A method for constructing a high performance liquid chromatography (HPLC) characteristic spectrum of a traditional Chinese medicine preparation for treating chronic liver disease and a quality detection method

Abstract

The application belongs to the technical field of medicine quality control, and relates to a construction method and quality detection method of high-performance liquid chromatography characteristic spectrum of a traditional Chinese medicine preparation for treating chronic liver disease; comprising the following steps: step S1, preparation of reference solution: morroniside control product is weighed, precisely weighed and determined, and 50% methanol is used to prepare the reference solution; preparation of test solution: the prepared traditional Chinese medicine preparation is finely ground, mixed with 50% methanol, ultrasonically treated, and then diluted, filtered and obtained; step S2, high-performance liquid chromatography detection is performed on the reference solution and the test solution respectively, and the characteristic spectrum of the traditional Chinese medicine preparation for treating chronic liver disease is obtained; the detection wavelength is 240 nm; the characteristic spectrum comprises eight characteristic peaks, wherein peak 1 is 5-hydroxymethyl furfural, peak 2 (S) is morroniside, peak 3 is loganin, peak 5 is spinooside, peak 6 is calycosin-7-glucoside, peak 7 is styrine, and peak 8 is salvianolic acid B.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug quality control, and relates to a method for constructing a high-performance liquid phase characteristic spectrum and a quality detection method for a traditional Chinese medicine preparation for treating chronic liver disease. Background Art

[0002] A TCM characteristic spectrum / characteristic spectrum refers to a spectrum of common peaks that characterize the TCM after appropriate processing and analytical methods. This spectrum is specific, encompassing not only the analysis of several known components but also the relative content analysis of known components and various unknown components.

[0003] A traditional Chinese medicine preparation for the treatment of chronic liver disease, comprising eight medicinal ingredients, including Astragalus, Polygonum multiflorum, Salvia miltiorrhiza, Cornus officinalis, Ziziphus jujuba seed, Pseudostellaria baicalensis, and Lonicera japonica, and / or their processed products, lacks a complete profile of these medicinal ingredients. Furthermore, conventional thin-layer scanning (TLC) is used to determine the content, which can lead to frequent testing and unsatisfactory results due to the use of specialized equipment and numerous interference factors. The existing TLC scanning method for determining ursolic acid in Cornus officinalis is inconsistent with the control parameters of the medicinal ingredient standard and the finished drug standard. Furthermore, the dual-wavelength scanning method suffers from low accuracy and operational difficulty.

[0004] Therefore, it is very necessary to establish a detection and control technology that is faster, more effective, simpler in process, and more accurate in results. Summary of the Invention

[0005] The purpose of the present invention is to address the above-mentioned problems existing in the prior art and to propose a method for constructing a high-performance liquid chromatography characteristic spectrum and a quality detection method for a traditional Chinese medicine preparation for treating chronic liver disease.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A method for constructing a characteristic spectrum of a traditional Chinese medicine preparation for treating chronic liver disease comprises the following steps:

[0008] Step S1, preparation of reference solution: accurately weigh the morroniside reference solution, and add 50% methanol to prepare the reference solution;

[0009] Preparation of test solution: Take the prepared Chinese medicine preparation, grind it into powder, add 50% methanol to mix, then ultrasonicate it, make up the volume, and filter it;

[0010] Step S2, performing high performance liquid chromatography on the reference solution and the test solution to obtain a characteristic spectrum of the traditional Chinese medicine preparation for treating chronic liver disease;

[0011] The detection wavelength is 240nm;

[0012] The characteristic spectrum includes 8 characteristic peaks, wherein peak 1 is 5-hydroxymethylfurfural, peak 2 (S): morroniside, peak 3 is loganin, peak 5 is spinosin, peak 6 is calycosin isoflavone glucoside, peak 7 is diphenylethylene glucoside, and peak 8 is salvianolic acid B.

