Method for constructing specific chromatogram of Shuangjin gastrointestinal capsule

Abstract

The invention discloses a construction method of a specific chromatogram of a Shuangjin gastrointestinal capsule, and belongs to the technical field of quality control and management of compound traditional Chinese medicine preparations. The preparation method comprises the following steps: taking different batches of Shuangjin gastrointestinal capsules as test samples, and taking chlorogenic acid, hesperidin, daidzein, nardosinone, caffeic acid, linarin, dehydrocostus lactone, rutaecarpin, limonin, nobiletin, tangeretin, epiberberine, genistin, daidzein, succinic acid and uridine as reference substances; high performance liquid chromatography and mass spectrometry are combined for detection, and the specific chromatogram of the double-jin gastrointestinal capsule is obtained. The method is simple and easy to implement, high in precision, good in reproducibility, accurate and reliable, at least 16 components are confirmed at a time under the same chromatographic condition, time and cost are saved, and the detection efficiency is improved; the method can accurately, clearly, objectively, quickly and comprehensively monitor the quality of a plurality of characteristic components of the Shuangjin stomach and intestine capsule at the same time, and has important effects and values for controlling the quality of the Shuangjin stomach and intestine capsule and ensuring the clinical curative effect.

Description

Technical Field

[0001] The invention belongs to the technical field of quality control management of compound Chinese medicine preparations, and particularly relates to a method for constructing a characteristic spectrum of Shuangjin gastrointestinal capsules. Background Art

[0002] Chinese patent medicine is made into dosage forms with certain specifications according to the prescribed prescriptions and standards under the guidance of traditional Chinese medicine theory, using Chinese herbal medicine slices as raw materials. It inherits the theory and experience of traditional Chinese medicine and absorbs the advanced technology of modern pharmaceutical industry. It has the characteristics of accurate dosage, convenient use and storage, and rich dosage forms. The main ingredients of Shuangjin Gastrointestinal Capsule (National Medicine Standard No.: Z20040055) include natural Chinese herbal medicines such as sea cucumber, white schizonepeta, Chuandongzi, Corydalis, Coptis chinensis, Evodia rutaecarpa, Cyperus rotundus, Nardostachys grandiflora, Curcuma aromatica, Curcuma zedoaria, Tangerine peel, Pinellia tuber, and Gallus gallus domesticus. These ingredients are scientifically proportioned and play a variety of functions such as soothing the liver and stomach, reducing acid and relieving pain. Shuangjin Gastrointestinal Capsule effectively relieves and treats symptoms of common gastrointestinal diseases such as gastric and duodenal ulcers, chronic gastritis, stomach bloating and stomach pain, inhalation hiccups, noisy pantothenic acid, and loss of appetite by inhibiting gastric acid secretion, promoting gastrointestinal motility, and protecting gastric mucosa.

[0003] Currently, there are few quality inspection methods for Shuangjin Gastrointestinal Capsules, and most of them are studies on the composition of a single herb in the prescription, which cannot reflect the overall composition information of Shuangjin Gastrointestinal Capsules. Summary of the invention

[0004] In order to overcome the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a method for constructing a characteristic spectrum of Shuangjin Gastrointestinal Capsules, so as to solve the technical problem that the existing quality detection method cannot reflect the overall composition information of Shuangjin Gastrointestinal Capsules.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: The first aspect of the present invention discloses a method for constructing a characteristic spectrum of Shuangjin gastrointestinal capsule, comprising the following steps: S1. Different batches of Shuangjin Gastrointestinal Capsules were used as test samples, and chlorogenic acid, hesperidin, daidzein, naringenin, caffeic acid, montanol, dehydrocostus lactone, evodiamine, limonin, nobiletin, tangeretin, epiberberine, genistin, glycitein, succinic acid and uridine were used as reference substances; S2. Perform HPLC analysis on the test sample and reference sample under the same conditions and record the corresponding chromatograms; S3, perform similarity analysis on the chromatogram of the test sample obtained in S2 to confirm the reliability of the result; S4, performing high-resolution mass spectrometry analysis 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. Compare the chromatogram of the test sample and the chromatogram of the reference sample obtained in S2, the total ion current obtained in S4, and the mass spectra of each chemical component to obtain a characteristic spectrum of Shuangjin Gastrointestinal Capsule.

[0006] Preferably, in S1, the contents of Shuangjin Gastrointestinal Capsules from different batches are weighed respectively, methanol solution is added, and reflux extraction is performed to obtain Shuangjin Gastrointestinal Capsules test solution.

[0007] Further preferably, every 30 mL of methanol solution contains 2.0 g of Shuangjin Gastrointestinal Capsule contents.

[0008] Preferably, the methanol solution is a pure methanol solution.

[0009] Preferably, the extraction is performed under reflux for 1 h.

