchased from the market, Xiaoyin capsule is produced by Shaanxi Momed Qixuehe Pharmaceutical Co., Ltd., and Xiaoyin granule is produced by Shaanxi Kanghui Pharmaceutical Co., Ltd. The reagents acetonitrile methanol, phosphoric acid and formic acid used are analytical grade chromatographic grade, and are produced by Tianjin Tianli Chemical Reagent Co., Ltd. and Hangzhou Wahaha Group Co., Ltd.

[0029] 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%), were all purchased from China Food and Drug Inspection Institutes; paeoniflorin (batch number: 22040205, purity: 99.7%) and quercetin (batch number: 21122007, purity: 98.96%) were all purchased from Chengdu Pufeide Biotechnology Co., Ltd. Isovitexin (batch number: PS001065, purity: >98%), benzoic acid (batch number: PS012647, purity:), matrine (batch number: PS000539, purity: >95%) were purchased from Chengdu Pusi Biotechnology Co., Ltd.; 5-O-methylvisaminol glycoside (batch number: 22040205, purity: >99.7%), chlorobactone (batch number: AZBG0405, purity: 98%) were purchased from Chengdu Aifa 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%) were purchased from Jiangsu Yongjian Pharmaceutical Technology Co., Ltd.

[0030] The present invention provides a HPLC characteristic fingerprint of Xiaoyin capsule / granule, and the specific construction method is as follows: S1. Prepare the test sample Xiaoyin capsule / granule solution using different batches of Xiaoyin capsule / granule as test samples; prepare the single reference solution using chlorogenic acid, gallic acid, hydroxysafflor yellow A, paeoniflorin, rutin, isovitexin, 5-O-methylvisaminol glycosides, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, benzoic acid, quercetin, paeonol, matrine, and phellodendron as reference substances; S2, performing HPLC analysis on the test Xiaoyin capsule / granule solution and the reference solution under the same conditions, and recording the corresponding chromatograms; S3, importing the chromatograms of the test sample and the reference sample obtained in S2 into the similarity evaluation system for the chromatographic characteristic spectra of traditional Chinese medicine and performing similarity analysis to confirm the reliability of the results; S4, performing high-resolution mass spectrometry analysis based on the chromatogram of the test sample obtained in S2 to obtain a total ion current diagram; performing data analysis based on the total ion current diagram and the peak conditions of the chromatogram of the test sample obtained in S2 to obtain a mass spectrometry result diagram of each chemical component; S5, comparing the chromatograms of the test sample and the reference sample obtained in S2, the total ion flow diagram obtained in S4, and the mass spectra of each chemical component, to obtain an HPLC characteristic fingerprint of the Xiaoyin capsule / granules consisting of common characteristic peaks.

[0031] Among them, the preparation method of the test solution in S1 is to weigh 1.0 g of the contents of 15 batches of Xiaoyin Capsules respectively, put them into a stoppered conical flask, add 25 mL of pure methanol solution, ultrasonically extract for 30 min, filter, and filter the filtrate with a 0.45 µm microporous filter membrane to obtain the Xiaoyin Capsule test solution.

[0032] The preparation method of the Xiaoyin granule test solution is as follows: 2.0 g of 15 batches of Xiaoyin granules are weighed respectively, placed in a stoppered conical flask, 25 mL of pure methanol solution is added, ultrasonic extraction is performed for 30 min, filtered, the filtrate is evaporated to dryness, 5 mL of methanol is added to the residue to dissolve it, filtered, and then filtered with a 0.45 µm microporous filter membrane to obtain the Xiaoyin granule test solution.

[0033] The preparation method of the standard solution in S2 is to accurately weigh chlorogenic acid, gallic acid, hydroxysafflor yellow A, paeoniflorin, rutin, isovitexin, 5-O-methylvisaminol, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, benzoic acid, quercetin, paeonol, matrine and phellodendron, and dissolve them in pure methanol solution to make each mL contain 88 μg of chlorogenic acid, 75 μg of gallic acid, 96 μg of hydroxysafflor yellow A, 83 μg of paeoniflorin, 68 μg of rutin, 95 μg of isovitexin, 123 μg of 5-O-methylvisaminol, 107 μg of 3,5-di-O-caffeoylquinic acid, 99 μg of 4,5-di-O-caffeoylquinic acid and 85 μg of benzoic acid. μg, a single reference solution containing 79 μg quercetin, 64 μg paeonol, 136 μg matrine, and 142 μg phellodendron.