[0013] Preferably, the reference peak of the HPLC characteristic spectrum of the traditional Chinese medicine preparation for treating chronic liver disease is the morroniside peak.

[0014] Preferably, in the high performance liquid chromatography detection process, octadecylsilane bonded silica gel is used as the filler, acetonitrile is used as the mobile phase A, and phosphoric acid aqueous solution is used as the mobile phase B.

[0015] Furthermore, the concentration of the phosphoric acid aqueous solution is 0.2% to 0.4%.

[0016] Furthermore, the concentration of the phosphoric acid aqueous solution is 0.3%.

[0017] Preferably, the elution mode of the high performance liquid chromatography is a gradient system, and the mobile phase A: mobile phase B is (4% to 35%): (65% to 96%)

[0018] Furthermore, in the initial stage, the ratio of mobile phase A to mobile phase B is (4% to 8%): (92% to 96%).

[0019] Furthermore, in the initial stage, the ratio of mobile phase A to mobile phase B was 6% to 94%.

[0020] Furthermore, the gradient elution conditions of the HPLC detection process are:

[0021] 0-20 min, 6% mobile phase A, 94% mobile phase B;

[0022] 20-60 minutes, 6% → 30% mobile phase A, 94% → 70% mobile phase B;

[0023] 60-70 minutes, 30% mobile phase A, 70% mobile phase B.

[0024] Preferably, the test solution in step S1 is a traditional Chinese medicine preparation for treating chronic liver disease, which is ground into powder and accurately weighed, placed in a 50 ml volumetric flask, and an appropriate amount of 50% methanol is added. The solution is ultrasonically treated for 30 minutes, cooled, and diluted to the mark with 50% methanol.

[0025] Furthermore, the ultrasonic treatment power is 300W and the frequency is 40kHz.

[0026] Furthermore, in step S1, the weight-to-volume ratio of the traditional Chinese medicine preparation for chronic liver disease to the test solution is 1 mg: (8-12) ml; and each 1 ml of the reference solution contains 10 μg of morroniside.

[0027] Furthermore, in step S1, the weight-to-volume ratio of the traditional Chinese medicine preparation for chronic liver disease to the test sample solution is 1 mg:10 ml.

[0028] Preferably, the reference peak of the HPLC characteristic spectrum of the traditional Chinese medicine preparation for treating chronic liver disease is the morroniside peak.

[0029] Furthermore, the peak corresponding to the morroniside reference was taken as the S peak, and the specified values ​​of the relative retention times of the characteristic peaks and the S peak were: 0.346 (peak 1), 1.000 (peak 2S), 1.564 (peak 3), 2.032 (peak 4), 2.088 (peak 5), 2.119 (peak 6), 2.147 (peak 7), and 2.714 (peak 8).

[0030] Furthermore, during the high performance liquid chromatography detection process, the theoretical plate number calculated based on the morroniside peak should be no less than 3000.

[0031] Preferably, the Chinese medicine preparation for treating chronic liver disease comprises raw materials including Cornus officinalis, Ziziphus jujuba seed, Tribulus terrestris, Polygonum multiflorum, Astragalus membranaceus, Pseudostellaria baicalensis, Salvia miltiorrhiza, and Lonicera japonica; the above raw materials are medicinal materials, and / or decoction pieces, and / or processed products, and / or powders of medicinal materials, and / or formula granules of medicinal materials, and / or powders of decoction pieces, and / or formula granules of decoction pieces, and / or powders of processed products, and / or formula granules of processed products.

[0032] Furthermore, the raw materials of the traditional Chinese medicine preparation for treating chronic liver disease include, by weight: 10-20 parts of Cornus officinalis, 1-20 parts of Ziziphus jujuba seeds, 5-20 parts of Tribulus terrestris, 7-15 parts of Polygonum multiflorum, 5-25 parts of Astragalus membranaceus, 5-30 parts of Pseudostellaria baicalensis, 5-20 parts of Salvia miltiorrhiza, and 5-20 parts of Lonicera japonica.

