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

[0002] Chinese patent medicines are formulated under the guidance of Traditional Chinese Medicine (TCM) theory using Chinese herbal medicine slices as raw materials, and formulated according to prescribed prescriptions and standards to meet specific dosage forms. They inherit the theories and practices of traditional TCM and incorporate advanced technologies from the modern pharmaceutical industry. They offer precise dosage, convenient use and storage, and a wide variety of dosage forms. Shuangjin Gastrointestinal Capsules (National Drug Approval Number: Z20040055) contain natural Chinese herbal remedies such as 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. These ingredients, scientifically formulated, work together to soothe the liver and stomach, reduce acidity, and alleviate pain. Shuangjin Gastrointestinal Capsules effectively relieve and treat symptoms of common gastrointestinal conditions, including gastric and duodenal ulcers, chronic gastritis, bloating and pain, hiccups, heartburn, and loss of appetite, by inhibiting gastric acid secretion, promoting gastrointestinal motility, and protecting the gastric mucosa.

[0003] Currently, there are few quality inspection methods for Shuangjin Gastrointestinal Capsules, and most of them are studies on the ingredients of single herbs in the prescription, which cannot reflect the overall ingredient 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 map 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:

[0006] The first aspect of the present invention discloses a method for constructing a characteristic map of Shuangjin Gastrointestinal Capsule, comprising the following steps:

[0007] S1. Shuangjin Gastrointestinal Capsules from different batches were used as test samples, and chlorogenic acid, hesperidin, daidzein, naringenone, caffeic acid, montanol, dehydrocostus lactone, evodiamine, limonin, nobiletin, tangeretin, epiberberine, genistin, glycitein, succinic acid, and uridine were used as reference substances.

[0008] S2. Perform HPLC analysis on the test sample and reference sample under the same conditions and record the corresponding chromatograms;

[0009] S3. Perform similarity analysis on the chromatograms of the test sample obtained in S2 to confirm the reliability of the results;

[0010] S4. Perform high-resolution mass spectrometry analysis on the chromatogram of the test sample obtained in S2 to obtain a total ion current diagram; perform 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 mass spectrometry results of each chemical component;

[0011] S5. Compare the chromatograms of the test sample and 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.

[0012] 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.

[0013] More preferably, each 30 mL of methanol solution contains 2.0 g of the contents of the Shuangjin Gastrointestinal Capsule.

[0014] Preferably, the methanol solution is a pure methanol solution.

[0015] Preferably, the extraction is carried out under reflux for 1 h.

[0016] 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.

[0017] Further preferably, in the single reference solution of chlorogenic acid, each 1 mL of methanol solution contains 0.65 mg of chlorogenic acid; in the single reference solution of hesperidin, each 1 mL of methanol solution contains 0.46 mg of hesperidin; in the single reference solution of naringenin, each 1 mL of methanol solution contains 0.67 mg of naringenin; in the single reference solution of daidzein, each 1 mL of methanol solution contains 0.60 mg of daidzein; in the single reference solution of caffeic acid, each 1 mL of methanol solution contains 0.75 mg of caffeic acid; in the single reference solution of limonene, each 1 mL of methanol solution contains 0.72 mg of limonene; in the single reference solution of dehydrocostus lactone, each 1 mL of methanol solution contains 0.78 mg of dehydrocostus lactone; in the single reference solution of evodiamine, each 1 mL of methanol solution contains 0.49 mg of rutaecarpine. g evodiamine; the limonin single reference solution contains 0.55 mg of limonin per 1 mL of methanol solution; the nobiletin single reference solution contains 0.59 mg of nobiletin per 1 mL of methanol solution; the tangeretin single reference solution contains 0.55 mg of tangeretin per 1 mL of methanol solution; the epiberberine single reference solution contains 0.65 mg of epiberberine per 1 mL of methanol solution; the genistin single reference solution contains 0.97 mg of genistin per 1 mL of methanol solution; the glycitein single reference solution contains 0.93 mg of glycitein per 1 mL of methanol solution; the succinic acid single reference solution contains 0.57 mg of succinic acid per 1 mL of methanol solution; and the uridine single reference solution contains 0.72 mg of uridine per 1 mL of methanol solution.

[0018] Preferably, in S2, the detection wavelength is 220 nm.