[0034] The liquid chromatography conditions in S3 are as follows: chromatographic column: Shim-pack VP-ODS (250 mm×4.6 mm, 5 μm); detection wavelength: 225 nm; volume 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, the elution program is shown in Table 1: Table 1: HPLC elution program

[0035] The present invention provides an optimization process for the HPLC characteristic fingerprint detection of the above-mentioned Xiaoyin capsule / granule: (1) Optimization of test solution preparation This example experimentally investigates different extraction methods (ultrasound, reflux, and immersion). Figure 1 As shown in the attached Figure 1 The data show that the chromatogram obtained by ultrasonic extraction has relatively comprehensive components and good separation, so the ultrasonic extraction method is adopted; The extraction effects of different extraction solvents (pure ethanol solution, 70% methanol solution, 50% methanol solution, pure methanol solution) were compared. Figure 2 As shown, it was found that when pure methanol solution was used as the extraction solvent, the extract chromatogram had the most information and the highest component content, so pure methanol solution was selected for extraction.

[0036] The extraction effects of different extraction times (30 min, 45 min, 1 h) were compared. Figure 3 As shown in the figure, it was found that the chromatogram obtained when ultrasonic extraction was performed for 30 min had the highest component content, good peak shape, and high separation, so 30 min was selected as the ultrasonic extraction time.

[0037] Based on the optimization results of the test solution of Xiaoyin Capsule, an experimental comparison of different extraction methods was conducted. Figure 4 As shown, ultrasonic extraction was performed followed by filtration, and the filtrate was evaporated to dryness and reconstituted with methanol before filtration. The components in the chromatogram of the obtained Xiaoyin granule test solution were relatively comprehensive and had good separation.

[0038] (2) Optimization of chromatographic conditions The detection wavelength was investigated using a UV-visible absorption detector, and the chromatograms at 220 nm, 225 nm, 230 nm, 275 nm, and 290 nm were extracted. Figure 5 As shown, when the detection wavelength condition is 225 nm, the chromatogram contains the most comprehensive information and the baseline is stable, so this method is selected as the detection wavelength condition.

[0039] The column temperature (25℃, 30℃, 35℃) was screened. For details, see the attached Figure 6 As shown, when the column temperature is maintained at 30°C, the peak elution is optimal and the separation effect of each component is better, so the column temperature is selected to be 30°C.

[0040] The flow rates (0.6 mL / min, 0.8 mL / min, 1.0 mL / min) were screened. For details, see the attached Figure 7 As shown in the figure, the best peak was obtained when the flow rate was 1.0 mL / min, and the separation effect of each component was better, so the final flow rate was 1.0 mL / min.

[0041] After determining the optimal detection wavelength, column temperature and flow rate, the present invention screened the mobile phase through a large number of experiments. Figure 8 As shown, acetonitrile-0.1% phosphoric acid was finally selected as the mobile phase, and the elution program was optimized. Part of the elution program is as follows: Table 2: Elution Program 1

[0042] Table 3: Elution Program 2

[0043] Table 4: Elution Program 3

[0044] Table 5: Elution Program 4

[0045] Table 6: Elution Program 5

[0046] Table 7: Elution Program 6

[0047] Test results such as Fig. 9 As shown, elution program 6 has good separation, high peak height, stable baseline and complete chromatographic information. Therefore, elution program 6 is selected as the optimal elution program.

[0048] Example 1 This embodiment provides a method for constructing an HPLC characteristic spectrum of Xiaoyin capsules / granules.