[0010] Preferably, in S1, chlorogenic acid, hesperidin, daidzein, naringenone, caffeic acid, montanol, dehydrocostus lactone, evodiamine, limonin, nobiletin, tangeretin, epiberberine, genistein, glycitein, succinic acid and uridine are respectively taken and dissolved in methanol solution to obtain a single reference solution.

[0011] Further preferably, in the single reference solution of chlorogenic acid, 0.65 mg of chlorogenic acid is contained in each 1 mL of methanol solution; in the single reference solution of hesperidin, 0.46 mg of hesperidin is contained in each 1 mL of methanol solution; in the single reference solution of naringenin, 0.67 mg of naringenin is contained in each 1 mL of methanol solution; in the single reference solution of daidzein, 0.60 mg of daidzein is contained in each 1 mL of methanol solution; in the single reference solution of caffeic acid, 0.75 mg of caffeic acid is contained in each 1 mL of methanol solution; in the single reference solution of limonin, 0.72 mg of limonin is contained in each 1 mL of methanol solution; in the single reference solution of dehydrocostus lactone, 0.78 mg of dehydrocostus lactone is contained in each 1 mL of methanol solution; in the single reference solution of evodiamine, 0.49 mg of evodiamine is contained in each 1 mL of methanol solution; in the single reference solution of limonin, 0.55 mg limonin; in the single reference solution of nobiletin, each 1 mL of methanol solution contains 0.59 mg nobiletin; in the single reference solution of tangeretin, each 1 mL of methanol solution contains 0.55 mg tangeretin; in the single reference solution of epiberberine, each 1 mL of methanol solution contains 0.65 mg epiberberine; in the single reference solution of genistin, each 1 mL of methanol solution contains 0.97 mg genistin; in the single reference solution of glycitein, each 1 mL of methanol solution contains 0.93 mg glycitein; in the single reference solution of succinic acid, each 1 mL of methanol solution contains 0.57 mg succinic acid; in the single reference solution of uridine, each 1 mL of methanol solution contains 0.72 mg uridine.

[0012] Preferably, in S2, the detection wavelength is 220 nm.

[0013] Preferably, in S2, the column temperature is 30°C.

[0014] Preferably, in S2, the flow rate is 0.6 mL / min.

[0015] Preferably, in S2, the mobile phase is acetonitrile-0.1% phosphoric acid.

[0016] Preferably, in S2, the gradient elution procedure is as follows: .

[0018] Preferably, S3 specifically comprises: importing the test sample chromatogram obtained in S2 into a similarity evaluation system for characteristic chromatograms of traditional Chinese medicine, selecting chromatographic peaks existing in chromatograms of different batches of test samples as common peaks, generating a control spectrum of Shuangjin Gastrointestinal Capsules by using an 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 a similarity result table between chromatograms of different batches of test samples and the common peak pattern; and confirming the reliability of the result based on the similarity result table and the test sample chromatogram.

[0019] Preferably, in S4, the detection conditions of the high-resolution mass spectrometry are: electrospray ionization, the spray voltage is 3500 V, the sheath gas flow rate is 40 arb, the auxiliary gas flow rate is 10 arb, the capillary temperature is 300°C, the auxiliary gas temperature is 300°C, the scanning mode is the full scan mode, and the mass-to-charge ratio scanning range m / z is 100-1500.

[0020] Preferably, S5 is specifically: according to the chromatogram of the test sample and the chromatogram of the reference substance obtained in S2, in combination with the total ion flow diagram obtained by high-resolution mass spectrometry in S4 and the mass spectrum result diagram of the chemical components, comparison is performed, and peak 2 uridine is used as the reference peak, and peak 3 in the chromatogram is identified as succinic acid, peak 6 as naringenone, peak 7 as chlorogenic acid, peak 10 as caffeic acid, peak 15 as epiberberine, peak 16 as genistein, peak 17 as hesperidin, peak 24 as daidzein, peak 25 as limonene, peak 27 as glycitein, peak 34 as limonin, peak 35 as nobiletin, peak 39 as tangeretin, peak 40 as evodia rutaecarpine, and peak 42 as dehydrocostus lactone, to obtain the characteristic spectrum of Shuangjin Gastrointestinal Capsules.

[0021] Preferably, after obtaining the characteristic spectrum of Shuangjin Gastrointestinal Capsule, attribution analysis is performed on the characteristic spectrum results of Shuangjin Gastrointestinal Capsule.

[0022] The second aspect of the present invention discloses a characteristic spectrum of Shuangjin gastrointestinal capsule constructed by the above method.

[0023] The third aspect of the present invention discloses a method for identifying Shuangjin Gastrointestinal Capsules, which is performed using the characteristic spectrum of Shuangjin Gastrointestinal Capsules obtained by the above-mentioned construction method.