[0033] Furthermore, the raw materials of the traditional Chinese medicine preparation include: 15 parts of Cornus officinalis, 15 parts of Ziziphus jujuba seeds, 15 parts of Tribulus terrestris, 10 parts of Polygonum multiflorum, 15 parts of Astragalus, 15 parts of Pseudostellaria baicalensis, 15 parts of Salvia miltiorrhiza, and 15 parts of Lonicera japonica.

[0034] Furthermore, the preparation of the Chinese medicine preparation includes: combining and decocting the raw materials, concentrating and drying them to make a Chinese medicine composition; or combining and decocting part of them, decocting part of them separately, and then combining and concentrating and drying them to make a Chinese medicine composition; or decocting all of them separately, combining them, concentrating and drying them to make a Chinese medicine composition; or combining and decocting part of them, crushing part of them into fine powder, decocting part of the medicinal flavors separately, and then combining them, concentrating and drying them to make a Chinese medicine composition; directly crushing all of them into fine powder to make a Chinese medicine composition; making preparations from the Chinese medicine composition, the preparations include but are not limited to tablets, pills, granules, and oral liquids.

[0035] A high-performance liquid chromatography quality detection method for a traditional Chinese medicine preparation for treating chronic liver disease comprises obtaining a spectrum of a test sample solution according to a high-performance liquid chromatography detection method, and comparing and analyzing the spectrum with a standard characteristic spectrum obtained by a method for constructing a high-performance liquid chromatography characteristic spectrum of a traditional Chinese medicine preparation for treating chronic liver disease.

[0036] Preferably, the relative retention time of the qualified test sample is ±10% of the value specified in the standard characteristic spectrum.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] 1. The high-performance liquid chromatography characteristic spectrum determination method of the present invention can detect 8 peaks, which is conducive to the efficient identification of traditional Chinese medicine preparations for treating chronic liver disease, including Cornus officinalis, Ziziphus jujuba seeds, Tribulus terrestris, Polygonum multiflorum, Astragalus membranaceus, Pseudostellaria baicalensis, Salvia miltiorrhiza, and Lonicera japonica, and obtains the peaks of 5-hydroxymethylfurfural, morroniside, loganin, spinosin, calycosin isoflavone glucoside, stilbene glucoside, and salvianolic acid B.

[0039] 2. The present invention uses the HPLC-UV method to explore the characteristic spectrum of the tablets of traditional Chinese medicine preparations for treating chronic liver disease containing Cornus officinalis, Ziziphus jujuba seeds, Tribulus terrestris, Polygonum multiflorum, Astragalus membranaceus, Pseudostellaria baicalensis, Salvia miltiorrhiza, and Lonicera japonica for treating chronic liver disease. The chromatograms of 8 flavor-deficient negative samples were collected for comparison. The characteristic peak regions of each component were associated and found to be Cornus officinalis (peak 1, peak 2, peak 3, peak 4), Lonicera japonica (peak 3), Ziziphus jujuba seeds (peak 5), Astragalus membranaceus (peak 6), Polygonum multiflorum processed (peak 7), and Salvia miltiorrhiza (peak 8); and since thin layer identification quality control items for Cornus officinalis, Lonicera japonica, and Ziziphus jujuba seeds have not been established, the establishment of the characteristic spectrum of the present invention can supplement the quality control of the corresponding medicinal flavors and better reflect the quality of the preparation.

[0040] 3. The characteristic spectrum constructed by the method for constructing the HPLC characteristic spectrum of the traditional Chinese medicine preparation for treating chronic liver disease of the present invention is combined with the HPLC quality detection method of the traditional Chinese medicine preparation for treating chronic liver disease to efficiently detect the qualified rate of the preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The liquid chromatography of the present invention adopts ultraviolet full gradient wavelength collection chart.

