A quality detection method for Wumeizhili granules

Abstract

The present invention discloses a quality detection method for Wumeizhili granules, belonging to the technical field of quality detection of traditional Chinese medicines. The method includes characteristic spectrum identification, and specifically comprises the following steps: (1) preparing a reference solution of hyperoside, a reference solution of coumarin, a reference solution of codonopsis pilosula, a reference solution of cinnamic acid, a reference solution of psoralen, a reference solution of isopsoralen, and a reference solution of limonin; (2) preparing a test solution using Wumeizhili granules; and (3) performing liquid chromatography detection on the seven reference solutions and the test solution. The quality detection method of the present invention has high accuracy, strong specificity, and reliable detection results, achieving effective quality control of Wumeizhili granules, meeting the needs of modern production, and having important significance for quality detection and clinical use of traditional Chinese medicines.

Description

Technical Field

[0001] The present invention relates to the technical field of quality detection of traditional Chinese medicines, and more particularly to a quality detection method for Wumeizhili granules. Background Art

[0002] In the Wumei Zhili Granules formula, Wumei is sour and warm, astringing yin, astringing the intestines and stopping diarrhea, and is the main medicine; Aconitum, Evodia, and cinnamon are pungent and hot, and Psoralea corylifolia is pungent and warm, tonifying kidney yang to warm and nourish the spleen and earth, and are collectively the ministerial medicines; Zanthoxylum bungeanum, Asarum, and cinnamon twig are pungent and warm, and dried ginger is very pungent and hot, warming the internal organs and dispelling cold, supporting yang and suppressing yin; Schisandra chinensis is sour and warm, consolidating the kidney and replenishing qi, astringing the essence and stopping diarrhea; ginseng and angelica nourish qi and blood, and support positive energy, and are collectively the adjuvant medicines; stir-fried barley sprouts are sweet and mild, and can help digestion and stimulate appetite, and are the guiding medicines.

[0003] Wumei Zhili Granules warm the yang and dispel cold, nourish the yin and stop diarrhea, and harmonize cold and heat. They are primarily used for chronic diarrhea characterized by a mixture of cold and heat, such as chronic enteritis, irritable bowel syndrome, and other intestinal disorders. Symptoms include prolonged diarrhea, abdominal distension, intestinal rumbling, dry and bitter mouth, and symptoms of heat (recurrent sore throat, mouth ulcers, hot flashes, night sweats, burning mouth syndrome, restlessness, and insomnia).

[0004] Currently, there is no quality testing for Wumei Zhili Granules, making it difficult to fully reflect the efficacy of the drug. Simultaneous multi-component analysis is not fully implemented, making it difficult to comprehensively evaluate and control the manufacturing process of the finished product. Therefore, it is urgent to establish more comprehensive quality testing methods to ensure quality control of Wumei Zhili Granules. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a quality detection method for Wumei Zhili Granules. The quality detection method has high accuracy, strong specificity, and reliable detection results, thereby achieving effective control of the quality of Wumei Zhili Granules.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A quality detection method for Wumeizhili granules, including characteristic spectrum identification, specifically comprises the following steps:

[0008] (1) Prepare hyperoside reference solution, coumarin reference solution, codonopsis pilosula reference solution, cinnamic acid reference solution, psoralen reference solution, isopsoralen reference solution, and limonin reference solution respectively;

[0009] (2) Prepare the test solution with Wumei Zhili Granules;

[0010] (3) Perform liquid chromatography on the above 7 reference substance solutions and test sample solutions respectively.

[0011] In summary, the present invention has the following beneficial effects:

[0012] Wumeizhili Granules were tested through characteristic spectrum identification, psoralen content determination, isopsoralen content determination, cinnamic acid content determination, thin layer identification, etc., and a more comprehensive and complete quality inspection method was established. This quality inspection method has high accuracy, strong specificity, and reliable test results. It achieves effective control of the quality of Wumeizhili Granules, adapts to modern production needs, and is of great significance to the quality inspection and clinical use of traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Chromatogram of the hyperoside reference solution obtained to verify the positioning of the characteristic spectrum determination method and system applicability of Wumeizhili Granules (peak 1 in the figure is the hyperoside peak);

[0014] Figure 2 Chromatogram of the coumarin reference solution obtained to verify the positioning and system suitability of the Wumeizhili Granules characteristic spectrum determination method (peak 1 in the figure is the coumarin peak);

[0015] Figure 3 Chromatogram of the dangshen glycoside reference solution obtained to verify the positioning and system suitability of the characteristic spectrum determination method of Wumeizhili Granules (peak 1 in the figure is the dangshen glycoside peak);

[0016] Figure 4 Chromatogram of the cinnamic acid reference solution obtained to verify the positioning and system suitability of the Wumeizhili Granules characteristic spectrum determination method (peak 1 in the figure is the cinnamic acid peak);

[0017] Figure 5 Chromatogram of the psoralen reference solution obtained to verify the positioning and system suitability of the Wumeizhili Granules characteristic spectrum determination method (peak 1 in the figure is the psoralen peak);

[0018] Figure 6 Chromatogram of the isopsoralen reference substance solution obtained to verify the positioning and system suitability of the Wumeizhili Granules characteristic spectrum determination method (peak 1 in the figure is the isopsoralen peak);

[0019] Figure 7 Chromatogram of the limonin reference solution obtained to verify the positioning and system suitability of the Wumeizhili Granules characteristic spectrum determination method (peak 1 in the figure is the limonin peak);