[0024] Compared with the prior art, the present invention has the following beneficial effects: The method for constructing the characteristic spectrum of Shuangjin Gastrointestinal Capsule provided by the present invention adopts a method of combining high performance liquid chromatography with mass spectrometry, and systematically and reasonably controls the chromatographic conditions, and uses high performance liquid chromatography and selected reference substances to identify 16 characteristic components, which can be used to characterize the medicinal taste of cuttlebone, white schizonepeta, Chuandongzi, Corydalis, Coptis chinensis, Evodia rutaecarpa, Cyperus rotundus, Nardostachys grandiflora, Curcuma aromatica, Curcuma zedoaria, Tangerine peel, Pinellia tuber and Gallus gallus domestica, and are respectively: caffeic acid (peak 10) is derived from white schizonepeta; epiberberine (peak 15) is derived from Coptis chinensis; dehydrocostus lactone (peak 42), evodia rutaecarpine (peak 40), limonene (peak 41), linalool (peak 43), linalool (peak 44), linalool (peak 45), linalool (peak 46), linalool (peak 47), linalool (peak 48), linalool (peak 49), linalool (peak 50), linalool (peak 51), linalool (peak 52), linalool (peak 53), linalool (peak 54), linalool (peak 55), linalool (peak 57), linalool (peak 58), linalool (peak 59), linalool (peak 51), linalool (peak 52), linalool (peak 53), linalool (peak 54), linalool (peak 55), linalool (peak 56), linalool (peak 57), Chlorogenic acid (peak 7), nobiletin (peak 6), and montmorillonin (peak 25) are derived from nardus; hesperidin (peak 20), nobiletin (peak 35), and tangeretin (peak 39) are derived from dried orange peel; succinic acid (peak 3) and uridine (peak 2) are derived from pinellia tuber; daidzein (peak 26), genistein (peak 17), and glycitein (peak 27) are derived from chicken gizzard lining. By simultaneously identifying 16 common peaks, the full picture of the characteristic spectrum of Shuangjin Gastrointestinal Capsules is formed, and the purpose of controlling the original medicinal materials in Shuangjin Gastrointestinal Capsules is achieved. The method and the characteristic spectrum constructed are simple and easy, with high precision, good reproducibility, accuracy and reliability. At least 16 components can be confirmed at one time under the same chromatographic conditions, which saves time, saves inspection costs and improves inspection efficiency. The method can accurately, clearly, objectively, quickly, comprehensively and simultaneously monitor the quality of multiple characteristic components of Shuangjin Gastrointestinal Capsules, which not only improves the quality control level of medicinal materials such as cuttlebone, Bletilla striata, and Chuandongzi, but also improves the intrinsic quality of Shuangjin Gastrointestinal Capsules and reduces the inspection workload. The method maintains the consistency of the quality of cuttlebone, Bletilla striata, Chuandongzi, Corydalis yanhusuo, Coptis chinensis, Evodia rutaecarpa, Cyperus rotundus, Nardostachys grandiflora, Curcuma aromatica, Curcuma zedoaria, Tangerine peel, Pinellia ternata, and Gallus gallus domesticus with Shuangjin Gastrointestinal Capsules, provides raw materials that meet the requirements of Shuangjin Gastrointestinal Capsules for clinical use, provides a guarantee for the safety of Shuangjin Gastrointestinal Capsules, and plays an important role and value in controlling the quality of Shuangjin Gastrointestinal Capsules and ensuring clinical efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a chromatogram obtained by optimizing the extraction method during the preparation of the test solution 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 present invention; Figure 3 This is a chromatogram obtained by optimizing the extraction time during the preparation of the test solution of the present invention; Figure 4 The chromatogram obtained by optimizing the detection wavelength under the chromatographic conditions of the present invention; Figure 5 The chromatogram obtained by optimizing the column temperature in the chromatographic conditions of the present invention; Figure 6The chromatogram obtained by optimizing the flow rate under the chromatographic conditions of the present invention; Figure 7 The chromatogram obtained by optimizing the mobile phase composition under the chromatographic conditions of the present invention; Figure 8 A chromatogram obtained by optimizing the elution procedure in the chromatographic conditions of the present invention; Fig. 9 is the chromatogram of epignadone of the present invention; Fig.10 This is the chromatogram of epimerin of the present invention; Fig.11 is the chromatogram of glycitein of the present invention; Fig.12 is the chromatogram of epiberberine of the present invention; Fig.13 is the chromatogram of hesperidin of the present invention; Fig.14 is the chromatogram of nobiletin of the present invention; Fig.15 is a chromatogram of daidzein of the present invention; Fig.16 is the succinic acid chromatogram of the present invention; Fig.17 This is the chromatogram of tangeretin of the present invention; Fig.18 is the caffeic acid chromatogram of the present invention; Fig.19 is the uridine chromatogram of the present invention; Fig. 20 is the chromatogram of limonin of the present invention; Fig.21 This is the chromatogram of dehydrocostus lactone of the present invention; Fig. 22 It is the chromatogram of genistin of the present invention; Fig.23 This is the chromatogram of Evodia rutaecarpa alkaloids of the present invention; Fig.24 is the chromatogram of chlorogenic acid of the present invention; Fig.25 This is the mass spectrum of the Shuangjin gastrointestinal capsule of the present invention in positive ion mode; Fig.26 This is the mass spectrum of the Shuangjin gastrointestinal capsule of the present invention in the negative ion mode.