[0019] Preferably, in S2, the column temperature is 30°C.

[0020] Preferably, in S2, the flow rate is 0.6 mL / min.

[0021] Preferably, in S2, the mobile phase is acetonitrile-0.1% phosphoric acid.

[0022] Preferably, in S2, the gradient elution procedure is as follows:

[0023]

[0024] Preferably, S3 is specifically as follows: importing the test sample chromatogram obtained in S2 into the Chinese medicine chromatographic fingerprint 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.

[0025] 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.

[0026] Preferably, 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 current map obtained by high-resolution mass spectrometry in S4 and the mass spectrum result map of the chemical components, 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 genistin, peak 17 as hesperidin, peak 24 as daidzein, peak 25 as lizitan, peak 27 as glycitein, peak 34 as limonin, peak 35 as nobiletin, peak 39 as tangeretin, peak 40 as evodiamine, and peak 42 as dehydrocostus lactone, to obtain the characteristic spectrum of Shuangjin Gastrointestinal Capsules.

[0027] Preferably, after obtaining the characteristic spectrum of Shuangjin Gastrointestinal Capsule, attribution analysis is performed on the characteristic spectrum results of Shuangjin Gastrointestinal Capsule.

[0028] The second aspect of the present invention discloses the application of the Shuangjin Gastrointestinal Capsule characteristic spectrum constructed by the above method in the quality detection, quality evaluation or quality control of Shuangjin Gastrointestinal Capsule and its corresponding medicine.

[0029] Preferably, the quality marker is one or more of chlorogenic acid, hesperidin, daidzein, naringenone, caffeic acid, limonene, dehydrocostus lactone, evodiamine, limonin, nobiletin, tangeretin, epiberberine, genistein, glycitein, succinic acid and uridine.

[0030] Preferably, the material basis of Shuangjin Gastrointestinal Capsule includes the following detected components: chlorogenic acid, hesperidin, daidzein, naringenone, caffeic acid, montanol, dehydrocostus lactone, evodiamine, limonin, nobiletin, tangeretin, epiberberine, genistein, glycitein, succinic acid and uridine.

[0031] 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.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 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. The 16 characteristic components are identified by high performance liquid chromatography and selected reference substances, 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 ternata and Gallus gallus domestica, and are respectively: caffeic acid (peak 10) derived from white schizonepeta; epiberberine (peak 15) derived from Coptis chinensis; dehydrocostus lactone (peak 42), evodiamine (peak 40), limonene (peak 41), maltitol (peak 43), maltitol (peak 44), maltitol (peak 45), maltitol (peak 46), maltitol (peak 47), maltitol (peak 48), maltitol (peak 49), maltitol (peak 50), maltitol (peak 51), maltitol (peak 52), maltitol (peak 53), maltitol (peak 54), maltitol (peak 55), maltitol (peak 57), maltitol (peak 58), maltitol (peak 59), maltitol (peak 51), maltitol (peak 52), maltitol (peak 53), maltitol (peak 54), maltitol (peak 55), maltitol (peak 56), maltitol (peak 57), maltitol (peak Chlorogenic acid (peak 7), nobiletin (peak 6), and montanadin (peak 25) are derived from naringa; hesperidin (peak 20), nobiletin (peak 35), and tangeretin (peak 39) are derived from dried tangerine peel; succinic acid (peak 3) and uridine (peak 2) are derived from pinellia; 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 to use, 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, reduces inspection costs and improves inspection efficiency. The method can accurately, clearly, objectively, quickly and comprehensively monitor the quality of multiple characteristic components of Shuangjin Gastrointestinal Capsules at the same time, which not only improves the quality control level of medicinal materials such as cuttlebone, Bletilla striata, and Chuanxiongzi, but also improves the intrinsic quality of Shuangjin Gastrointestinal Capsules and reduces the inspection workload. The method maintains the consistency of the quality of medicinal materials such as cuttlebone, Bletilla striata, Chuanxiongzi, Corydalis yanhusuo, Coptis chinensis, Evodia rutaecarpa, Cyperus rotundus, Nardostachys grandiflora, Curcuma aromatica, Curcuma zedoaria, Citrus reticulata, Pinellia ternata, and Gallus gallus domestica with Shuangjin Gastrointestinal Capsules, provides clinical raw materials that meet the requirements of Shuangjin Gastrointestinal Capsules, provides a guarantee for the safe use 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