[0049] S1. Preparation of test solution: 1.0 g of the contents of 15 batches of Xiaoyin Capsules (Xiaoyin Capsules are produced by Shaanxi Momed Qixuehe Pharmaceutical Co., Ltd., batch numbers: 230501, 230306, 230704, 230902, 231201, 221103, 220806, 220701, 220502, 220307, ​​211201, 211008, 210804, 210607, 210305) were weighed respectively, placed in a stoppered conical flask, added with 25 mL of pure methanol solution, ultrasonically extracted for 30 min, filtered, and the filtrate was filtered through a 0.45 µm microporous filter membrane to obtain the Xiaoyin Capsule test solution.

[0050] 2.0 g of 15 batches of Xiaoyin Granules (Xiaoyin Granules are produced by Shaanxi Kanghui Pharmaceutical Co., Ltd., batch numbers: 230304, 230405, 230801, 201205, 201019, 210708, 210501, 230801, 210304, 210808, 210607, 221108, 220604, 220306, 220901) were weighed respectively, placed in a stoppered conical flask, added with 25 mL of pure methanol solution, ultrasonically extracted for 30 min, filtered, the filtrate was evaporated to dryness, 5 mL of methanol was added to the residue to dissolve it, filtered, and then filtered with a 0.45 µm microporous filter membrane to obtain the Xiaoyin Granule test solution.

[0051] S2. Preparation of reference solution Chlorogenic acid, gallic acid, hydroxysafflor yellow A, paeoniflorin, rutin, isovitexin, 5-O-methylvisaminol, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, benzoic acid, quercetin, paeonol, matrine, and phellodendron were accurately weighed and dissolved in pure methanol solution. Each mL contained 88 μg of chlorogenic acid, 75 μg of gallic acid, 96 μg of hydroxysafflor yellow A, 83 μg of paeoniflorin, 68 μg of rutin, 95 μg of isovitexin, 123 μg of 5-O-methylvisaminol, 107 μg of 3,5-di-O-caffeoylquinic acid, 99 μg of 4,5-di-O-caffeoylquinic acid, 85 μg of benzoic acid, and 79 μg of quercetin. μg, a single reference solution containing 64 μg of paeonol, 136 μg of matrine, and 142 μg of phellodendron.

[0052] S3. Chromatographic analysis The test solution in S1 and the reference solution in S2 were injected into a high performance liquid chromatograph for chromatographic analysis, and the detection was performed under the same conditions, using an UltiMate 3000 chromatograph. The liquid chromatography conditions were as follows: chromatographic column: Shim-pack VP-ODS (250 mm×4.6 mm, 5 μm); detector: diode array detector, detection wavelength 225 nm; volume 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, using the preferred elution program 6 parameters, the chromatograms of different single reference solutions are shown in Figure 2. Figure 10~Figure 23 shown.

[0053] S4. High performance liquid chromatography-mass spectrometry analysis According to the chromatogram of the Xiaoyin capsule / granule test solution and the chromatogram of the single reference solution obtained in S3, the Xiaoyin capsule / granule test solution was analyzed by high-resolution mass spectrometry. The high-resolution mass spectrometry detection conditions were: electrospray ionization, spray voltage 3500 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 300°C, auxiliary gas temperature 300°C, full scan mode, mass-to-charge ratio scanning range m / z 100-1500, and the total ion current ( Fig.24 and Fig.25 ). The total ion flow chart was imported into Xcalibur software, and the mass spectrum result chart of each chemical component was obtained according to the peak of the chromatogram of the test solution obtained in S3 ( Figure 26~Figure 40 ).