[0042] Figure 2 It is the characteristic peak diagram of liquid chromatography 230nm-280nm in the characteristic spectrum construction method of the present invention.

[0043] Figure 3 The chromatograms are the characteristic spectrum construction methods of different mobile phases of the present invention.

[0044] Figure 4 Construct method chromatograms for characteristic profiles at different column temperatures.

[0045] Figure 5 This is a comparison chart of the common peaks in the characteristic spectrum and the UV absorption spectrum of the standard sample, in which the reference sample solution and the test sample solution are detected by high performance liquid chromatography in the construction of the standard characteristic spectrum of the present invention.

[0046] Figure 6 This is a superimposed comparison diagram of the negative HPLC spectra of each component in the traditional Chinese medicine composition preparation used in the construction of the standard characteristic spectrum of the present invention.

[0047] Figure 7 This is a test diagram specific to the characteristic spectrum construction method of the present invention.

[0048] Figure 8 It is the control characteristic feature map of the present invention. DETAILED DESCRIPTION

[0049] The following are specific embodiments of the present invention, which further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments.

[0050] (1) Construction of HPLC characteristic spectrum

[0051] The instruments, reagents, reference substances, and samples used in the present invention are as follows:

[0052] (1) Instruments:

[0053]

[0054] (2) Reagents:

[0055] Product Name level batch number factory Acetonitrile Chromatographically pure 22015122 TEDIA water ------ 220207 Wahaha Drinking Purified Water (Bottled) phosphoric acid analytically pure C13465060 Maclean Methanol Chromatographically pure 22025303 TEDIA

[0056] (3) Reference substances:

[0057] Product Name batch number purity source Morroniside 111998-202104 96.8％ China Food and Drug Inspection Institute

[0058] (4) Acquisition of samples of traditional Chinese medicine preparations:

[0059] Prepare wine-soaked cornus fruit slices, stir-fried spinach seed slices, stir-fried tribulus terrestris slices, astragalus slices, Pseudostellaria pseudoginseng slices, Salvia miltiorrhiza slices, honeysuckle vine slices, and processed Polygonum multiflorum slices according to the proportions described in Table 1.

[0060] The wine-cooked cornus pieces were placed in a sterilizer and sterilized with circulating steam at 120°C for 40 minutes, dried, and ground into cornus powder with a particle size of 200 mesh.

[0061] The stir-fried pieces of Chinese jujube seeds were put into an extraction tank and decocted three times with water. The first time, 10 times the amount of water was added and the decocting time was 2 hours. The second time, 8 times the amount of water was added and the decocting time was 1.5 hours. The third time, 7.5 times the amount of water was added and the decocting time was 1.5 hours. The decoction was filtered, concentrated, and microwave-dried to prepare the first extract powder, 100 mesh.

[0062] Put the fried Tribulus terrestris slices, Astragalus membranaceus slices, Pseudostellaria chinensis slices, Salvia miltiorrhiza slices, Lonicera japonica slices, and Polygonum multiflorum slices into an extraction tank, add water and boil twice, adding water twice, 8 times and 6 times respectively, for 1.5 hours each time, filter the two decoctions, concentrate, and microwave dry them to make the second extract powder, 100 mesh;

[0063] 15 parts of Cornus officinalis powder were added to 1.6 parts of the first extract powder and 10.2 parts of the second extract powder (without adding excipients), and the mixture was granulated with 92% ethanol. The wet granules were placed in a circulating drying oven and dried at 60°C. After drying, the granules were sized, 1% magnesium stearate was added and mixed, and the mixture was pressed into plain tablets to obtain a sample of a traditional Chinese medicine tablet.