[0034] Figure 1This is a chromatogram obtained by optimizing the extraction method during the preparation of the test solution of the present invention;

[0035] Figure 2 This is a chromatogram obtained by optimizing the extraction solvent during the preparation of the test solution of the present invention;

[0036] Figure 3 This is a chromatogram obtained by optimizing the extraction time during the preparation of the test solution of the present invention;

[0037] Figure 4 This is the DAD full-wavelength 3D scanning diagram (190nm~400nm) of the test sample during the prior art investigation of the present invention;

[0038] Figure 5 The chromatogram obtained by optimizing the detection wavelength under the chromatographic conditions of the present invention;

[0039] Figure 6 The chromatogram obtained by optimizing the column temperature in the chromatographic conditions of the present invention;

[0040] Figure 7 The chromatogram obtained by optimizing the flow rate under the chromatographic conditions of the present invention;

[0041] Figure 8 The chromatogram obtained by optimizing the mobile phase composition under the chromatographic conditions of the present invention;

[0042] Figure 9 The chromatogram obtained by optimizing the elution procedure under the chromatographic conditions of the present invention;

[0043] Figure 10 The chromatogram (A) of epigallocatechin-3-one, the UV spectrum (B) of epigallocatechin-3-one, and the full-wavelength 3D scanning diagram (C) of epigallocatechin-3-one of the present invention are shown;

[0044] Figure 11 The chromatogram of lignoside (A), UV spectrum of lignoside (B), and full-wavelength 3D scanning diagram of lignoside (C) of the present invention are shown;

[0045] Figure 12 The chromatogram of glycitein (A), the UV spectrum of glycitein (B), and the full-wavelength 3D scanning image of glycitein (C) of the present invention are shown;

[0046] Figure 13 The chromatogram of epiberberine (A), the UV spectrum of epiberberine (B), and the full-wavelength 3D scanning diagram of epiberberine (C) of the present invention are shown;

[0047] Figure 14 The chromatogram of hesperidin (A), the UV spectrum of hesperidin (B), and the full-wavelength 3D scanning image of hesperidin (C) of the present invention are shown;

[0048] Figure 15The chromatogram of nobiletin (A), the UV spectrum of nobiletin (B), and the full-wavelength 3D scanning image of nobiletin (C) of the present invention are shown;

[0049] Figure 16 The chromatogram of daidzein of the present invention (A), the UV spectrum of daidzein (B), and the full-wavelength 3D scanning image of daidzein (C);

[0050] Figure 17 The succinic acid chromatogram (A), succinic acid UV spectrum (B), and succinic acid full-wavelength 3D scanning diagram (C) of the present invention are shown;

[0051] Figure 18 The chromatogram of tangeretin (A), the UV spectrum of tangeretin (B), and the full-wavelength 3D scanning image of tangeretin (C) of the present invention are shown;

[0052] Figure 19 The caffeic acid chromatogram (A), caffeic acid UV spectrum (B), and caffeic acid full wavelength 3D scanning diagram (C) of the present invention are shown;

[0053] Figure 20 The uridine chromatogram (A), uridine UV spectrum (B), and uridine full wavelength 3D scanning diagram (C) of the present invention are shown;

[0054] Figure 21 The limonin chromatogram (A), limonin UV spectrum (B), and limonin full wavelength 3D scanning diagram (C) of the present invention are shown;

[0055] Figure 22 The chromatogram of dehydrocostus lactone (A), the UV spectrum of dehydrocostus lactone (B), and the full-wavelength 3D scanning diagram of dehydrocostus lactone (C) of the present invention are shown;

[0056] Figure 23 The chromatogram of genistin (A), the UV spectrum of genistin (B), and the full-wavelength 3D scanning image of genistin (C) of the present invention are shown;

[0057] Figure 24 The chromatogram of Evodia rutaecarpine (A), the UV spectrum of Evodia rutaecarpine (B), and the full-wavelength 3D scanning diagram of Evodia rutaecarpine (C) of the present invention are shown;

[0058] Figure 25 The chlorogenic acid chromatogram (A), chlorogenic acid UV spectrum (B), and chlorogenic acid full wavelength 3D scanning diagram (C) of the present invention;