[0026] Fig. 27 is the mass spectrum of syringic acid of the present invention; Fig.28 is the mass spectrum of cinnamic acid of the present invention; Fig.29 is the mass spectrum of genistin of the present invention; Fig.30 This is the mass spectrum of limonene glycosides of the present invention; Fig.31 is the mass spectrum of caffeic acid of the present invention; Fig.32 This is the mass spectrum of chuandongsu / isochuandongsu of the present invention; Fig.33 This is the mass spectrum of naringenone of the present invention; Fig.34 This is the hesperidin spectrum of the present invention; Fig.35 This is the mass spectrum of dehydrocostus lactone of the present invention; Fig.36 This is the mass spectrum of chlorogenic acid of the present invention; Fig.37 is the mass spectrum of bamipine of the present invention; Fig.38 This is the mass spectrum of protocatechuic acid of the present invention; Fig.39 This is the mass spectrum of lemon bitterness of the present invention; Fig.40 This is the mass spectrum of pomelo peel of the present invention; Fig.41 is the mass spectrum of p-hydroxybenzoic acid of the present invention; Fig.42 is the mass spectrum of uracil of the present invention; Fig.43 This is the mass spectrum of Evodia rutaecarpine of the present invention; Fig.44 This is the characteristic spectrum of 15 batches of Shuangjin Gastrointestinal Capsules test samples of the present invention; Fig.45 This is the characteristic spectrum of Shuangjin Gastrointestinal Capsule. DETAILED DESCRIPTION

[0027] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific words used in the text are the common meanings understood by those skilled in the art for the present invention. When there is a conflict, the definition in this specification shall prevail. The theories or mechanisms described and disclosed herein, whether right or wrong, should not limit the scope of the present invention in any way, that is, the content of the present invention can be implemented without being limited by any specific theory or mechanism.

[0028] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

[0029] The present invention provides a method for constructing a characteristic spectrum of Shuangjin gastrointestinal capsule, comprising the following steps: S1. Weigh appropriate amounts of the contents of Shuangjin Gastrointestinal Capsules from different batches, add methanol solution, and reflux for extraction to obtain Shuangjin Gastrointestinal Capsules test solution; S2, dissolving chlorogenic acid, hesperidin, daidzein, naringenone, caffeic acid, montanol, dehydrocostus lactone, evodiamine, limonin, nobiletin, tangeretin, epiberberine, genistein, glycitein, succinic acid, and uridine in methanol respectively to obtain corresponding single reference substance solutions; S3, inject the test solution in S1 and the reference solution in S2 into a high performance liquid chromatograph for chromatographic analysis, perform detection under the same conditions, and record the corresponding chromatograms; S4, perform high-resolution mass spectrometry analysis on the test solution to obtain a total ion current diagram. The total ion current diagram is imported into Xcalibur software, and data analysis is performed based on the peaks of the chromatogram of the test solution obtained in S3 to obtain the mass spectrum result diagram of each chemical component, and the mass spectrum results are attributed and analyzed; S5. Import the chromatograms of the test solutions of different batches of Shuangjin Gastrointestinal Capsules obtained in S3 into the Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System (2012 version); select the chromatographic peaks existing in the chromatograms of the test solutions of 15 batches of Shuangjin Gastrointestinal Capsules as common peaks, generate the control spectrum of Shuangjin Gastrointestinal Capsules by the average value calculation method, and calculate the relative retention time and relative peak area of ​​each common peak; perform similarity analysis after data import, multi-point correction and data matching; obtain and export the similarity result table between the chromatograms of the test solutions of different batches of Shuangjin Gastrointestinal Capsules and the common peak pattern; confirm the reliability of the results based on the similarity result table and the chromatogram of the test solution of Shuangjin Gastrointestinal Capsules; S6. Based on the chromatogram of the Shuangjin Gastrointestinal Capsule test solution and the chromatogram of the single reference solution obtained in S3, the total ion flow diagram obtained by high-resolution mass spectrometry and the mass spectrum result diagram of the chemical components are compared to obtain a characteristic spectrum of the Shuangjin Gastrointestinal Capsule. The characteristic spectrum results of the Shuangjin Gastrointestinal Capsule are subjected to attribution analysis to determine the chemical components of each peak in the characteristic spectrum.

