A method for constructing a characteristic spectrum of a traditional Chinese medicine composition or a double-shanzhai-ginseng-lingzhi preparation and application thereof

Abstract

The present application relates to a kind of traditional Chinese medicine composition or its preparation Shuangshenling granules characteristic map construction method and its application, the method uses high performance liquid chromatography method.Chromatographic conditions are as follows: chromatographic column: octadecylsilane bonding silica gel chromatographic column: mobile phase: with acetonitrile as mobile phase A, water or formic acid aqueous solution as mobile phase B, gradient elution is carried out.The method provided by the present application can obtain characteristic map with large information amount and high peak response value, avoids the singleness and one-sidedness of quality control, can effectively control the key quality attributes of Shuangshenling granules and other preparations, has rapid, simple, comprehensive reproducibility and stability, and is conducive to industrial industry popularization, and has important significance for Shuangshenling granules quality control standard.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine quality control technology, specifically relating to a method for constructing a characteristic spectrum of a traditional Chinese medicine composition or its preparation, Shuangshenling Granules, and its application. Background Technology

[0002] Traditional Chinese medicine has certain advantages in treating chronic renal failure. According to market research, there are currently few types of oral Chinese medicine for the clinical treatment of chronic renal failure. The main ones include Haikun Shenxi Capsules, Bailin Capsules, Huangkui Capsules, Shenshuining Capsules, and Uremic Clearing Granules, which are used to treat chronic renal failure with damp-turbidity syndrome, lung and kidney deficiency, chronic nephritis with damp-heat syndrome, spleen and stomach qi deficiency, internal obstruction of damp-turbidity, and imbalance of ascending and descending qi.

[0003] For chronic renal failure with spleen and kidney yang deficiency, there are currently no traditional Chinese medicine preparations on the market specifically targeting this syndrome, indicating significant market potential. Chinese patent CN 200410021299.5 discloses a drug for treating uremia, kidney yang deficiency, and renal failure. This traditional Chinese medicine composition consists of thirteen herbs: Astragalus membranaceus, red ginseng, deer antler, yam, cornus officinalis, poria cocos, rhubarb, salvia miltiorrhiza, chuanxiong rhizome, cinnamon, eucommia ulmoides, achyranthes bidentata, and angelica sinensis. It has the effects of warming and tonifying the spleen and kidney, promoting blood circulation and removing blood stasis, and eliminating dampness and turbidity. It is mainly suitable for patients with chronic renal failure and spleen and kidney yang deficiency, characterized by symptoms such as nausea and vomiting, loss of appetite, fatigue, weakness, lower back and knee pain, edema, aversion to cold, cold limbs, oliguria, and loose stools. The granule formulation of this composition is called "Shuangshenling Granules". Shuangshenling Granules are an empirical formula composed of "Wenpi Decoction" from Sun Simiao's "Essential Prescriptions Worth a Thousand Gold Pieces" from the Tang Dynasty and "Yougui Pill" from Zhang Jingyue's "Complete Works of Jingyue" from the Ming Dynasty, combined with clinical practice, using modifications and selections from these two formulas.

[0004] Due to the complexity of traditional Chinese medicine (TCM) components, qualitative and quantitative analysis of single indicator components is insufficient to comprehensively reflect the quality of the tested products. Therefore, holistic quality control of multiple components should be strengthened. TCM characteristic chromatograms are a multi-indicator quality control model that can comprehensively reflect the types and quantities of chemical components, thereby enabling comprehensive reflection and monitoring of TCM quality and holistic control and evaluation of TCM preparations. However, a comprehensive characteristic chromatogram detection method has not yet been developed in this field for TCM compositions or Shuangshenling granule preparations.

[0005] Therefore, there is an urgent need to develop a characteristic chromatographic detection method for this traditional Chinese medicine composition or Shuangshenling granules to obtain characteristic chromatograms with a large amount of information and the ability to identify multiple components, thereby improving the quality control level of this traditional Chinese medicine composition or its preparation products such as Shuangshenling granules. Summary of the Invention

[0006] One objective of this invention is to overcome the shortcomings of existing technologies and provide a method for detecting the characteristic chromatograms of a specific traditional Chinese medicine composition or its preparation, Shuangshenling Granules. This method has the advantages of being comprehensive, rapid, and reliable, and can obtain relatively comprehensive characteristic chromatogram information of multiple components. This method provides a basis for quality control research on this specific traditional Chinese medicine composition or its preparation, Shuangshenling Granules, and provides a method for comprehensively and scientifically evaluating product quality, which is of great significance to the quality control standards of Shuangshenling Granules.

[0007] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0008] This invention provides a method for constructing characteristic chromatograms of a traditional Chinese medicine composition or its preparation, Shuangshenling Granules, wherein the method is high-performance liquid chromatography (HPLC).

[0009] The chromatographic conditions are as follows:

[0010] The chromatographic column is an octadecylsilane-bonded silica column, with acetonitrile as mobile phase A and water or formic acid aqueous solution as mobile phase B, and gradient elution is performed.

[0011] The chromatographic conditions were further optimized as follows:

[0012] Chromatographic column: Octadecylsilane-bonded silica gel column; column length 250 mm, diameter 4.6 mm, particle size 5 μm;

[0013] Mobile phase: Gradient elution was performed using acetonitrile as mobile phase A and formic acid aqueous solution as mobile phase B;

[0014] Detection wavelength: 190nm-400nm;

[0015] Column temperature: 30~40℃.

[0016] The chromatographic conditions are further optimized as follows:

[0017] Column: Selected from Inertsil ODS-3-C18 and Phenomenex Omega Polar or UltimateXB-C18; Inertsil ODS-3-C18 is the preferred choice.

[0018] Mobile phase: Gradient elution was performed using acetonitrile as mobile phase A and formic acid aqueous solution as mobile phase B;

[0019] Detection wavelength: 250nm.

[0020] The method is a high-performance liquid chromatography (HPLC) method, which includes the following steps:

[0021] (1) Prepare a test solution of Shuangshenling granules or its intermediate extract;

[0022] The traditional Chinese medicine composition or its preparation, Shuangshenling Granules, is prepared from the following medicinal ingredients: Astragalus membranaceus, red ginseng, deer antler powder, yam, Cornus officinalis, Poria cocos, rhubarb, Salvia miltiorrhiza, Ligusticum chuanxiong, cinnamon, Eucommia ulmoides, Achyranthes bidentata, and Angelica sinensis.

[0023] (2) Pipette the test solution into the liquid chromatograph, determine it according to the high performance liquid chromatography method, record the chromatogram, and obtain the result;

[0024] The conditions for the high-performance liquid chromatography method include: using an octadecylsilane-bonded silica column, with acetonitrile as mobile phase A and formic acid aqueous solution as mobile phase B, and performing gradient elution.