[0059] Figure 26 This is the mass spectrum of the Shuangjin Gastrointestinal Capsule of the present invention in positive ion mode;

[0060] Figure 27 This is the mass spectrum of the Shuangjin Gastrointestinal Capsule of the present invention in negative ion mode;

[0061] Figure 28 This is the mass spectrum of syringic acid of the present invention;

[0062] Figure 29 This is the mass spectrum of cinnamic acid of the present invention;

[0063] Figure 30 is the mass spectrum of genistin of the present invention;

[0064] Figure 31 This is the mass spectrum of limonene glycosides of the present invention;

[0065] Figure 32 This is the mass spectrum of caffeic acid of the present invention;

[0066] Figure 33 This is the mass spectrum of taenanthene / isotaenanthene of the present invention;

[0067] Figure 34 This is the mass spectrum of naringenone of the present invention;

[0068] Figure 35 This is the hesperidin spectrum of the present invention;

[0069] Figure 36 This is the mass spectrum of dehydrocostus lactone of the present invention;

[0070] Figure 37 This is the mass spectrum of chlorogenic acid of the present invention;

[0071] Figure 38 This is the mass spectrum of palmatine of the present invention;

[0072] Figure 39 This is the mass spectrum of protocatechuic acid of the present invention;

[0073] Figure 40 This is the mass spectrum of lemon bitter of the present invention;

[0074] Figure 41 This is the mass spectrometry of pomelo peel of the present invention;

[0075] Figure 42 This is the mass spectrum of p-hydroxybenzoic acid of the present invention;

[0076] Figure 43 is the mass spectrum of uracil of the present invention;

[0077] Figure 44 This is the mass spectrum of the Evodia rutaecarpa alkaloid of the present invention;

[0078] Figure 45 This is the chromatogram of the test solution of 15 batches of Shuangjin Gastrointestinal Capsules of the present invention;

[0079] Figure 46 This is the characteristic spectrum of Shuangjin Gastrointestinal Capsule. DETAILED DESCRIPTION

[0080] To help those skilled in the art understand the features 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 terms used herein have the ordinary meanings understood by those skilled in the art regarding the present invention. In the event of any conflict, the definitions in this specification shall prevail. The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not in any way limit the scope of the present invention. That is, the present invention can be implemented without being limited by any particular theory or mechanism.

[0081] In this document, for the sake of brevity, not all possible combinations of the various technical features in each embodiment or example are described. Therefore, as long as there are no contradictions in the combination of these technical features, the various technical features in each embodiment or example can be combined in any way, and all possible combinations should be considered to be within the scope of this specification.

[0082] The present invention provides a method for constructing a characteristic spectrum of Shuangjin gastrointestinal capsule, comprising the following steps:

[0083] S1. Weigh appropriate amounts of the contents of Shuangjin Gastrointestinal Capsules from different batches, add methanol solution, and reflux extraction to obtain Shuangjin Gastrointestinal Capsules test solution;

[0084] S2. Dissolve chlorogenic acid, hesperidin, daidzein, glycitein, caffeic acid, montanol, dehydrocostus lactone, evodiamine, limonin, nobiletin, tangeretin, epiberberine, genistin, glycitein, succinic acid, and uridine in methanol to obtain corresponding single reference solution;

[0085] S3. Inject the test solution in S1 and the reference solution in S2 into a high performance liquid chromatograph for chromatographic analysis, perform the test under the same conditions, and record the corresponding chromatograms;

[0086] S4. Perform high-resolution mass spectrometry on the test solution to obtain a total ion current (TIC). Import the TIC into Xcalibur software and perform data analysis based on the peaks in the chromatogram of the test solution obtained in S3 to obtain the mass spectrometry results of each chemical component and perform attribution analysis on the mass spectrometry results.