[0054] A total of 20 chemical components were analyzed by high-resolution mass spectrometry analysis of the material benchmark of Xiaoyin Capsule. Combined with the HPLC-MS component analysis results of extracts of 13 medicinal materials in the preparation prescription, including Rehmannia root, Paeonia suffruticosa, Paeonia lactiflora, Angelica sinensis, Sophora flavescens, Honeysuckle, Scrophularia ningpoensis, Arctium lappa, Periostracum cicadae, Dictamni root, Saposhnikovia divaricata, Folium Isatidis, and Carthamus tinctorius, the 20 chemical components were attributed and analyzed, covering 11 medicinal materials. The results showed that paeoniflorin was a common chemical component of Paeonia suffruticosa and Paeonia lactiflora; gallic acid was a common chemical component of Paeonia lactiflora, Angelica sinensis, and Carthamus tinctorius; verbascoside was a common chemical component of Rehmannia root and Scrophularia ningpoensis; green Original acid is a common chemical component of Angelica sinensis, honeysuckle and arctium fruit; paeoniflorin is a common chemical component of honeysuckle and isatis indigotica; rutin is a common chemical component of honeysuckle and safflower; oxidized paeoniflorin comes from Paeonia suffruticosa bark; matrine and normatrine come from Sophora flavescens; 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, neochlorogenic acid and isochlorogenic acid C come from honeysuckle; angoside C comes from Scrophularia ningpoensis; isochlorogenic acid A and cryptochlorogenic acid come from Arctium fruit; phellodendron ketone comes from Dictamni cortex; isogerminin and isovitexin come from isatis indigotica; hydroxysafflor yellow A comes from safflower.

[0055] S5. Reliability confirmation The 15 batches of Xiaoyin capsule test solutions obtained in S3 were introduced into the Chinese medicine chromatographic characteristic spectrum similarity evaluation system (2012 A version); the chromatograms of 15 batches of Xiaoyin capsule / granule test solutions ( Figure 41-42 ) were taken as common peaks, and the control spectrum of Xiaoyin Capsule was generated by the average value calculation method, and the relative retention time and relative peak area of ​​each common peak were calculated; similarity analysis was performed after data import, multi-point correction and data matching; the similarity result table between the chromatograms of the test solutions of Xiaoyin Capsule from different batches and the common peak patterns was obtained and exported; the reliability of the results was confirmed based on the similarity result table and the chromatogram of the test solution of Xiaoyin Capsule.

[0056] S6, Xiaoyin capsule / granule characteristic fingerprint According to the chromatogram of the test solution of Xiaoyin capsule / granules and the chromatogram of the single reference solution obtained in S3, combined with the total ion flow obtained by high-resolution mass spectrometry and the mass spectrum results of chemical components, chlorogenic acid (peak 4) was used as the reference peak, and peak 1 in the chromatogram was identified as gallic acid, peak 5 as hydroxysafflor yellow A, peak 6 as paeoniflorin, peak 8 as rutin, peak 9 as isovitexin, peak 10 as 5-O-methylvisaminol glycoside, peak 11 as 3,5-di-O-caffeoylquinic acid, peak 12 as 4,5-di-O-caffeoylquinic acid, peak 13 as benzoic acid, peak 15 as quercetin, peak 19 as paeonol, peak 21 as matrine, and peak 22 as phellodendron, and the characteristic fingerprint of Xiaoyin capsule / granules was obtained, see Appendix. Fig.43 shown.

[0057] 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 medicinal materials including Rehmannia glutinosa, Paeonia suffruticosa, Paeonia lactiflora, Angelica sinensis, Sophora flavescens, Honeysuckle, Scrophularia ningpoensis, Arctium lappa, Periostracum Cicadae, Dictamni, Saposhnikovia divaricata, Folium Isatidis, and Carthamus tinctorius in the preparation prescription in the early stage of the present invention, the chemical components corresponding to the 14 characteristic peaks in the characteristic spectrum of Xiaoyin Capsules (granules) were attributed and analyzed. Among them, gallic acid (peak 1) is a common peak of red peony root, Chinese angelica root and safflower; chlorogenic acid (peak 4) is a common peak of Chinese angelica root and arctium fruit; rutin (peak 8) is a common peak of honeysuckle and safflower; paeoniflorin (peak 6) is derived from red peony root; paeonol (peak 19) is derived from peony bark; matrine (peak 21) is derived from sophora flavescens; 3,5-di-O-caffeoylquinic acid (peak 11) and 4,5-di-O-caffeoylquinic acid (peak 12) are derived from honeysuckle; phellodendron chinense (peak 22) is derived from Dictamni; 5-O-methylvisamin glycoside (peak 10) is derived from Saposhnikovia divaricata; isovitexin (peak 9) is derived from Isatis indigotica; hydroxysafflor yellow A (peak 5) and quercetin (peak 15) are derived from safflower.