[0025] This invention utilizes high-performance liquid chromatography (HPLC) with an octadecylsilane-bonded silica column and a specific combination of mobile phase A (acetonitrile) and mobile phase B (formic acid aqueous solution) to obtain characteristic spectra with high information content and peak response values. Sixteen common characteristic peaks were identified, allowing identification of seven medicinal herbs: rhubarb, eucommia, cornus officinalis, astragalus, angelica sinensis, chuanxiong, and salvia miltiorrhiza. Simultaneously, the characteristic peaks of fourteen substances were successfully identified: gallic acid, genipin, monoglucoside, loganin, verrucoside glucoside, ferulic acid, rosmarinic acid, salvianolic acid B, aloe-emodin, rhein, emodin, chrysophanol, emodin methyl ether, and tanshinone IIA.

[0026] It avoids the limitations and one-sidedness of quality control, effectively controls the key quality attributes of compound preparations, and has rapid, simple, comprehensive reproducibility and stability. It is also conducive to industrial promotion and has important significance for the quality control standards of Shuangshenling Granules.

[0027] Furthermore, the present invention demonstrates through methodological verification that the characteristic spectrum detection method has good repeatability and high feasibility, and exhibits good robustness to different instruments, different column temperatures (30-40℃), different flow rates (0.9-1.1 ml / min), and different mobile phase acidities (0.05%-0.15% formic acid aqueous solution).

[0028] In this invention, the intermediate extract refers to the intermediate product obtained by extracting, concentrating and drying the prescription medicinal ingredients, including but not limited to extract, concentrate or dry powder.

[0029] Preferably, step (1) includes: mixing the Shuangshenling granules or its intermediate extract with the extraction solvent, extracting, filtering, and obtaining the test solution.

[0030] Preferably, the extraction solvent includes methanol and / or n-butanol and ethyl acetate, with n-butanol being the most preferred.

[0031] The "methanol" mentioned in this invention includes not only pure methanol, but also methanol aqueous solutions of different concentrations, such as 80% methanol aqueous solution, 60% methanol aqueous solution, etc.

[0032] In the preferred embodiment of the present invention, n-butanol is selected as the extraction solvent. Compared with other alcohol solvents, the final characteristic spectrum has more information, can identify more medicinal flavors, and is more conducive to the quality control of Shuangshenling granules.

[0033] Preferably, the extraction method includes ultrasonic treatment, followed by purification with a corresponding extraction solvent.

[0034] Preferably, the sample to be tested is the intermediate extract of Shuangshenling granules, and the intermediate extract is a dry powder. Step (1) specifically includes: taking about 2.5g of the intermediate extract of Shuangshenling granules, accurately weighing it, placing it in a stoppered conical flask, accurately adding 50ml of methanol, weighing it, sonicating it (power 400W, frequency 40kHz) for 30 minutes, taking it out, cooling it, replenishing the lost weight with methanol, shaking it well, filtering it, evaporating the filtrate to dryness, adding 25ml of water to dissolve the residue, adding water-saturated n-butanol and shaking to extract it 3 times, 25ml each time, combining the n-butanol liquids, evaporating it to dryness, adding methanol to dissolve the residue, transferring it to a 25ml volumetric flask, adding methanol to dilute to the mark, shaking it well, filtering it, and taking the filtrate to obtain the final product.

[0035] Preferably, in step (2), the gradient elution procedure of the high-performance liquid chromatography is as follows:

[0036] Within 0 to 10 minutes, the volume ratio of mobile phase A to the total mobile phase gradually changes from 2% to 10% at a constant rate.

[0037] Within 10–15 minutes, the volume ratio of mobile phase A to the total mobile phase is 10% isocratic.

[0038] Within 15 to 25 minutes, the volume ratio of mobile phase A to the total mobile phase gradually changes from 10% to 20% at a constant rate.

[0039] Within 25 to 50 minutes, the volume ratio of mobile phase A to the total mobile phase gradually changes from 20% to 25% at a constant rate.

[0040] Within 50–65 minutes, the volume ratio of mobile phase A to the total mobile phase gradually and uniformly changes from 25% to 30%.

[0041] Within 65–85 minutes, the volume ratio of mobile phase A to the total mobile phase gradually changes from 30% to 70% at a constant rate.

[0042] Within 85 to 100 minutes, the volume ratio of mobile phase A to the total mobile phase gradually changes from 70% to 85% at a constant rate.

[0043] Within 100–105 min, the volume ratio of mobile phase A to the total mobile phase is 85%.

[0044] The present invention further preferably uses the above-mentioned specific gradient for elution, resulting in a more stable baseline and better separation of each peak in the chromatogram.

[0045] Preferably, in step (2), the concentration of the formic acid aqueous solution is 0.05%-0.15%, for example, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, etc. In this invention, the concentration of the formic acid solution refers to the volume concentration.

[0046] Preferably, in step (2), the detection wavelength of the high performance liquid chromatography is 190nm-400nm, such as 190nm, 230nm, 250nm, 270nm, 310nm, etc., with 250nm being the most preferred.

[0047] The present invention further optimizes the detection wavelength to 190nm-400nm, which can obtain chromatograms with more information. Among them, the response values ​​of each chromatographic peak are the highest and the information content is the largest when the wavelength is 250nm.

[0048] Preferably, in step (2), the flow rate of the high performance liquid chromatography is 0.9-1.1 ml / min, for example, 0.9, 1.0, 1.1 ml / min, etc.

[0049] Preferably, in step (2), the column temperature of the high performance liquid chromatography is 30 to 40°C, such as 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, etc.

[0050] Preferably, in step (2), the injection volume of the high performance liquid chromatography is 10 μL.

[0051] Preferably, in the chromatogram, salvianolic acid B is used as a reference peak, and the relative retention time of each peak is calculated using the reference peak. The relative retention time of the characteristic peak of gallic acid is 0.18–0.22, the relative retention time of the characteristic peak of genipin is 0.25–0.31, the relative retention time of the characteristic peak of monoglucoside is 0.35–0.43, the relative retention time of the characteristic peak of loganin is 0.46–0.56, the relative retention time of the characteristic peak of verrucoside isoflavone glucoside is 0.55–0.67, and the relative retention time of the characteristic peak of ferulic acid is... The relative retention times of the peaks are 0.58–0.70, the relative retention times of the characteristic peaks of rosmarinic acid are 0.73–0.89, the relative retention times of the characteristic peaks of aloe-emodin are 1.33–1.63, the relative retention times of the characteristic peaks of rhein are 1.37–1.67, the relative retention times of the characteristic peaks of emodin are 1.46–1.78, the relative retention times of the characteristic peaks of chrysophanol are 1.56–1.90, the relative retention times of the characteristic peaks of emodin methyl ether are 1.61–1.97, and the relative retention times of the characteristic peaks of tanshinone IIA are 1.64–2.00.