[0030] The present invention provides an optimization process for detecting the characteristic spectrum of the Shuangjin gastrointestinal capsule: S1. Optimization of test solution preparation: The present invention experimentally investigated different extraction methods (ultrasound, reflux, immersion), and the experimental results are as follows Figure 1 shown.

[0031] The results showed that the chromatogram components of reflux extraction were relatively comprehensive and had good separation, so reflux extraction was selected as the method for further operation; The present invention compares the extraction effects of different extraction solvents (50% methanol solution, 70% methanol solution, pure methanol solution, water, ethanol), and the experimental results are as follows: Figure 2 shown.

[0032] The results show that when pure methanol solution is used as the extraction solvent, the extract chromatogram has the most information and the highest component content, so pure methanol solution is selected for extraction; This paper compares the extraction effects of different extraction times (30 min, 45 min, 1 h). The experimental results are as follows: Figure 3 shown.

[0033] The results show that the chromatogram obtained when the reflux extraction is performed for 1 h has the highest component content, the best peak shape, and high separation, so 1 h is selected as the reflux extraction time.

[0034] S2. Optimize the chromatographic conditions: The present invention uses a diode array detector to investigate the detection wavelength, and scans the sample at 210 nm, 220 nm, 230 nm, and 245 nm wavelengths. The experimental results are as follows: Figure 4 shown.

[0035] The results show that the chromatogram contains the most comprehensive information and a stable baseline when the detection wavelength is 220 nm, so this method is selected as the detection wavelength condition; The present invention screened the column temperature (25°C, 30°C, 35°C), and the experimental results were as follows: Figure 5 shown.

[0036] The results showed that the peak elution was best when the column temperature was maintained at 30°C, and the separation effect of each component was better, so the column temperature was finally selected to be 30°C; The present invention screened the flow rates (0.6 mL / min, 0.8 mL / min, 1.0 mL / min), and the experimental results were as follows: Figure 6 shown.

[0037] The results showed that the peak was best when the flow rate was maintained at 0.6 mL / min, and the separation effect of each component was good, so the flow rate was finally selected as 0.6 mL / min; The present invention compares the elution effects of different elution systems such as acetonitrile-0.1% phosphoric acid, methanol-water, acetonitrile-water, methanol-0.1% phosphoric acid under different gradients. The experimental results are as follows: Figure 7 shown.

[0038] The results showed that when acetonitrile-0.1% phosphoric acid was used as the mobile phase, the separation effect of the components in Shuangjin Gastrointestinal Capsules was better, so acetonitrile-0.1% phosphoric acid was finally selected as the mobile phase.

[0039] After determining the optimal mobile phase composition, the present invention screened the best gradient elution program through a large number of experiments, and some elution programs are shown in Tables 1 to 6: Table 1 Elution procedure 1

[0040] Table 2 Elution procedure 2

[0041] Table 3 Elution procedure 3

[0042] Table 4 Elution procedure 4

[0043] Table 5 Elution procedure 5

[0044] Table 6 Elution procedure 6

[0045] Test results such as Figure 8 As shown. Figure 8 It can be seen that elution program 4 has good separation, high peak height, stable baseline, and complete chromatographic information. Therefore, elution program 4 is selected as the optimal elution program.

[0046] 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 are not intended 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 fall within the scope limited by the appended claims of the application equally.

[0047] The instruments used in the following examples are shown in Table 7. The different batches of Shuangjin Gastrointestinal Capsule samples used were purchased from Shaanxi Momede Qixuehe Pharmaceutical Co., Ltd. (Batch No.: 220801, 220802, 220806, 230801, 231101, 230101, 230304, 230302, 230301, 231001, 230201, 230401, 230403, 230501, 230503) Reference substance: Caffeic acid reference substance (Batch No.: 110885-20170 3, purity: 99.7%)), epiberberine reference substance (batch number: 112118-202401, purity: 96%), dehydrocostus lactone reference substance (batch number: 111525-202313, purity: 99.8%), evodia rutaecarpine reference substance (batch number: 110801-202109: 99.3%), limonin reference substance (batch number: 110800-202308: 98.8%), chlorogenic acid reference substance (batch number: 110753-202119, purity: 96.3%), and galanthinone Reference substance (batch number: 111832-202205: 99.6%), hesperidin reference substance (batch number: 110721-202220, purity: 97.2%), nobiletin reference substance (batch number: 112055-202102, purity: 99.7%), tangeretin reference substance (batch number: 112054-202102, purity: 99.7%), succinic acid reference substance (batch number: 100362-202404, purity: 97.3%), uridine reference substance (batch number: 100635-2019 02, purity: 99.9%), daidzein reference substance (batch number: 111502-202304, purity: 99.3%), mongoside reference substance (batch number: 111528-202213, purity: 94.6%) 111528-202213 genistin reference substance (batch number: 111709-202303, purity: 99.6%), all purchased from China Food and Drug Inspection Institute; glycitein reference substance (batch number: G2785, purity: 99.8%) was purchased from Jiangsu Yongjian Pharmaceutical Technology Co., Ltd. The reagents used are shown in Table 8. If not specified, the raw materials and instruments and equipment all use conventional commercial products or conventional instruments and equipment in this field, and their specifications are conventional specifications in this field. The experimental methods for which specific conditions are not indicated in the following examples are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0048] Table 7 Instruments