[0087] S5. Import the chromatograms of the different batches of Shuangjin Gastrointestinal Capsule test solution obtained in S3 into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012A Version); select the chromatographic peaks present in the chromatograms of the 15 batches of Shuangjin Gastrointestinal Capsule test solution as common peaks, generate a reference spectrum of Shuangjin Gastrointestinal Capsule using 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 a similarity result table between the chromatograms of the different batches of Shuangjin Gastrointestinal Capsule test solution and the common peak pattern; confirm the reliability of the result based on the similarity result table and the chromatogram of the Shuangjin Gastrointestinal Capsule test solution;

[0088] S6. Compare the chromatograms of the Shuangjin Weichang Capsule test solution and the single reference solution obtained in S3 with the total ion current (TIC) obtained by high-resolution mass spectrometry and the mass spectrometry results of the chemical components to obtain a characteristic spectrum of the Shuangjin Weichang Capsule. Perform attribute analysis on the characteristic spectrum of the Shuangjin Weichang Capsule to determine the chemical components of each peak in the characteristic spectrum.

[0089] The present invention provides an optimization process for detecting the characteristic spectrum of the Shuangjin Gastrointestinal Capsule:

[0090] S1. Optimization of test solution preparation:

[0091] The present invention conducted experimental investigations on different extraction methods (ultrasound, reflux, and immersion), and the experimental results are as follows Figure 1 shown.

[0092] 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;

[0093] 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.

[0094] 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;

[0095] The present invention compares the extraction effects of different extraction times (30min, 45min, 1h), and the experimental results are as follows Figure 3 shown.

[0096] The results show that the chromatogram obtained when the reflux extraction is carried out for 1 hour has the highest component content, the best peak shape and the highest separation degree, so 1 hour is selected as the reflux extraction time.

[0097] S2. Optimize chromatographic conditions:

[0098] The present invention uses a diode array detector to investigate the detection wavelength, performs a full wavelength scan of 190-400 nm on the sample, and extracts the chromatogram at 210 nm, 220 nm, 230 nm, and 245 nm. The experimental results are as follows Figure 4 、 Figure 5 shown.

[0099] The results show that the chromatogram contains the most comprehensive information and has a stable baseline when the detection wavelength is 220 nm, so this method is selected as the detection wavelength condition.

[0100] The present invention screened the column temperature (25°C, 30°C, 35°C), and the experimental results were as follows: Figure 6 shown.

[0101] 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 of 30°C was finally selected.

[0102] The present invention screened the flow rate (0.6mL / min, 0.8mL / min, 1.0mL / min), and the experimental results were as follows: Figure 7 shown.

[0103] The results showed that the peak elution was best when the flow rate was maintained at 0.6 mL / min, and the separation effect of each component was better, so the flow rate was finally selected as 0.6 mL / min.

[0104] 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 8 shown.

[0105] 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.

[0106] After determining the optimal mobile phase composition, the present invention screened the best gradient elution program through a large number of experiments. Some elution programs are shown in Tables 1 to 6:

[0107] Table 1 Elution procedure 1

[0108]

[0109] Table 2 Elution procedure 2

[0110]

[0111] Table 3 Elution procedure 3

[0112]

[0113] Table 4 Elution procedure 4

[0114]

[0115] Table 5 Elution procedure 5

[0116]

[0117] Table 6 Elution procedure 6

[0118]

[0119]

[0120] Test results such as Figure 9 As shown. Figure 9 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.

[0121] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, 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 equally within the scope limited by the appended claims of the application.

[0122] The instruments used in the following examples are shown in Table 7. The different batches of Shuangjin Gastrointestinal Capsules samples used were purchased from Shaanxi Momede Qixuehe Pharmaceutical Co., Ltd. (batch numbers: 230101, 230301, 230302, 230304, 220802, 230801, 231101, 231001, 230201, etc.); reference substances: caffeic acid reference substance (batch number: 110885-201703, purity: 99.7%), epiberberine reference substance (batch number: 112118-202 401, purity: 96%), dehydrocostus lactone reference substance (lot number: 111525-202313, purity: 99.8%), evodiamine reference substance (lot number: 110801-202109, purity: 99.3%), limonin reference substance (lot number: 110800-202308, purity: 98.8%), chlorogenic acid reference substance (lot number: 110753-202119, purity: 96.3%), and galantone reference substance (lot number: 111832-20220 5, purity: 99.6%), hesperidin reference substance (lot number: 110721-202220, purity: 97.2%), nobiletin reference substance (lot number: 112055-202102, purity: 99.7%), tangeretin reference substance (lot number: 112054-202102, purity: 99.7%), succinic acid reference substance (lot number: 100362-202404, purity: 97.3%), uridine reference substance (lot number: 100635-201902, purity: 99 .9%), daidzein reference substance (batch number: 111502-202304, purity: 99.3%), limonene reference substance (batch number: 111528-202213, purity: 94.6%), 111528-202213 genistin reference substance (batch number: 111709-202303, purity: 99.6%), were 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. Unless otherwise specified, raw materials and instruments and equipment are 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.