[0052] Preferably, in step (2), the characteristic peaks of rhubarb, eucommia, cornus officinalis, astragalus, angelica sinensis, chuanxiong and salvia miltiorrhiza can be identified in the obtained chromatogram.

[0053] Preferably, in the chromatogram, salvianolic acid B is used as a reference peak, which is also the S peak. The relative retention times of each peak are calculated using the reference peak. The relative retention times of the characteristic peaks of rhubarb are 0.18–0.22 min, 1.04–1.26 min, 1.33–1.63 min, 1.37–1.67 min, 1.46–1.78 min, 1.56–1.90 min, and 1.61–1.97 min. The retention times of the characteristic peaks of eucommia are 0.25–0.31 min. The retention times of the characteristic peaks of cornus officinalis are 0.35–0.43 min and 0.46–0.56 min. The retention times of the characteristic peaks of astragalus membranaceus are 0.55–0.67 min. The retention times of the characteristic peaks of angelica sinensis and chuanxiong are 0.58–0.70 min and 0.74–0.90 min, respectively. The retention time of the characteristic peak of salvia miltiorrhiza is 1.64–2.00 min.

[0054] More preferably, the characteristic spectrum detection method for the Shuangshenling granules or its intermediate extract specifically includes the following steps:

[0055] (1) Mix the Shuangshenling granules or its intermediate extract with the extraction solvent, extract, filter, and purify to obtain the test solution;

[0056] (2) Pipette the test solution into the liquid chromatograph, determine it according to the high performance liquid chromatography method, record the chromatogram, and obtain the result;

[0057] The conditions for the high-performance liquid chromatography (HPLC) method include: an octadecylsilane-bonded silica column, a detection wavelength of 190 nm-400 nm, a flow rate of 0.9-1.1 ml / min, a column temperature of 30-40 °C, an injection volume of 10 μL, acetonitrile as mobile phase A, and 0.05%-0.15% formic acid aqueous solution as mobile phase B, with elution performed according to the following gradient:

[0058] Within 0 to 10 minutes, the volume ratio of mobile phase A to the total mobile phase gradually changes from 2% to 10% at a constant rate.

[0059] Within 10–15 minutes, the volume ratio of mobile phase A to the total mobile phase is 10% isocratic.

[0060] Within 15 to 25 minutes, the volume ratio of mobile phase A to the total mobile phase gradually changes from 10% to 20% at a constant rate.

[0061] Within 25 to 50 minutes, the volume ratio of mobile phase A to the total mobile phase gradually changes from 20% to 25% at a constant rate.

[0062] Within 50–65 minutes, the volume ratio of mobile phase A to the total mobile phase gradually and uniformly changes from 25% to 30%.

[0063] Within 65–85 minutes, the volume ratio of mobile phase A to the total mobile phase gradually changes from 30% to 70% at a constant rate.

[0064] Within 85 to 100 minutes, the volume ratio of mobile phase A to the total mobile phase gradually changes from 70% to 85% at a constant rate.

[0065] Within 100–105 min, the volume ratio of mobile phase A to the total mobile phase is 85%.

[0066] In the preferred embodiment of the present invention, by comprehensively considering and analyzing the combined effects of conditions such as the mobile phase gradient elution program, flow rate, and column temperature on separation and detection, the detection results are optimized.

[0067] The second objective of this invention is to provide a quality control method for Shuangshenling granules, wherein the quality control method includes the characteristic spectrum detection method described in the first objective.

[0068] The quality control method specifically includes: using a characteristic chromatogram detection method obtained from one of the objectives to perform quality control on Shuangshenling granules: the test sample chromatogram should show 16 characteristic peaks, and the retention times should correspond to the 16 characteristic peaks in the reference characteristic chromatogram, wherein peak 9 should be consistent with the retention time of the reference peak of salvianolic acid B. The peak corresponding to the reference peak of salvianolic acid B is taken as peak S, and the relative retention times of peaks 1 to 16 are calculated. Their relative retention times should be within ±10% of the specified value. The specified values ​​are: 0.20 (peak 1), 0.28 (peak 2), 0.39 (peak 3), 0.51 (peak 4), 0.61 (peak 5), 0.64 (peak 6), 0.81 (peak 7), 0.82 (peak 8), 1.00 (peak 9), 1.15 (peak 10), 1.48 (peak 11), 1.52 (peak 12), 1.62 (peak 13), 1.73 (peak 14), 1.79 (peak 15), and 1.82 (peak 16). Products that meet the above conditions are considered qualified.

[0069] Compared with the prior art, the present invention has the following beneficial effects:

[0070] (1) This invention utilizes high performance liquid chromatography (HPLC) and selects a specific combination of mobile phase A and mobile phase B to obtain characteristic spectra with high information content and peak response values. Sixteen common characteristic peaks were identified, which can identify seven medicinal herbs: rhubarb, eucommia, cornus officinalis, astragalus, angelica sinensis, chuanxiong, and salvia miltiorrhiza. At the same time, the characteristic peaks of fourteen substances, including gallic acid, genipin, monoglucoside, loganin, verrucoside glucoside, ferulic acid, rosmarinic acid, salvianolic acid B, aloe-emodin, rhein, emodin, chrysophanol, emodin methyl ether, and tanshinone IIA, were successfully identified. This avoids the singleness and one-sidedness of quality control and can effectively control the key quality attributes of Shuangshenling granules. It has rapid, simple, comprehensive reproducibility and stability, and is conducive to industrial promotion. It is of great significance to the quality control standard of Shuangshenling granules.

[0071] (2) Furthermore, the present invention has demonstrated through method verification that the feature map detection method has good repeatability, high feasibility, high specificity, and high sensitivity. Attached Figure Description

[0072] Figure 1 This is the HPLC chromatogram of Example 1.

[0073] Figure 2 This is the HPLC chromatogram of Example 2.

[0074] Figure 3 This is the HPLC chromatogram of Example 3.

[0075] Figure 4 This is the HPLC chromatogram of Example 4.

[0076] Figure 5 This is the HPLC chromatogram (190 nm) of Example 5.

[0077] Figure 6 This is the HPLC chromatogram (230 nm) of Example 6.

[0078] Figure 7 This is the HPLC chromatogram (250 nm) of Example 7.

[0079] Figure 8 This is the HPLC chromatogram (270 nm) of Example 8.

[0080] Figure 9 This is the HPLC chromatogram (310 nm) of Example 9.