[0064] (5) Selection of measurement wavelength

[0065] The above-mentioned Chinese medicine preparation sample (batch number: S0601) was used for testing, and the UV full gradient wavelength was used for acquisition (see Figure 1 ) found that in the short wavelength range (190nm ~ 230nm), the baseline in the chromatogram fluctuates greatly, and the interference with the chromatographic peak is also greater; after 280nm, the number of chromatographic peaks is small, which is not conducive to the identification of characteristic peaks. Therefore, the measurement wavelength is selected between 230nm and 280nm, and the six wavelengths of 230nm, 240nm, 250nm, 260nm, 270nm, and 280nm are selected for measurement and comparison (see Figure 2 ), and finally selected a wavelength of 240 nm as the measurement wavelength of the characteristic spectrum.

[0066] (6) Selection of mobile phase parameters

[0067] The column temperature was set to 40°C, the flow rate to 1.0 ml / min, the injection volume to 20 μl, and the detection wavelength to 240 nm. Octadecylsilane bonded silica gel was used as the filler (ACE Excel 5C18-AR 250×4.6 mm, 5 μm). The theoretical plate number, calculated based on the morroniside peak, should be no less than 3000.

[0068] The elution process is carried out according to the following 5 methods. The elution process diagram is shown in Figure 3

[0069] Method 1:

[0070] Time (min) Acetonitrile (%) Methanol (%) 0～20 6 94 20～65 6→20 94→80 65～70 20 80

[0071] Method 2:

[0072]

[0073] Method 3:

[0074] Time (min) Acetonitrile (%) 0.3% phosphoric acid (%) 0～20 6 94 20～65 6→30 94→70 65～70 30 70

[0075] Method 4:

[0076] Time (min) Acetonitrile (%) 0.3% phosphoric acid (%) 0～20 6 94 20～60 6→30 94→70 60～70 30 70

[0077] Method 5:

[0078] Time (min) Acetonitrile (%) 0.1% phosphoric acid (%) 0～20 6 94 20～60 6→30 94→70 60～70 30 70

[0079] The mobile phase gradient elution program under different conditions leads to changes in the chromatogram baseline, chromatographic peak shape, and separation degree from adjacent peaks. Figure 3 Select the parameters in Method 4.

[0080] (7) Selection of column temperature

[0081] Select different column temperatures (20℃, 30℃, 40℃) to examine the HPLC spectrum of the test sample. The results are shown in Figure 4 The results showed that the peak shape and separation of morroniside components were better at 40℃. As the column temperature decreased, the peak shape and separation decreased. The column temperature was selected as 40℃.

[0082] (8) Construction of standard feature maps:

[0083] Preparation of reference solution: Weigh the morroniside reference and add 50% methanol to prepare the reference solution (concentration: 10 μg / ml);

[0084] Preparation of test solution: Grind the prepared Chinese medicine preparation sample (batch number: S0601) finely, accurately weigh 0.5 g, place it in a 50 ml volumetric flask, add an appropriate amount of 50% methanol, ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes, let cool, dilute to the scale with 50% methanol, shake well, filter, and take the filtrate.

[0085] Set up the HPLC parameters:

[0086] Chromatographic column: ACE Excel 5C18-AR (4.6×250mm, 5um)

[0087] Filler: Octadecylsilane bonded silica gel

[0088] Mobile phase:

[0089] Time (min) A (acetonitrile)% B (0.3% phosphoric acid solution)% 0～20 6 94 20～60 6→30 94→70 60～70 30 70

[0090] Column temperature: 40°C, flow rate: 1.0 ml / min, injection volume: 20 μl, detection wavelength: 240 nm; the theoretical plate number calculated based on the morroniside peak should be no less than 3000.

[0091] The reference solution and the test solution were detected by high performance liquid chromatography. The common peaks in the characteristic spectrum were compared with the UV absorption spectrum of the standard. Figure 5 As shown; HPLC-UV method and UPLC-Obitrap high-resolution mass spectrometry were used to identify the 8 characteristic peaks in the characteristic spectrum and attribute the medicinal taste, and compared with the corresponding reference substances for confirmation. It can be seen that the attribution of each chemical component is as shown in Table 1, and the negative HPLC spectrum of each component is superimposed and compared as shown in the figure below. Figure 6 .