[0123] Table 7 Instruments

[0124]

[0125] Table 8 Reagents

[0126]

[0127]

[0128] Example 1 Construction of the characteristic spectrum of Shuangjin gastrointestinal capsule

[0129] S1. Preparation of Shuangjin Gastrointestinal Capsule test solution:

[0130] 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.

[0131] S2. Preparation of reference solution:

[0132] Chlorogenic acid, hesperidin, daidzein, daidzein, caffeic acid, montanadine, 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 daidzein, 0.60 mg of caffeic acid, 0.75 mg of montanadine, 0.72 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.

[0133] S3. Chromatographic analysis:

[0134] The test solution in S1 and the reference solution in S2 were 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, elution program as shown in Table 4, record the corresponding chromatograms, different single reference solution chromatograms as shown in Table 4. Figures 10 to 25 shown.

[0135] S4. High performance liquid chromatography coupled with mass spectrometry analysis:

[0136] 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 3500V, sheath gas flow rate 40arb, auxiliary gas flow rate 10arb, capillary temperature 300℃, auxiliary gas temperature 300℃, full scan mode, mass-to-charge ratio scanning range m / z 100-1500, and total ion current ( Figure 26 and Figure 27 Import the total ion current graph into Xcalibur software, perform data analysis based on the peaks of the chromatogram of the test solution obtained in S3, and obtain the mass spectrum results of each chemical component ( Figures 28 to 44 ).

[0137] High-resolution mass spectrometry analysis of the Shuangjin Gastrointestinal Capsule test solution revealed 18 chemical components, which were then assigned to their respective constituents. The results showed that uracil originated from cuttlebone; caffeic acid, cinnamic acid, protocatechuic acid, and p-hydroxybenzoic acid originated from Bletilla striata; chondrin and isochondrin originated from Toosendan fruit; palmatine originated from Coptis chinensis; dehydrocostus lactone, evodiamine, and limonin originated from Evodia rutaecarpa; syringic acid, linalool, naringenone, and chlorogenic acid originated from naringensis; hesperidin and naringenin originated from dried tangerine peel; and genistin originated from Gallus gallus domesticus.

[0138] S5. Reliability confirmation:

[0139] The chromatograms of the test solution of 15 batches of Shuangjin Weichang Capsules obtained in S3 were imported into the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012A Version); the chromatograms of the test solution of 15 batches of Shuangjin Weichang Capsules were selected ( Figure 45 ) 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; a similarity result table between the chromatograms of the test solutions of Shuangjin Gastrointestinal Capsules from different batches and the common peak pattern 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.

[0140] S6, Shuangjin Gastrointestinal Capsule Characteristic Spectrum:

[0141] According to the chromatogram of the test solution of Shuangjin Weichang Capsule and the chromatogram of the single reference solution obtained in S3, and combined with the total ion current obtained by high-resolution mass spectrometry and the mass spectrometry results of the chemical components, uridine (peak 2) was used as the reference peak, and peak 3 in the chromatogram 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 mongoside, peak 27 as glycitein, peak 34 as limonin, peak 35 as nobiletin, peak 39 as tangeretin, peak 40 as evodiamine, and peak 42 as dehydrocostus lactone, and the characteristic spectrum of Shuangjin Weichang Capsule was obtained ( Figure 46 ).

[0142] 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 naringensis; hesperidin (peak 20), nobiletin (peak 35), and tangeretin (peak 39) were derived from dried tangerine peel; succinic acid (peak 3) and uridine (peak 2) were derived from Pinellia ternata; daidzein (peak 26), genistin (peak 17), and glycitein (peak 27) were derived from Gallus gallus domesticus.

[0143] Example 2 Methodological Study of Characteristic Spectrum Detection Method

[0144] S1. Similarity Study

[0145] 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 test samples had relatively good similarity. The results are shown in Table 9.

[0146] Table 9 Similarity between each batch of samples and the common chromatographic peak pattern

[0147]

[0148] 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.