[0081] Figure 10 This is a comparison of HPLC chromatograms for different extraction solvents.

[0082] Figure 11 This is the HPLC chromatogram of Comparative Example 1.

[0083] Figure 12 This is the HPLC chromatogram of Comparative Example 2.

[0084] Figure 13 This is the HPLC chromatogram of Comparative Example 3.

[0085] Figure 14 This is the HPLC chromatogram of Comparative Example 4.

[0086] Figure 15 This is the HPLC chromatogram of Comparative Example 5.

[0087] Figures 16-17 This is the HPLC chromatogram of the medicinal flavor peaks.

[0088] Figure 18 This is the HPLC chromatogram of the reference standard.

[0089] Figure 19 These are the UPLC-UV chromatograms and UPLC-MS total ion currents of Shuangshenling Granules.

[0090] Figure 20 HPLC comparison chart of blank solvent, test sample, and reference standard.

[0091] Figure 21 It is an overlay of instrument precision results.

[0092] Figure 22 This is a superimposed graph of instrument stability results.

[0093] Figure 23 It is a superimposed image of repetitive results.

[0094] Figure 24 This is a superimposed HPLC graph at different column temperatures.

[0095] Figure 25 It is an HPLC overlay graph at different flow rates.

[0096] Figure 26 These are HPLC overlay chromatograms of different types of chromatographic columns.

[0097] Figure 27 These are HPLC overlay chromatograms of columns with different serial numbers.

[0098] Figure 28 These are overlay HPLC graphs from different instruments.

[0099] Figure 29 It is a common pattern diagram of the characteristic spectra of 10 batches of samples.

[0100] Figure 30 It is a comparative feature map. Detailed Implementation

[0101] The present invention will be further described in detail below through embodiments. These embodiments are only used to illustrate the present invention and do not limit the scope of the present invention.

[0102] The test sample used in the following examples and comparative examples is Shuangshenling granules, and its prescription and preparation method are as follows:

[0103] Prescription: Astragalus membranaceus 211g, red ginseng 176g, deer antler powder 14g, Poria cocos 106g, Dioscorea opposita 141g, Cornus officinalis 106g, Eucommia ulmoides 106g, Cinnamomum cassia 106g, Angelica sinensis 106g, Ligusticum chuanxiong 106g, Salvia miltiorrhiza 141g, Rheum palmatum 106g, Achyranthes bidentata 106g.

[0104] Preparation method: The above thirteen ingredients are prepared by adding an equal weight of 75% ethanol to the deer antler powder and allowing it to soak for 24 hours, then drying, pulverizing, and passing through an 80-mesh sieve to obtain deer antler powder for later use; Angelica sinensis, cinnamon, and Ligusticum chuanxiong are crushed separately, added with 8 times the amount of water, and extracted by steam distillation for 6 hours, collecting the aromatic water, filtering the decoction, concentrating the filtrate appropriately, and reserving the concentrate and residue; the aromatic water is encapsulated with beta-cyclodextrin (aromatic water: beta-cyclodextrin 10:1, ground for 20 minutes), refrigerated for 24 hours, filtered, the filter cake is dried at 50℃, pulverized, and the encapsulated compound is reserved; Poria cocos, Dioscorea opposita, and Eucommia ulmoides are decocted with 10 times the amount of water for 1.5 hours, filtered, and the filtrate is concentrated appropriately, and the concentrate and residue are reserved; Astragalus membranaceus, Panax ginseng, Rheum palmatum, Cornus officinalis, Achyranthes bidentata, and Ligusticum chuanxiong are also prepared. Extract twice with 10 times the volume of 75% ethanol, reflux for 1.5 hours each time. Filter, recover the ethanol from the filtrate and concentrate to a relative density of 1.05-1.10 (60℃). Reserve the concentrate and residue. Combine the residues, add 10 times the volume of water, decoct for 1.5 hours, filter, and concentrate the filtrate appropriately. Reserve the concentrate. Combine the concentrates and concentrate under reduced pressure to a clear extract with a relative density of 1.10-1.15 (60℃). Add 94g of dextrin, stir evenly, spray dry to obtain dry extract powder. Add the above deer antler powder and betacyclodextrin inclusion complex powder, mix well, add 3g of aspartame and an appropriate amount of dextrin, mix well, granulate, dry, and make 1000g of granules to obtain Shuangshenling granules.

[0105] The instruments and reagents used in the following examples and comparative examples are as follows:

[0106] High-performance liquid chromatographs: Shimadzu LC-20AT high-performance liquid chromatograph (DAD detector); Agilent 1260 high-performance liquid chromatograph (DAD detector).

[0107] Electronic analytical balances: Tianjin Tianma Hengji Instrument Co., Ltd. TD5002C; Mettler Toledo MS204TS; Mettler Toledo XPE105.

[0108] Chromatographic columns: Inertsil ODS-3-C18 (4.6×250mm, 5μm), Innoval ODS-2 (4.6×250mm, 5μm), Ultimate LP-C18 (4.6×250mm, 5μm), Agilent ZORBAX SB-C18 (4.6×250mm, 5μm), Inevllil ODS-LP (4.6×250mm, 5μm), Phenomenex Omega Polar (4.6×250mm, 5μm), Ultimate XB-C18 (4.6×250mm, 5μm).

[0109] Reagents: Methanol was analytical grade, produced by Tianjin Damao Chemical Reagent Co., Ltd.; Acetonitrile was chromatographic grade, produced by Concord Co., Ltd.; Formic acid was chromatographic grade, produced by Tianjin Damao Chemical Reagent Co., Ltd.; n-Butanol was analytical grade, produced by Tianjin Xinbote Chemical Co., Ltd.; Water was Wahaha purified water.

[0110] Reference standard:

[0111] Tanshinone B reference standard (batch number: 111562-201917, purity: 96.6%);

[0112] Rhein reference standard (batch number: 110796-201922, purity: 99.4%);

[0113] Gallic acid reference standard (batch number: 110831-201906, purity: 91.5%);

[0114] Genipin glycoside reference standard (batch number: 111828-201805, purity: 98.1%);

[0115] Monoglycine reference standard (batch number: 111998-202104, purity: 96.8%);

[0116] Loganin reference standard (batch number: 111640-201808, purity: 99.0%);

[0117] Verbena isoflavone glucoside reference standard (batch number: 111920-201606, purity: 97.6%);

[0118] Ferulic acid reference standard (batch number: 110773-201905, purity: 99.4%);

[0119] Rosmarinic acid reference standard (batch number: 111871-201706, purity: 90.5%);

[0120] Aloe-emodin reference standard (batch number: 110795-202011, purity: 97.5%);

[0121] Rhein reference standard (batch number: 110757-201607, purity: 99.3%);

[0122] Rhein reference standard (batch number: 110756-201913, purity: 96.0%);

[0123] Rhein methyl ether reference standard (batch number: 110758-201817, purity: 99.2%);

[0124] Tanshinone IIA reference standard (batch number: 110766-202022, purity: 98.9%)

[0125] All the reference standards mentioned above were purchased from the National Institutes for Food and Drug Control.