[0092] Table 1. Corresponding chemical composition classification table

[0093]

[0094] according to Figure 6 As shown in Table 1, the chromatogram of the test sample should show 8 characteristic peaks, such as Figure 7 As shown, 7 peaks correspond to the known 5-hydroxymethylfurfural, morroniside, loganin, spinosin, calycosin glucoside, stilbene glucoside, and salvianolic acid B; peak 4 has not been located and its composition is unknown.

[0095] The retention time of peak 2 is consistent with that of the reference substance. The peak corresponding to the morroniside reference substance is designated as the S peak. The relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.346 (peak 1), 1.000 (peak 2S), 1.564 (peak 3), 2.032 (peak 4), 2.088 (peak 5), 2.119 (peak 6), 2.147 (peak 7), and 2.714 (peak 8).

[0096] According to the obtained standard characteristic spectrum, the sample is tested:

[0097] Test 1: Specificity test

[0098] Preparation of blank solvent: 50% methanol, denoted as S1;

[0099] Preparation of reference solution: Weigh the morroniside reference and add 50% methanol to prepare a reference solution (concentration of 10 μg / ml), recorded as S2;

[0100] Preparation of test solution: Weigh 0.5 g of the prepared traditional Chinese medicine preparation sample (batch number: S0601), add 50% methanol, mix well, and then ultrasonically treat to constant volume and filter to obtain the solution, which is recorded as S3.

[0101] The S1, S2 and S3 prepared above were tested under the same chromatographic conditions as above. Figure 8 ,according to Figure 3 It can be seen that the blank solvent (50% methanol solution) has no interference with the determination of the reference substance and the sample, and the method has good specificity.

[0102] Test 2: Precision test

[0103] The test solution of the traditional Chinese medicine preparation sample (batch number: S0601) was injected continuously 6 times under the same chromatographic conditions as above, and its characteristic spectrum was recorded. The RSD of the relative retention time and relative peak area of ​​the common peak and the internal reference peak (morroniside peak) were examined. The results are shown in Table 2 below.

[0104] Table 2. Precision test data table

[0105]

[0106]

[0107] According to the above table, the RSDs of the relative retention times and relative peak areas of the eight common peaks of the six test solutions and the internal reference peak (morroniside peak) were all less than 3%, demonstrating that the method had good precision.

[0108] Test 3: Repeatability test

[0109] The test solution of the traditional Chinese medicine preparation sample (batch number: S0601) was injected continuously 6 times under the same chromatographic conditions as above, and its characteristic spectrum was recorded. The RSD of the relative retention time and relative peak area of ​​the common peak and the internal reference peak (morroniside peak) were examined. The results are shown in Table 3 below.

[0110] Table 3. Repeatability test data table

[0111]

[0112]

[0113] According to the above table, the RSDs of the relative retention times and relative peak areas of the eight common peaks of the six test solutions and the internal reference peak (morroniside peak) were all less than 3%, demonstrating that the method had good repeatability.

[0114] Test 4: Stability test

[0115] The test solution of the traditional Chinese medicine preparation sample (batch number: S0601) was taken, and the chromatographic conditions were the same as above. The samples were injected and analyzed at 0h, 2h, 4h, 8h, 12h, 16h, 20h, and 24h, and the characteristic spectrum was recorded. The relative retention time and relative peak area RSD of the common peak and the internal reference peak (morroniside peak) were examined. The results are shown in Table 4 below.

[0116] Table 4. Stability test data

[0117]

[0118]

[0119] According to the above table, within 0h, 2h, 4h, 8h, 12h, 16h, 20h, and 24h, the RSDs of the relative retention times and relative peak areas of the eight common peaks and the internal reference peak (morroniside peak) were all less than 3%, demonstrating that the method had good stability.