[0058] Example 2: Methodological study of characteristic spectrum detection method (1) Similarity study At the same time, the present invention uses the automatically generated reference spectrum R to generate a common chromatographic peak pattern. Analysis and calculation show that the common chromatographic peaks of 15 batches of Xiaoyin Capsules and Xiaoyin Granules have relatively good similarity, indicating that the characteristic spectrum 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 Tables 8-9.

[0059] Table 8: Similarity between each batch of Xiaoyin capsule samples and the common chromatographic peak pattern

[0060] Table 9: Similarity between each batch of Xiaoyin granule samples and the common chromatographic peak pattern

[0061] (2) Precision study The test solution prepared above was taken and analyzed according to the optimized detection method. The sample was injected 6 times in parallel with an injection volume of 10 μL. Chlorogenic acid (peak 4) was used as the reference peak. The peak area and retention time of the common peak were analyzed and the RSD value was calculated. See Table 10 for details. It can be seen from the data in Table 10 that the RSD of the relative retention time is less than 0.87%, and the RSD of the relative peak area is less than 2.73%, indicating that the parallel injection precision of the equipment is good.

[0062] Table 10: Peak area and retention time of each batch of Xiaoyin granule samples and common peaks and calculated RSD values

[0063] (3) Stability study The prepared test solution was taken and analyzed according to the optimized detection method. The sample was injected at different times of 0, 2, 6, 12, 18, and 24 h, and the injection volume was 10 μL. Chlorogenic acid (peak 4) was used as the reference peak. The peak area and retention time of the common peak of the HPLC characteristic spectrum of the sample were analyzed and the RSD value was calculated. See Table 11 for details. The results showed that the RSD of the relative retention time was less than 0.66%, and the RSD of the relative peak area was less than 1.88%, indicating that the chromatographic peak of the Xiaoyin Capsule test solution within 24 h had almost no change and good stability.

[0064] Table 11: Peak area and retention time of common peaks of Xiaoyin granules samples at different times and calculated RSD values

[0065] (4) Reproducibility study Six batches of sample solutions were prepared according to the above method. With reference to the optimal chromatographic conditions, the injection volume was 10 μL, and chlorogenic acid (peak 4) was used as the reference peak. The peak area and retention time of the common peaks of the sample HPLC characteristic spectrum were analyzed and the RSD value was calculated, as shown in Table 12. The results showed that the RSD of the relative retention time was less than 0.86%, and the RSD of the relative peak area was less than 2.72%, indicating that the sample chromatographic peak reproducibility was good and the repeatability of the method was good.

[0066] Table 12: Peak area and retention time of common peaks of each batch of Xiaoyin granule samples and calculated RSD value

[0067] The above experimental results indicate that the characteristic spectrum construction method of Xiaoyin capsules / granules provided by the present invention has the characteristics of good stability, high precision and good repeatability, and can comprehensively and objectively evaluate the quality of Xiaoyin capsules / granules, providing quality assurance for clinical efficacy.

[0068] The above embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention. The protection scope of the present invention is defined by the claims. The above contents are only used as examples and explanations of the concept of the present invention. Various modifications or supplements made by technicians in the technical field to the described specific embodiments or replacements in similar ways shall fall within the protection scope of the present invention as long as they do not deviate from the concept of the invention or exceed the scope defined by the claims.

Claims

1. A method for constructing a characteristic fingerprint of Xiaoyin capsules / granules by HPLC, characterized in that: include: S1, using different batches of Xiaoyin capsules / granules as test samples to prepare test sample Xiaoyin capsules / granules solutions; Using chlorogenic acid, gallic acid, hydroxysafflor yellow A, paeoniflorin, rutin, isovitexin, 5-O-methylvisaminol glycosides, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, benzoic acid, quercetin, paeonol, matrine, and phellodendron as reference substances, a single reference substance solution was prepared; S2, performing HPLC analysis on the test Xiaoyin capsule / granule solution and the reference solution under the same conditions, and recording the corresponding chromatograms; S3, importing the chromatograms of the test sample and the reference sample obtained in S2 into the similarity evaluation system for the chromatographic characteristic spectra of traditional Chinese medicine and performing similarity analysis to confirm the reliability of the results; S4, performing high-resolution mass spectrometry analysis based on the chromatogram of the test sample obtained in S2 to obtain a total ion current diagram; performing data analysis based on the total ion current diagram and the peak conditions of the chromatogram of the test sample obtained in S2 to obtain a mass spectrometry result diagram of each chemical component; S5, comparing the chromatograms of the test sample and the reference sample obtained in S2, the total ion flow diagram obtained in S4, and the mass spectra of each chemical component, to obtain an HPLC characteristic fingerprint of the Xiaoyin capsule / granules consisting of common characteristic peaks.