[0126] Example 1

[0127] This embodiment provides a method for detecting the characteristic spectrum of Shuangshenling granules, as detailed below:

[0128] (1) Preparation of the test solution of Shuangshenling Granules:

[0129] Take 1g of the test sample, accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of 80% methanol, seal tightly, weigh it, sonicate (power 250W, frequency 40kHz) for 30 minutes, remove it, cool it, weigh it again, make up the lost weight with 80% methanol, shake well, filter it, and take the filtrate to obtain the test sample.

[0130] (2) Determination by high performance liquid chromatography:

[0131] Chromatographic conditions: Inertsil ODS-3 (4.6×250mm, 5μm) column was used; acetonitrile was used as mobile phase A and 0.1% formic acid solution was used as mobile phase B, and gradient elution was performed according to the specifications in the table below; the detection wavelength was 250nm; the flow rate was 1mL / min; the column temperature was 35℃; and the injection volume was 10μL.

[0132] Time (min) Mobile phase A (%) Mobile phase B (%) 0～35 2→25 98→75 35～37 25→30 75→70 37～60 30→35 70→65 60～75 35→70 65→30 75～90 70→85 30→15

[0133] The chromatogram obtained in Example 1 is as follows: Figure 1 As shown, the chromatogram contains a large amount of information and has a high peak response value, which can be used for the quality control of Shuangshenling granules. However, the resolution at 8-12 and 40-50 min still needs to be improved, and there are fewer chromatographic peaks at 55-70 min.

[0134] Example 2

[0135] The only difference from Example 1 is that the elution gradient is as shown in the table below:

[0136] Time (min) Mobile phase A (%) Mobile phase B (%) 0～20 2→10 98→90 20～40 10→20 90→80 40～55 20→25 80→75 55～70 25→70 75→30 70～80 70→85 30→15 80～100 85 15

[0137] The chromatogram obtained in Example 2 is as follows Figure 2 As shown, the chromatogram contains a large amount of information and has a high peak response value, which can be used for the quality control of Shuangshenling granules. However, the resolution needs to be improved at 60-70 min, and there are no chromatographic peaks at 85-100 min.

[0138] Example 3

[0139] The only difference from Example 1 is that the elution gradient is as shown in the table below:

[0140] Time (min) Mobile phase A (%) Mobile phase B (%) 0～10 2→10 98→90 10～20 10→13 90→87 20～25 13→20 87→80 25～55 20→30 80→70 55～60 30→80 70→20 60～80 80→85 20→15 80～90 85 15

[0141] The chromatogram obtained in Example 3 is as follows: Figure 3 As shown, the chromatogram contains a large amount of information and has a high peak response value, making it suitable for quality control of Shuangshenling granules. Its resolution is better than that of Examples 1-2. Figure 1-2 Slightly better, but the separation efficiency at 60-70 min still needs improvement.

[0142] Example 4

[0143] The only difference from Example 1 is that the elution gradient is as shown in the table below:

[0144] Time (min) Mobile phase A (%) Mobile phase B (%) 0～10 2→10 98→90 10～15 10 90 15～25 10→20 90→80 25～50 20→25 80→75 50～65 25→30 75→70 65～85 30→70 70→30 85～100 70→85 30→15 100～105 85 15

[0145] The chromatogram obtained in Example 4 is as follows Figure 4 As shown, this spectrum is different from that of Examples 1-3 ( Figure 1-3 The baseline is more stable and the separation of each peak is better. Therefore, the present invention further prefers to use the elution gradient of Example 4.

[0146] Examples 5-9

[0147] Based on Example 4, chromatograms obtained by different detection wavelengths were compared. The detection wavelengths were 190nm (Example 5), 230nm (Example 6), 250nm (Example 7), 270nm (Example 8), and 310nm (Example 9).

[0148] The chromatograms obtained in Examples 5-9 above are respectively as follows: Figures 5-9 As shown in the comparison, it can be seen that setting the detection wavelength in the range of 190 to 400 nm (Examples 5-9) can obtain a spectrum with more information. Among them, the response values ​​of each chromatographic peak are the highest and the information content is the largest when the wavelength is 250 nm.

[0149] Examples 10-11

[0150] The only difference from Example 4 is that the extraction solvent in step (1) is replaced by 60% methanol (Example 10) and 80% methanol (Example 11).

[0151] Example 12

[0152] The only difference from Example 4 is that step (1) is as follows:

[0153] Take the sample, grind it into a fine powder, take about 5g, accurately weigh (2 portions), accurately add 50ml of methanol, weigh, sonicate (power 400W, frequency 40kHz) for 30 minutes, remove, cool, weigh again, make up the lost weight with methanol, shake well, filter, evaporate the filtrate to dryness, add 25ml of water to dissolve the residue, extract one portion with water-saturated n-butanol three times, 25ml each time, and extract the other portion with ethyl acetate three times, 25ml each time, combine the n-butanol solution and ethyl acetate solution separately, evaporate to dryness, add methanol to dissolve the residue, transfer to a 25ml volumetric flask, dilute with methanol to the mark, shake well, filter, and take the filtrate to obtain the final product.

[0154] Chromatographic comparison diagrams of Examples 4, 10-12 are shown below. Figure 10 As shown.

[0155] Comparative chromatograms obtained from Examples 4 and 10-11 Figure 10 As can be seen, different preparation methods of the test sample solution resulted in different chromatograms. There were no significant differences in the peak response values ​​and the number of peaks in the test samples prepared using methanol, 60% methanol-water solution, and 80% methanol-water solution as solvents. In Example 12, the test sample prepared using methanol extraction followed by ethyl acetate extraction showed partial absence of peaks at 10-30 minutes, and the peak response values ​​were low. The test sample prepared in Example 12 using methanol extraction followed by n-butanol extraction exhibited large peak response values, conforming to the principle of maximizing information. Therefore, this invention further optimizes the use of n-butanol as the solvent for preparing the test sample solution to obtain a characteristic chromatogram with greater information content.