[0120] Test 4: Inter-batch test

[0121] Table 6. Performance data of different batches

[0122]

[0123] According to the above table, the standard characteristic spectrum obtained by the construction method of the HPLC characteristic spectrum of the present invention can effectively detect the traditional Chinese medicine preparation for treating chronic liver disease of the present invention, which includes Astragalus, Polygonum multiflorum, Salvia miltiorrhiza, Cornus officinalis, Ziziphus jujuba seed, Pseudostellaria baicalensis, and Lonicera japonica; and the quality control method proves that the traditional Chinese medicine preparation of the present invention has a good pass rate.

[0124] Examples 1 to 9

[0125] Set the HPLC parameters:

[0126] Chromatographic column: ACE Excel 5C18-AR (4.6×250mm, 5um)

[0127] Filler: Octadecylsilane bonded silica gel

[0128] Mobile phase:

[0129] Time (min) A (acetonitrile)% B (0.3% phosphoric acid solution)% 0～20 6 94 20～60 6→30 94→70 60～70 30 70

[0130] Column temperature: 40°C, flow rate: 1.0 ml / min, injection volume: 20 μl, detection wavelength: 240 nm; the theoretical plate number calculated based on the morroniside peak should be no less than 3000.

[0131] HPLC was used to determine the characteristic spectrum of the test sample, wherein the test sample solution was a traditional Chinese medicine preparation for treating chronic liver disease prepared according to the raw materials and their proportions in Table 7; the test sample was compared with the standard characteristic spectrum to detect whether it was qualified. The results are shown in Table 9.

[0132] Table 7, Raw material ratio table

[0133] Cornus officinalis Suanzaorensis seeds Tribulus terrestris Astragalus Pseudostellaria heterophylla Salvia miltiorrhiza Honeysuckle vine Polygonum multiflorum 10～20 1～20 5～20 5～25 5～30 5～20 5～20 7～15 Application Example 1 15 18 20 13 10 20 17 10 Application Example 2 13 20 20 10 16 13 19 13 Application Example 3 12 16 11 20 11 10 20 12 Application Example 4 15 15 15 15 10 10 10 10 Application Example 5 15 15 15 15 15 15 15 15 Application Example 6 21 15 15 15 15 15 15 12 Application Example 7 8 15 15 15 15 15 15 8 Application Example 8 17 15 15 15 15 15 15 17 Application Example 9 10 15 15 15 15 15 15 15

[0134] Examples 10 to 16

[0135] Compared with Example 1, the difference is that the test solution is a traditional Chinese medicine preparation for treating chronic liver disease prepared according to the proportions in Table 8; it is compared with the standard characteristic spectrum to test whether it is qualified, and the results are shown in Table 9.

[0136] Table 8. Raw material ratio table

[0137]

[0138] Table 9. Comparison results with standard characteristic maps

[0139]

[0140]

[0141] Note: The values ​​in the above table are maximum values.

[0142] In summary, the method for constructing a high-performance liquid chromatography characteristic spectrum of the present invention obtains a characteristic spectrum of a traditional Chinese medicine preparation for treating chronic liver disease composed of astragalus, polygonum multiflorum, salvia miltiorrhiza, cornus officinalis, spinach seed, pseudostellaria, and honeysuckle vine. The relative retention time of a qualified test sample should be within ±10% of the specified value; and with reference to the standard characteristic spectrum, an efficient quality detection method is obtained. The traditional Chinese medicine preparation for treating chronic liver disease obtained by the method of the present invention has a qualified rate.

[0143] The specific embodiments described herein are merely illustrative of the spirit of the present invention. Persons skilled in the art may make various modifications, additions, or substitutions to the described specific embodiments without departing from the spirit of the present invention or exceeding the scope of the appended claims.