[0049] Table 8 Reagents

[0050] Example 1 Construction of the characteristic spectrum of Shuangjin gastrointestinal capsule S1. Preparation of Shuangjin Gastrointestinal Capsule Test Solution: Weigh 2.0 g of the contents of 15 batches of Shuangjin Gastrointestinal Capsules respectively, place them in a stoppered conical flask, add 30 mL of pure methanol solution, reflux extract for 1 h, filter, and pass the filtrate through a 0.45 µm microporous filter membrane twice to obtain the Shuangjin Gastrointestinal Capsule test solution.

[0051] S2. Preparation of reference solution: Chlorogenic acid, hesperidin, daidzein, naringenin, caffeic acid, montanol, dehydrocostus lactone, evodipine, limonin, nobiletin, tangeretin, epiberberine, genistin, glycitein, succinic acid, and uridine were accurately weighed and dissolved in methanol to prepare a single reference solution containing 0.65 mg of chlorogenic acid, 0.46 mg of hesperidin, 0.67 mg of naringenin, 0.60 mg of daidzein, 0.75 mg of caffeic acid, 0.72 mg of montanol, 0.78 mg of dehydrocostus lactone, 0.49 mg of evodipine, 0.55 mg of limonin, 0.59 mg of nobiletin, 0.55 mg of tangeretin, 0.65 mg of epiberberine, 0.97 mg of genistin, 0.93 mg of glycitein, 0.57 mg of succinic acid, and 0.72 mg of uridine per 1 mL.

[0052] S3. Chromatographic analysis: The test solution in S1 and the reference solution in S2 were respectively injected into a high performance liquid chromatograph for chromatographic analysis and detected under the same conditions. The liquid chromatography detection conditions were as follows: chromatographic column: Shim-pack VP-ODS (250 mm×4.6 mm, 5 μm) chromatographic column; detector: diode array detector, detection wavelength 220 nm; volume flow rate: 0.6 mL / min; injection volume: 10 μL; mobile phase: acetonitrile (A)-0.1% phosphoric acid aqueous solution (B), gradient elution, the elution program is shown in Table 4, and the corresponding chromatograms are recorded. The chromatograms of different single reference solutions are shown in Table 4. Figure 19 to Figure 24 shown.

[0053] S4. High performance liquid chromatography-mass spectrometry analysis: The test solution of Shuangjin Weichang Capsule was analyzed by high-resolution mass spectrometry. The high-resolution mass spectrometry detection conditions were as follows: 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 total ion current diagram ( Fig.25 and Fig.26). The total ion flow chart was imported into Xcalibur software, and data analysis was performed based on the peaks of the chromatogram of the test solution obtained in S3 to obtain the mass spectrum result chart of each chemical component ( Figure 27 to Figure 43 ).

[0054] Through high-resolution mass spectrometry analysis of the test solution of Shuangjin Gastrointestinal Capsules, a total of 18 chemical components were analyzed and their attribution was analyzed. The results showed that uracil was derived from Cuttlebone; caffeic acid, cinnamic acid, protocatechuic acid, and p-hydroxybenzoic acid were derived from Bletilla striata; chuandongsu and isochuandongsu were derived from Chuandongzi; palmatine was derived from Coptis chinensis; dehydrocostus lactone, evodipine, and limonin were derived from Evodia rutaecarpa; syringic acid, montanol, naringone, and chlorogenic acid were derived from naringa; hesperidin and naringenin were derived from dried orange peel; and genistein was derived from Gallus gallus domesticus.

[0055] S5. Reliability confirmation: The chromatograms of the test solutions of 15 batches of Shuangjin Gastrointestinal Capsules obtained in S3 were imported into the Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System (2012 version); the chromatograms of the test solutions of 15 batches of Shuangjin Gastrointestinal Capsules were selected ( Fig.44 ) were taken as common peaks, and the control spectrum of Shuangjin Gastrointestinal Capsules 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 Shuangjin Gastrointestinal Capsules 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 Shuangjin Gastrointestinal Capsules.