[0156] Comparative Example 1

[0157] The only difference from Example 4 is that water is used as the mobile phase B, and the elution gradient is shown in the table below:

[0158] Time (min) Mobile phase A (%) Mobile phase B (%) 0～10 2→10 98→90 10～15 10 90 15～25 10→20 90→80 25～50 20→25 80→75 50～65 25→30 75→70 65～85 30→70 70→30 85～100 70→85 30→15

[0159] The chromatogram of Comparative Example 1 is as follows: Figure 11 As shown in the figure, by comparing the chromatograms obtained in Example 4 and Comparative Example 1, it can be seen that the gradient elution using acetonitrile as mobile phase A and formic acid aqueous solution as mobile phase B in this invention yields a large amount of chromatographic information and higher peak response values; while Comparative Example 1, using water as mobile phase B, produces fewer chromatographic peaks and lower peak response values. Therefore, this invention, by selecting a specific combination of mobile phases for gradient elution, obtains characteristic chromatographic chromatograms that can acquire more chromatographic peak information, thereby identifying more medicinal components and providing a foundation for the quality control of Shuangshenling granules.

[0160] Comparative Examples 2-5

[0161] The only difference from Example 4 is that the chromatographic column was replaced by Inertsil ODS-3 (4.6×250mm, 5μm) with Innoval ODS-2 (4.6×250mm, 5μm) (Comparative Example 2), Ultimate LP-C18 (4.6×250mm, 5μm) (Comparative Example 3), Agilent ZORBAX SB-C18 (4.6×250mm, 5μm) (Comparative Example 4) and Inevllil ODS-LP (4.6×250mm, 5μm) (Comparative Example 5).

[0162] The chromatograms obtained from Comparative Examples 2-5 are as follows: Figure 12-15 As shown. By comparing the chromatograms obtained in Example 4 and Comparative Examples 2-5, it can be seen that the chromatogram obtained in Comparative Example 2 (…) Figure 12 The separation was poor in the 30-45 min range, compared to Comparative Example 3. Figure 13 The chromatograms obtained showed poor resolution at 30-45 min and 55-60 min, compared to Comparative Example 4. Figure 14 The chromatograms obtained showed poor resolution at 30-35 min, compared to Comparative Example 5. Figure 15 The obtained chromatograms showed poor resolution within the 25-38 min range. Therefore, it can be seen that the characteristic chromatograms obtained by selecting a specific chromatographic column in this invention can achieve better resolution, providing a foundation for the quality control of Shuangshenling granules.

[0163] Example 13: Characteristic Spectrum Analysis of Shuangshenling Granules

[0164] The following analysis is performed on the feature map obtained in Example 4:

[0165] 1. Assignment of chromatographic peaks of medicinal herbs

[0166] (1) Weigh out 0.52g of Poria cocos, 0.70g of Dioscorea opposita, 0.52g of Eucommia ulmoides, 0.52g of Angelica sinensis, 0.52g of Ligusticum chuanxiong, and 0.52g of Cinnamomum cassia. Add 25ml of water to each, decoct for 30 minutes, filter, add water-saturated n-butanol to the filtrate and shake to extract 3 times, 25ml each time. Combine the n-butanol extracts, evaporate to dryness, dissolve the residue in methanol and transfer to a 25ml volumetric flask, dilute to the mark, shake well, and the product is obtained.

[0167] (2) Weigh out 1.05g of Astragalus membranaceus, 0.88g of Panax ginseng, 0.52g of Rheum palmatum, 0.52g of Cornus officinalis, 0.52g of Achyranthes bidentata, and 0.70g of Salvia miltiorrhiza. Add 25ml of 75% ethanol to each herb, reflux for 30 minutes, filter, add 25ml of water to the residue, decoct for 30 minutes, filter, combine the two filtrates and evaporate to dryness, dissolve the residue in 25ml of water, extract three times with water-saturated n-butanol, 25ml each time, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in methanol and transfer to a 25ml volumetric flask, dilute to the mark, shake well, and the product is obtained.

[0168] (3) Inject 10 μl of each of the above-mentioned medicinal slices solutions into a high-performance liquid chromatograph and perform detection under the same chromatographic conditions as in Example 4 to obtain the chromatogram of the single-herb test sample solution.

[0169] (4) Compare the chromatogram of the single herb slices with the chromatogram of the test sample obtained in Example 4, such as... Figures 16-17 As shown, in the chromatogram of the test sample, rhubarb, eucommia bark, cornus fruit, astragalus root, angelica root, chuanxiong rhizome, and salvia root all have corresponding chromatographic peaks. Among them, peaks 1, 10, 11, 12, 13, 14, and 15 belong to rhubarb; peak 2 belongs to eucommia bark; peaks 3 and 4 belong to cornus fruit; peak 5 belongs to astragalus root; peaks 6 and 8 belong to angelica root and chuanxiong rhizome; and peaks 7, 9, and 16 belong to salvia root.

[0170] 2. Chromatographic peak identification

[0171] (1) Take appropriate amounts of each reference standard (emodin methyl ether, emodin, emodin, rhein, aloe-emodin, tanshinone IIA, salvianolic acid B, rosmarinic acid, ferulic acid, verrucoside glucoside, monoglucoside, loganin, genipin glycoside, gallic acid) and add methanol to prepare a solution containing 0.1 mg per ml, thus obtaining the reference standard solution.

[0172] (2) Inject 10 μl of the above reference solution into a high performance liquid chromatograph and perform detection under the same chromatographic conditions as in Example 4 to obtain the chromatogram of the reference solution.

[0173] (3) Compare the chromatograms of each reference solution with the chromatogram of the test sample obtained in Example 4, such as... Figure 18 As shown, after comparing the retention time with the reference standard, the characteristic spectrum of Shuangshenling Granules showed that peak 1 was gallic acid, peak 2 was genipin acid, peak 3 was monoglycine, peak 4 was loganin, peak 5 was verrucoside glucoside, peak 6 was ferulic acid, peak 7 was rosmarinic acid, peak 9 was salvianolic acid B, peak 11 was aloe-emodin, peak 12 was rhein, peak 13 was emodin, peak 14 was chrysophanol, peak 15 was emodin methyl ether, and peak 16 was tanshinone IIA.

[0174] (4) Qualitative analysis of the chemical components of Shuangshenling granules was performed using UPLC-Q-TOF-MS, and the characteristic chromatographic peaks were identified and deduced. UPLC-UV chromatograms and total ion chromatograms (positive mode) are shown below. Figure 19 As shown, since the liquid phase of the liquid chromatography-mass spectrometry (LC-MS) instrument is UPLC, its chromatogram differs somewhat from that of the HPLC fingerprint chromatogram. The specific peak correspondence is shown in Table 1.

[0175] Table 1. Characteristic map of Shuangshenling Granules: herbal flavor attribution and peak identification.