2. The method for constructing the HPLC characteristic fingerprint of Xiaoyin capsule / granule according to claim 1, characterized in that: In S1, the contents of different batches of Xiaoyin capsules / granules were taken, methanol solution was added, ultrasonic extraction was performed, and microporous filter membrane was used to obtain the test sample Xiaoyin capsule / granule solution.

3. The method for constructing the HPLC characteristic fingerprint of Xiaoyin capsule / granule according to claim 1, characterized in that: In S2, the HPLC detection wavelength is 210-290 nm, the column temperature is 25-35° C., and the flow rate is 0.6-1.0 mL / min.

4. The method for constructing the HPLC characteristic fingerprint of Xiaoyin capsule / granule according to claim 1, characterized in that: In S2, the HPLC determination conditions are: 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: 。 5. The method for constructing the HPLC characteristic fingerprint of Xiaoyin capsule / granule according to claim 1, characterized in that: S3 specifically includes: importing the chromatograms of the test solutions of different batches of Xiaoyin capsules / granules obtained in S2 into the similarity evaluation system of traditional Chinese medicine chromatographic characteristic spectra; selecting the chromatographic peaks existing in the chromatograms of the test solutions of different batches of Xiaoyin capsules / granules as common peaks, generating a control spectrum of Xiaoyin capsules / granules by the average value calculation method, and calculating the relative retention time and relative peak area of ​​each common peak; performing similarity analysis after data import, multi-point correction and data matching; obtaining and exporting the similarity result table between the chromatograms of the test solutions of Xiaoyin capsules from different batches and the common peak pattern; confirming the reliability of the results based on the similarity result table and the chromatogram of the test solution of Xiaoyin capsules.

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

7. The method for constructing the HPLC characteristic fingerprint of Xiaoyin capsule / granule according to claim 1, characterized in that: S5 is specifically as follows: according to the chromatograms of the test sample and the reference sample obtained in S3, combined with the total ion flow diagram obtained by high-resolution mass spectrometry in S5 and the mass spectrum result diagram of the chemical components, comparison is performed, and peak 4 chlorogenic acid is used as the reference peak. Peak 1 in the chromatogram is identified as gallic acid, peak 5 as hydroxysafflor yellow A, peak 6 as paeoniflorin, peak 8 as rutin, peak 9 as isovitexin, peak 10 as 5-O-methylvisaminol glycoside, peak 11 as 3,5-di-O-caffeoylquinic acid, peak 12 as 4,5-di-O-caffeoylquinic acid, peak 13 as benzoic acid, peak 15 as quercetin, peak 19 as paeonol, peak 21 as matrine, and peak 22 as phellodendron to obtain the characteristic spectrum of Xiaoyin capsules / granules.

8. The method for constructing the HPLC characteristic fingerprint of Xiaoyin capsules / granules according to claim 1, characterized in that: After obtaining the characteristic spectrum of Xiaoyin capsules / granules, attribute analysis is performed on the characteristic spectrum results of Xiaoyin capsules / granules.

9. The HPLC characteristic fingerprint of Xiaoyin capsule / granules obtained according to the construction method according to any one of claims 1 to 8.

10. A method for detecting the quality of Xiaoyin capsules / granules, characterized in that: The quality of the Xiaoyin capsule / granule sample is detected by using the HPLC characteristic fingerprint of the Xiaoyin capsule / granule obtained by the construction method described in any one of claims 1 to 8.

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