[0056] S6, Shuangjin Gastrointestinal Capsule Characteristic Spectrum: According to the chromatogram of the test solution of Shuangjin Weichang Capsule and the chromatogram of the single reference solution obtained in S3, combined with the total ion current obtained by high-resolution mass spectrometry and the mass spectrum result of the chemical components, uridine (peak 2) was used as the reference peak, and peak 3 was identified as succinic acid, peak 6 as naringenone, peak 7 as chlorogenic acid, peak 10 as caffeic acid, peak 15 as epiberberine, peak 16 as genistin, peak 17 as hesperidin, peak 24 as daidzein, peak 25 as montanol, peak 27 as glycitein, peak 34 as limonin, peak 35 as nobiletin, peak 39 as tangeretin, peak 40 as evodia rutaecarpine, and peak 42 as dehydrocostus lactone in the chromatogram, and the characteristic spectrum of Shuangjin Weichang Capsule was obtained ( Fig.45 ).

[0057] The 16 chromatographic results of the characteristic spectrum of Shuangjin Gastrointestinal Capsule were attributed and analyzed. The results showed that caffeic acid (peak 10) was derived from Bletilla striata; epiberberine (peak 15) was derived from Coptis chinensis; dehydrocostus lactone (peak 42), evodiamine (peak 40) and limonin (peak 34) were derived from Evodia rutaecarpa; chlorogenic acid (peak 7), naringenone (peak 6) and limonin (peak 25) were derived from Nardostachys chinensis; hesperidin (peak 20), nobiletin (peak 35) and tangeretin (peak 39) were derived from Citrus reticulatae; succinic acid (peak 3) and uridine (peak 2) were derived from Pinellia ternata; daidzein (peak 26), genistein (peak 17) and glycitein (peak 27) were derived from Gallus gallus domesticus.

[0058] Example 2 Methodological Study of Characteristic Spectrum Detection Method S1. Similarity Study The automatically generated reference spectrum R was used to generate the common chromatographic peak pattern. The analysis and calculation showed that the common chromatographic peaks of the 15 batches of Shuangjin Gastrointestinal Capsules tested had relatively good similarity. The results are shown in Table 9.

[0059] Table 9 Similarity between each batch of samples and the common chromatographic peak pattern

[0060] The results showed that the characteristic spectrum of Shuangjin Gastrointestinal Capsules established by this method can well detect the quality of Shuangjin Gastrointestinal Capsules and 15 batches of Shuangjin Gastrointestinal Capsules.

[0061] S2. Precision Study The test solution prepared by the method of Example 1 was taken and analyzed according to the detection method of Example 1. The samples were injected 6 times in parallel with an injection volume of 10 μL. Uridine, succinic acid, naringenone, chlorogenic acid, caffeic acid, epiberberine, genistin, hesperidin, limonin, daidzein, glycitein, limonin, nobiletin, tangeretin, evodiamine, and dehydrocostus lactone were used as reference peaks. The peak area and retention time were analyzed and the RSD value was calculated. Table 10 Peak area and retention time of precision study

[0062] The calculation results are shown in Table 10. The results show that the RSD of the relative retention time is less than 1.71%, and the RSD of the relative peak area is less than 2.90%, indicating that the parallel injection precision of the equipment is good.

[0063] S3. Stability study The test solution prepared by the method of Example 1 was taken and analyzed according to the detection method of Example 1. The sample was injected and analyzed at different times of 0, 2, 6, 12, 18, and 24 h. The injection volume was 10 μL. Uridine, succinic acid, naringenone, chlorogenic acid, caffeic acid, epiberberine, genistin, hesperidin, limonin, daidzein, glycitein, limonin, nobiletin, tangeretin, evodia rutaecarpine, and dehydrocostus lactone were used as reference peaks. The peak area and retention time of the common peaks of the HPLC characteristic spectrum of the sample were analyzed and the RSD value was calculated.

[0064] Table 11 Peak areas and retention times of stability studies

[0065] The calculation results are shown in Table 11. The results show that the RSD of the relative retention time is less than 0.47%, and the RSD of the relative peak area is less than 2.60%, indicating that the chromatographic peak of the Shuangjin Gastrointestinal Capsule test solution has almost no change within 24 hours and has good stability.

[0066] S4. Reproducibility Study Six batches of sample solutions were prepared according to the test solution method in Example 1. The chromatographic conditions of Example 1 were referred to, and the injection volume was 10 μL. Uridine, succinic acid, naringenone, chlorogenic acid, caffeic acid, epiberberine, genistin, hesperidin, limonene, daidzein, glycitein, limonin, nobiletin, tangeretin, evodiamine, and dehydrocostus lactone were used as reference peaks. The peak areas and retention times of the common peaks in the HPLC characteristic spectra of the samples were analyzed and the RSD values ​​were calculated. Table 12 Peak area and retention time of repeatability study

[0067] The calculation results are shown in Table 12. The results show that the RSD of the relative retention time is less than 0.71%, and the RSD of the relative peak area is less than 2.28%, indicating that the sample chromatographic peak has good reproducibility and the method has good repeatability.