Claims

1. A method for constructing a high performance liquid chromatography characteristic spectrum of a traditional Chinese medicine preparation for treating chronic liver disease, characterized in that: The method for determining the high performance liquid phase characteristic spectrum comprises the following steps: Step S1, preparation of reference solution: accurately weigh the morroniside reference substance and add 50% methanol to prepare a reference solution; each 1 ml of the reference solution contains 10 μg of morroniside; Preparation of test solution: Grind the prepared traditional Chinese medicine for treating chronic liver disease into powder, add 50% methanol to mix, ultrasonicate, dilute to volume, and filter to obtain the test solution; the weight-to-volume ratio of the test solution is 1 mg:10 ml; The raw materials of the traditional Chinese medicine preparation for treating chronic liver disease include cornus officinalis, spinach seed, tribulus terrestris, polygonum multiflorum, astragalus root, pseudoginseng, salvia miltiorrhiza, and honeysuckle vine; Step S2, performing high performance liquid chromatography on the reference solution and the test solution to obtain a characteristic spectrum of the traditional Chinese medicine preparation for treating chronic liver disease; During the high performance liquid chromatography detection process, octadecylsilane bonded silica gel was used as the filler, acetonitrile was used as the mobile phase A, and phosphoric acid aqueous solution was used as the mobile phase B; the concentration of the phosphoric acid aqueous solution was 0.3%; Set the column temperature to 40°C, flow rate to 1.0 ml / min, injection volume to 20 μL, and detection wavelength to 240 nm; the gradient elution conditions are: 0–20 min, 6% mobile phase A, 94% mobile phase B; 20-60 min, 6%→30% mobile phase A, 94%→70% mobile phase B; 60-70 min, 30% mobile phase A, 70% mobile phase B; The characteristic spectrum includes 8 characteristic peaks, the reference peak is the morroniside peak, among which peak 1: 5-hydroxymethylfurfural, peak 2: morroniside, peak 3: loganin, peak 5: spinosin, peak 6: calycosin isoflavone glucoside, peak 7: stilbene glucoside, and peak 8: salvianolic acid B; the characteristic peak regions of each component are associated and identified as follows: peak 1, peak 2, peak 3, and peak 4 belong to Cornus officinalis, peak 3 belongs to Lonicera japonica vine, peak 5 belongs to Ziziphus jujuba seed, peak 6 belongs to Astragalus membranaceus, peak 7 belongs to Polygonum multiflorum processed, and peak 8 belongs to Salvia miltiorrhiza.

2. The method for constructing the HPLC characteristic spectrum of the Chinese medicine preparation for treating chronic liver disease according to claim 1, characterized in that: The peak corresponding to the morroniside reference was designated as the S peak, and the specified values ​​of the relative retention times of peaks 1 to 8 and the S peak were 0.346, 1.000, 1.564, 2.032, 2.088, 2.119, 2.147, and 2.714, respectively.

3. The method for constructing a high performance liquid chromatography characteristic spectrum of a Chinese medicine preparation for treating chronic liver disease according to claim 1 or 2, characterized in that: A sample solution to be tested is taken and a spectrum is obtained according to the high performance liquid chromatography detection method. The spectrum is compared and analyzed with the standard characteristic spectrum obtained by the method for constructing the high performance liquid chromatography characteristic spectrum of the traditional Chinese medicine preparation for treating chronic liver disease; the relative retention time of the qualified sample to be tested is ±10% of the value specified by the standard characteristic spectrum.

4. A high performance liquid chromatography quality detection method for a traditional Chinese medicine preparation for treating chronic liver disease, characterized in that: The high-performance liquid chromatography quality detection method includes obtaining a spectrum of the test sample solution according to the high-performance liquid chromatography detection method, and comparing and analyzing the spectrum with the standard characteristic spectrum obtained by the high-performance liquid chromatography characteristic spectrum construction method of the traditional Chinese medicine preparation for treating chronic liver disease according to claim 1.

5. The high performance liquid chromatography quality detection method for a traditional Chinese medicine preparation for treating chronic liver disease according to claim 4, characterized in that: The relative retention time of qualified test samples is ±10% of the value specified in the standard characteristic spectrum.

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