[0176]

[0177] The results showed that, after comparison with the reference standard and each medicinal decoction piece, each medicinal decoction piece and its corresponding actual substance (test sample) had corresponding chromatographic peaks. Chromatographic peaks with good stability and suitable response values ​​in the characteristic chromatograms were selected as characteristic peaks, and a total of 16 characteristic peaks were identified. Among them, peaks 1, 10, 11, 12, 13, 14, and 15 are characteristic peaks of rhubarb; peak 2 is a characteristic peak of eucommia; peaks 3 and 4 are characteristic peaks of cornus officinalis; peak 5 is a characteristic peak of astragalus membranaceus; peaks 6 and 8 are characteristic peaks of angelica sinensis and chuanxiong rhizome; and peaks 7, 9, and 16 are characteristic peaks of salvia miltiorrhiza.

[0178] 3. Selection of reference point

[0179] Compared with the reference standard, the chromatographic peaks at 10.916 minutes and 15.126 minutes were gallic acid, genipin glycoside, 21.422 minutes were monoglycine, 27.411 minutes were loganin, 33.312 minutes were verbascoside glucoside, 34.532 minutes were ferulic acid, 44.395 minutes were rosmarinic acid, 56.075 minutes were salvianolic acid B, 80.459 minutes were aloe-emodin, 83.542 minutes were rhein, 88.461 minutes were emodin, 94.553 minutes were chrysophanol, 97.594 minutes were emodin methyl ether, and 99.285 minutes were tanshinone IIA. Among them, the absorption intensity and retention time of the chromatographic peak of salvianolic acid B are moderate and the response is stable, achieving baseline separation. Therefore, salvianolic acid B is selected as the reference peak and as the S peak. The relative retention of each peak is calculated using the reference peak for evaluation.

[0180] Example 14 Methodological Investigation

[0181] The following methodological investigations were conducted based on the sample preparation method and chromatographic conditions of Example 4:

[0182] 1. Examination of specificity and integrity

[0183] To investigate whether the blank solvent interfered with the characteristic chromatogram of Shuangshenling granules, the test solution, reference solution, and blank solvent (methanol) were precisely pipetted and analyzed according to the chromatographic conditions of Example 4. Simultaneously, the integrity of the sample was examined by extending the sampling time by 30 minutes (using the highest organic phase concentration in the gradient table, isocratic elution). The results showed that the blank solvent did not interfere with the common peak position, and there were essentially no chromatographic peaks after 105 minutes. The results generally met the principle of maximizing information content. Figure 20 As shown.

[0184] 2. Instrument precision test

[0185] Take the test sample, grind it finely, and accurately weigh approximately 5g. Perform the determination under the chromatographic conditions described in Example 4, injecting the sample six times consecutively. The characteristic chromatogram of the test sample should show a peak with the same retention time as the reference peak. Using the S peak as a reference, calculate the relative retention times and relative peak areas of the remaining peaks. See [link to specific determination results] for details. Figure 21 (The numbers 1-6 in the figure represent the numbering of 6 injections), Tables 2 and 3.

[0186]

[0187] 3. Stability test

[0188] Take the test sample, grind it finely, and accurately weigh approximately 5g. Perform the determination under the chromatographic conditions of Example 4, injecting the sample at 0, 2, 4, 8, 12, and 24 hours. The characteristic chromatogram of the test sample should show a peak with the same retention time as the reference peak. Using the S peak as a reference, calculate the relative retention times and relative peak areas of the remaining peaks. See [link to specific results] for details. Figure 22 Tables 4 and 5.

[0189]

[0190] 4. Repeatability test

[0191] Take the test sample, grind it finely, and accurately weigh approximately 5g (6 portions in total). Perform the determination under the chromatographic conditions described in Example 4. The characteristic chromatogram of the test sample should show a peak with the same retention time as the reference peak. Using peak S as a reference, calculate the relative retention times and relative peak areas of the remaining peaks. See [link to specific determination results] for details. Figure 23 (Numbers 1-6 in the figure represent the numbers of the 6 samples), Tables 6 and 7.

[0192]

[0193] 5. Durability test

[0194] The robustness of the test sample to the chromatographic conditions was investigated under different flow rates, column temperatures, column brands, different SN numbers of the same model column, and two instruments from different manufacturers. The characteristic chromatogram of the test sample should show a peak with the same retention time as the reference peak. Using the S peak as a reference, the relative retention times of the remaining peaks were calculated. Specific results are shown in Tables 8 to 12. Figures 24-28 ,in, Figure 24 This is a superimposed HPLC chromatogram at different column temperatures. Figure 25 This is a stacked HPLC graph showing different flow rates. Figure 26 HPLC overlay chromatograms of different column types: 1-Ultimate XB-C18 (4.6*250mm, 5μm); 2-Phenomenex Omega Polar(4.6*250mm,5μm); 3-Intersil ODS-3(4.6*250mm,5μm)), Figure 27 HPLC overlay chromatograms of columns with different serial numbers (1-Intersil ODS-3 (4.6*250mm, 5μm) SN:1A7177156; 2-Intersil ODS-3 (4.6*250mm, 5μm) SN:1A7187844). Figure 28 This is a superimposed HPLC chromatogram from different instruments.

[0195]

[0196]

[0197] 6. Method Validation

[0198] Ten batches of samples were analyzed using the chromatographic method described in Example 4. The corresponding peak of the salvianolic acid B reference was taken as the S peak. The relative retention times of each characteristic peak and the S peak in the ten batches of samples (211215-01, 211215-02, 211215-03, 211228-01, 211228-02, 211228-03, C-SSLKL-211201, C-SSLKL-220202, C-SSLKL-220203, C-SSLKL-220204) were calculated. Using the "Similarity Evaluation Software System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" issued by the National Pharmacopoeia Commission, the AIA files of the ten batches of HPLC chromatograms were exported. Chromatographic peaks were automatically matched to form a common pattern diagram, generating a reference chromatogram. The results are shown in […]. Figure 29 (R-compare characteristic maps, S1-211215-01, S2-211215-02, S3-211215-03, S4-211228-01, S5-211228-02, S6-211228-03, S7-C-SSLKL-211201, S8-C-SSLKL-220202, S9-C-SSLKL-220203, S10-C-SSLKL-220204) and Figure 30 (Peak 1: Gallic acid, Peak 2: Genipin glycoside, Peak 3: Monoside, Peak 4: Loganin, Peak 5: Versicolor isoflavone glucoside, Peak 6: Ferulic acid, Peak 7: Rosmarinic acid, Peak 9(S): Tanshinone B, Peak 11: Aloe-emodin, Peak 12: Rhein, Peak 13: Emodin, Peak 14: Rhein, Peak 15: Emodin methyl ether, Peak 16: Tanshinone IIA), and Table 13.