[0068] The above experimental results show that the characteristic spectrum construction method of Shuangjin Gastrointestinal Capsule provided by the present invention has the characteristics of good stability, high precision and good repeatability, can comprehensively and objectively evaluate the quality of Shuangjin Gastrointestinal Capsule, and provide quality assurance for clinical efficacy.

[0069] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A method for constructing a characteristic spectrum of Shuangjin gastrointestinal capsule, characterized in that: The following steps are involved: S1. Different batches of Shuangjin Gastrointestinal Capsules were used as test samples, and chlorogenic acid, hesperidin, daidzein, naringenin, caffeic acid, montanol, dehydrocostus lactone, evodiamine, limonin, nobiletin, tangeretin, epiberberine, genistin, glycitein, succinic acid and uridine were used as reference substances; S2. Perform HPLC analysis on the test sample and reference sample under the same conditions and record the corresponding chromatograms; S3, perform similarity analysis on the chromatogram of the test sample obtained in S2 to confirm the reliability of the result; S4, performing high-resolution mass spectrometry analysis on the chromatogram of the test sample obtained in S2 to obtain a total ion current diagram; Data analysis was performed based on the total ion flow chart and the peaks of the chromatogram of the test sample obtained in S2 to obtain the mass spectrum result chart of each chemical component; S5. Compare the chromatogram of the test sample and the chromatogram of the reference sample obtained in S2, the total ion current obtained in S4, and the mass spectra of each chemical component to obtain a characteristic spectrum of Shuangjin Gastrointestinal Capsule.

2. The method for constructing the characteristic spectrum of Shuangjin gastrointestinal capsule according to claim 1, characterized in that: In S1, the contents of Shuangjin Gastrointestinal Capsules from different batches were weighed, methanol solution was added, and reflux extraction was performed to obtain Shuangjin Gastrointestinal Capsules test solution.

3. The method for constructing the characteristic spectrum of Shuangjin gastrointestinal capsule according to claim 1, characterized in that: In S2, the detection wavelength was 220 nm, the column temperature was 30 °C, and the flow rate was 0.6 mL / min.

4. The method for constructing the characteristic spectrum of Shuangjin gastrointestinal capsule according to claim 1, characterized in that: In S2, the mobile phase was acetonitrile-0.1% phosphoric acid, and the gradient elution program was as follows: 。 5. The method for constructing the characteristic spectrum of Shuangjin gastrointestinal capsule according to claim 1, characterized in that: S3 specifically includes: importing the test sample chromatogram obtained in S2 into the Chinese medicine chromatographic characteristic spectrum similarity evaluation system, selecting the chromatographic peaks existing in the chromatograms of different batches of test samples as common peaks, generating a control spectrum of Shuangjin Gastrointestinal Capsules using 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 different batches of test samples and the common peak pattern; and confirming the reliability of the results based on the similarity result table and the test sample chromatogram.

6. The method for constructing the characteristic spectrum of Shuangjin gastrointestinal capsule according to claim 1, characterized in that: In S4, the detection conditions of high-resolution mass spectrometry were as follows: 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, full scan mode, and mass-to-charge ratio scanning range of m / z 100-1500.

7. The method for constructing the characteristic spectrum of Shuangjin gastrointestinal capsule according to claim 1, characterized in that: S5 is specifically as follows: according to the chromatogram of the test sample and the chromatogram of 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 result diagram of the chemical components, a comparison is performed, and peak 2 uridine is used as the reference peak, and peak 3 in the chromatogram is identified as succinic acid, peak 6 as naringenone, peak 7 as chlorogenic acid, peak 10 as caffeic acid, peak 15 as epiberberine, peak 16 as genistein, peak 17 as hesperidin, peak 24 as daidzein, peak 25 as lizonerin, peak 27 as glycitein, peak 34 as limonin, peak 35 as nobiletin, peak 39 as tangeretin, peak 40 as evodia rutaecarpine, and peak 42 as dehydrocostus lactone, and the characteristic spectrum of Shuangjin Gastrointestinal Capsules is obtained.

8. The method for constructing the characteristic spectrum of Shuangjin gastrointestinal capsule according to claim 1, characterized in that: After obtaining the characteristic spectrum of Shuangjin Gastrointestinal Capsules, attribution analysis was performed on the characteristic spectrum results of Shuangjin Gastrointestinal Capsules.

9. The characteristic spectrum of Shuangjin gastrointestinal capsule constructed by the method described in any one of claims 1 to 8.

10. A method for identifying Shuangjin gastrointestinal capsules, characterized in that: The Shuangjin gastrointestinal capsule is identified by using the characteristic spectrum obtained by the construction method described in any one of claims 1 to 8.

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