[0149] S2. Precision Study

[0150] 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 in parallel 6 times with an injection volume of 10 μL. Uridine, succinic acid, naringenone, chlorogenic acid, caffeic acid, epiberberine, genistein, hesperidin, limonin, daidzein, glycitein, limonin, nobiletin, tangeretin, evodiamine, and dehydrocostus lactone were used as reference peaks. The peak areas and retention times were analyzed and the RSD values ​​were calculated.

[0151] Table 10 Peak area and retention time of precision study

[0152]

[0153]

[0154] The calculation results are shown in Table 10. The results show that the RSDs of retention time are all less than 1.71%, and the RSDs of peak area are all less than 2.90%, indicating that the parallel injection precision of the device is good.

[0155] S3. Stability Study

[0156] 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 at different times of 0, 2, 6, 12, 18, and 24 h, and the injection volume was 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 of the common peak of the HPLC characteristic spectrum of the sample were analyzed and the RSD value was calculated.

[0157] Table 11 Peak areas and retention times in stability studies

[0158]

[0159]

[0160] The calculation results are shown in Table 11, which show that the RSD of retention time is less than 0.47%, and the RSD of 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.

[0161] S4. Reproducible Studies

[0162] Six batches of sample solutions were prepared according to the test solution method in Example 1. Referring to the chromatographic conditions of Example 1, the injection volume was 10 μL, and 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 area and retention time of the common peaks of the HPLC characteristic spectrum of the samples were analyzed and the RSD values ​​were calculated.

[0163] Table 12 Peak area and retention time of repeatability study

[0164]

[0165]

[0166] The calculation results are shown in Table 12. The results show that the RSDs of retention time are all less than 0.71%, and the RSDs of peak area are all less than 2.28%, indicating that the sample chromatographic peaks have good reproducibility and the repeatability of the method is good.

[0167] The above experimental results show that the characteristic spectrum construction method of Shuangjin Gastrointestinal Capsules 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 Capsules, and provide quality assurance for clinical efficacy.

[0168] The above content is 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 map of Shuangjin Gastrointestinal Capsules, characterized in that: Shuangjin Gastrointestinal Capsule is composed 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. The method for constructing its characteristic map includes: S1. Shuangjin Gastrointestinal Capsules from different batches were used as test samples, and chlorogenic acid, hesperidin, daidzein, naringenone, 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 chromatograms of the test sample obtained in S2 to confirm the reliability of the results; S4. Perform high-resolution mass spectrometry analysis on the chromatogram of the test sample obtained in S2 to obtain a total ion current diagram; perform 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 mass spectrometry results of each chemical component; S5. Compare the chromatograms of the test sample and 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: S3 specifically includes: importing the test sample chromatogram obtained in S2 into the Chinese medicine chromatographic fingerprint 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 chromatogram of the test sample.

5. 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.

6. 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 current 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. 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 lizitan, peak 27 as glycitein, peak 34 as limonin, peak 35 as nobiletin, peak 39 as tangeretin, peak 40 as evodiamine, and peak 42 as dehydrocostus lactone, and the characteristic spectrum of Shuangjin Gastrointestinal Capsules is obtained.

7. Use of the Shuangjin Gastrointestinal Capsule characteristic spectrum constructed by the method according to any one of claims 1 to 6 in the quality inspection, quality evaluation or quality control of Shuangjin Gastrointestinal Capsule and its counterparts.

8. The use according to claim 7, characterized in that The quality markers are one or more of chlorogenic acid, hesperidin, daidzein, naringenone, caffeic acid, montanol, dehydrocostus lactone, evodiamine, limonin, nobiletin, tangeretin, epiberberine, genistein, glycitein, succinic acid and uridine.

9. The use according to claim 7, characterized in that The material basis of Shuangjin Gastrointestinal Capsules includes the following detected ingredients: chlorogenic acid, hesperidin, daidzein, galangin, caffeic acid, montanadin, dehydrocostus lactone, evodiamine, limonin, nobiletin, tangeretin, epiberberine, genistein, glycitein, succinic acid and uridine.

10. A method for identifying Shuangjin Gastrointestinal Capsules, characterized in that: The Shuangjin gastrointestinal capsule is identified by the characteristic spectrum constructed using the method described in any one of claims 1 to 6.

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