[0199]

[0200] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for constructing a characteristic spectrum of a traditional Chinese medicine composition or its preparation, wherein the traditional Chinese medicine composition comprises Astragalus membranaceus, red ginseng, deer antler powder, Poria cocos, Dioscorea opposita, Cornus officinalis, Eucommia ulmoides, Cinnamomum cassia, Angelica sinensis, Ligusticum chuanxiong, Salvia miltiorrhiza, Rheum palmatum, and Achyranthes bidentata, characterized in that, The method employs high-performance liquid chromatography. The chromatographic conditions are as follows: Chromatographic column: Octadecylsilane-bonded silica gel column: Mobile phase: Acetonitrile is used as mobile phase A, and water or formic acid aqueous solution is used as mobile phase B for gradient elution.

2. The method for constructing the characteristic spectrum of a traditional Chinese medicine composition or its preparation as described in claim 1, Its features are, The chromatographic conditions are as follows: Chromatographic column: Octadecylsilane-bonded silica gel column; column length 250 mm, diameter 4.6 mm, particle size 5 μm; Mobile phase: Gradient elution was performed using acetonitrile as mobile phase A and formic acid aqueous solution as mobile phase B. Detection wavelength: 190nm-400nm; Column temperature: 30~40℃.

3. The method for constructing the characteristic spectrum of a traditional Chinese medicine composition or its preparation as described in claim 1 or 2, characterized in that, The chromatographic conditions are as follows: Column: Selected from Inertsil ODS-3-C18 and Phenomenex OmegaPolar or Ultimate XB-C18; preferred Inertsil ODS-3-C18; Mobile phase: Gradient elution was performed using acetonitrile as mobile phase A and formic acid aqueous solution as mobile phase B. Detection wavelength: 250nm; Column temperature: 30~40℃; The gradient elution procedure is as follows: Within 0 to 10 minutes, the volume ratio of mobile phase A to the total mobile phase gradually changes from 2% to 10% at a constant rate. Within 10–15 minutes, the volume ratio of mobile phase A to the total mobile phase is 10% isocratic. Within 15 to 25 minutes, the volume ratio of mobile phase A to the total mobile phase gradually changes from 10% to 20% at a constant rate. Within 25 to 50 minutes, the volume ratio of mobile phase A to the total mobile phase gradually changes from 20% to 25% at a constant rate. Within 50–65 minutes, the volume ratio of mobile phase A to the total mobile phase gradually changes from 25% to 30% at a uniform rate. Within 65–85 minutes, the volume ratio of mobile phase A to the total mobile phase gradually changes from 30% to 70% at a constant rate. Within 85 to 100 minutes, the volume ratio of mobile phase A to the total mobile phase gradually changes from 70% to 85% at a constant rate. Within 100–105 min, the volume ratio of mobile phase A to the total mobile phase is 85%.

4. A method for constructing the characteristic spectrum of a traditional Chinese medicine composition or its preparation as described in claim 1 or 2, comprising the following steps: (1) Prepare a test solution of a traditional Chinese medicine composition or its preparation; The traditional Chinese medicine composition is prepared from the following herbs: Astragalus membranaceus, red ginseng, deer antler powder, Poria cocos, Dioscorea opposita, Cornus officinalis, Eucommia ulmoides, Cinnamomum cassia, Angelica sinensis, Ligusticum chuanxiong, Salvia miltiorrhiza, Rheum palmatum, and Achyranthes bidentata; the traditional Chinese medicine composition or its preparation is mixed with an extraction solvent, extracted, filtered, and purified to obtain the test solution; (2) Take the test solution, inject it into the liquid chromatograph, determine it according to the high performance liquid chromatography method, and record the chromatogram.

5. The method for constructing a characteristic spectrum of a traditional Chinese medicine composition or its preparation according to claim 4, characterized in that, The extraction solvent in step (1) is selected from methanol and / or n-butanol, ethyl acetate, preferably n-butanol; the extraction method is ultrasonic treatment.

6. The feature map construction method according to claim 4, characterized in that, In step (2), the concentration of the formic acid aqueous solution is 0.05%-0.15%.

7. The feature map construction method according to claim 4, characterized in that, In step (2), the flow rate of the high performance liquid chromatography is 0.9-1.1 ml / min.

8. The feature map construction method according to claim 4, characterized in that, In step (2), the characteristic peaks of gallic acid, genipin, monoglucoside, loganin, verrucoside glucoside, ferulic acid, rosmarinic acid, salvianolic acid B, aloe-emodin, rhein, emodin, chrysophanol, emodin methyl ether and tanshinone IIA can be identified in the obtained chromatogram. Preferably, in the chromatogram, salvianolic acid B is used as a reference peak, and as peak S, the relative retention times of each peak are calculated using the reference peak. The relative retention time of the characteristic peak of gallic acid is 0.18–0.22, the relative retention time of the characteristic peak of genipin is 0.25–0.31, the relative retention time of the characteristic peak of monoglucoside is 0.35–0.43, the relative retention time of the characteristic peak of loganin is 0.46–0.56, the relative retention time of the characteristic peak of verrucoside isoflavone glucoside is 0.55–0.67, and the relative retention time of the characteristic peak of ferulic acid is… The relative retention times of the characteristic peaks of the following substances are as follows: 0.58–0.70, 0.73–0.89, 1.33–1.63, 1.37–1.67, 1.46–1.78, 1.56–1.90, 1.61–1.97, and 1.64–2.

00.

9. The feature map construction method according to claim 4, characterized in that, In step (2), the characteristic peaks of rhubarb, eucommia, cornus officinalis, astragalus, angelica sinensis, chuanxiong and salvia miltiorrhiza can be identified in the obtained chromatogram; Preferably, in the chromatogram, salvianolic acid B is used as a reference peak, which is also the S peak. The relative retention times of each peak are calculated using the reference peak. The relative retention times of the characteristic peaks of rhubarb are 0.18–0.22 min, 1.04–1.26 min, 1.33–1.63 min, 1.37–1.67 min, 1.46–1.78 min, 1.56–1.90 min, and 1.61–1.97 min. The retention times of the characteristic peaks of eucommia are 0.25–0.31 min. The retention times of the characteristic peaks of cornus officinalis are 0.35–0.43 min and 0.46–0.56 min. The retention times of the characteristic peaks of astragalus membranaceus are 0.55–0.67 min. The retention times of the characteristic peaks of angelica sinensis and chuanxiong are 0.58–0.70 min and 0.74–0.90 min, respectively. The retention time of the characteristic peak of salvia miltiorrhiza is 1.64–2.00 min.

10. A quality control method for Shuangshenling granules, characterized in that, The quality control method includes the feature map construction method according to any one of claims 1-9.

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