[0020] Figure 8 The chromatogram of the test sample solution was obtained to verify the positioning of the characteristic spectrum determination method and the suitability of the system;

[0021] Figure 9The chromatogram obtained to verify the column temperature durability at 25℃ of the characteristic spectrum determination method of Wumeizhili granules;

[0022] Figure 10 The chromatogram obtained to verify the column temperature durability at 35℃ for the determination method of Wumeizhili granules' characteristic spectrum;

[0023] Figure 11 The chromatogram was obtained to verify the flow rate durability of the Wumeizhili granule characteristic spectrum determination method at a flow rate of 0.18mL / min;

[0024] Figure 12 The chromatogram was obtained to verify the flow rate durability of the Wumeizhili granule characteristic spectrum determination method at a flow rate of 0.22 mL / min;

[0025] Figure 13 The chromatogram obtained to verify the wavelength durability of 205nm in the determination method of Wumeizhili granules' characteristic spectrum;

[0026] Figure 14 The chromatogram obtained to verify the wavelength durability of 215nm in the determination method of Wumeizhili granules' characteristic spectrum;

[0027] Figure 15 This is the chromatogram obtained by measuring the characteristic spectrum of Wumei Zhili Granules with batch number WMZL-K-250317-02;

[0028] Figure 16 Chromatogram of the reference solution obtained to verify the positioning and system suitability of the determination method of psoralen and isopsoralen in Wumeizhili granules (peak 1 in the figure is the psoralen peak);

[0029] Figure 17 Chromatogram of the reference solution obtained to verify the positioning and system suitability of the determination method of psoralen and isopsoralen in Wumeizhili granules (peak 1 in the figure is the isopsoralen peak);

[0030] Figure 18 The chromatogram of the test sample solution was obtained to verify the positioning and system suitability of the determination method of psoralen and isopsoralen in Wumeizhili granules;

[0031] Figure 19 The chromatogram was obtained to verify the column temperature durability at 25℃ for the determination of psoralen and isopsoralen contents in Wumeizhili granules;

[0032] Figure 20 The chromatogram was obtained to verify the column temperature durability at 35℃ for the determination of psoralen and isopsoralen in Wumeizhili granules.

[0033] Figure 21The chromatogram was obtained to verify the flow rate durability of the determination method of psoralen and isopsoralen in Wumeizhili granules at a flow rate of 0.18 mL / min;

[0034] Figure 22 The chromatogram was obtained to verify the flow rate durability of the determination method of psoralen and isopsoralen in Wumeizhili granules at a flow rate of 0.22 mL / min;

[0035] Figure 23 The chromatogram was obtained to verify the wavelength durability of 239nm in the determination method of psoralen and isopsoralen in Wumeizhili granules;

[0036] Figure 24 The chromatogram was obtained to verify the wavelength durability of 249nm in the determination method of psoralen and isopsoralen in Wumeizhili granules;

[0037] Figure 25 Chromatogram of the reference solution obtained to verify the positioning and system suitability of the determination method of cinnamic acid content in Wumeizhili granules;

[0038] Figure 26 The chromatogram of the test sample solution was obtained to verify the positioning and system suitability of the determination method of cinnamic acid content in Wumeizhili granules;

[0039] Figure 27 The chromatogram was obtained to verify the column temperature durability of the determination method of cinnamic acid in Wumeizhili granules at 25℃;

[0040] Figure 28 The chromatogram was obtained to verify the column temperature durability of the determination method of cinnamic acid in Wumeizhili granules at 35℃;

[0041] Figure 29 The chromatogram was obtained to verify the robustness of the determination method of cinnamic acid content in Wumeizhili granules at a flow rate of 0.18 mL / min;

[0042] Figure 30 The chromatogram was obtained to verify the robustness of the determination method of cinnamic acid content in Wumeizhili granules at a flow rate of 0.22 mL / min;

[0043] Figure 31 The chromatogram was obtained to verify the wavelength durability of 275nm in the determination method of cinnamic acid content in Wumeizhili granules;

[0044] Figure 32 The chromatogram was obtained to verify the wavelength durability of 285nm in the determination method of cinnamic acid content in Wumeizhili granules;

[0045] Figure 33 This is the chromatogram obtained from the durability test of Wumeizhili granules using thin layer chromatography identification method at 4°C;

[0046] Figure 34 This is the chromatogram obtained from the durability test of Wumeizhili Granules using the thin layer chromatography identification method at 30°C.

[0047] Figure 35 This is the chromatogram obtained from the durability test of Wumei Zhili Granules under 30% humidity using the thin layer chromatography identification method.

[0048] Figure 36 This is the chromatogram obtained from the durability test of Wumei Zhili Granules under 70% humidity using the thin layer chromatography identification method.

[0049] Figure 37 This is the chromatogram obtained by thin-layer identification test of Wumeizhili granules using thin-layer plates purchased from Qingdao Ocean Chemical Co., Ltd.

[0050] Figure 38 This is the chromatogram obtained by thin layer identification test of Wumeizhili granules using thin layer plates purchased from Qingdao Shuoyuan Silica Gel Technology Co., Ltd.

[0051] Figure 39 This is the chromatogram obtained by the thin layer identification test of Wumeizhili Granules using thin layer plates purchased from Qingdao Xinchanglai Silica Gel Co., Ltd.

[0052] Note: Figures 8 to 15 Peak 4 is the peak of hyperoside, peak 5 is the peak of coumarin, peak 7 is the peak of dangshenin, peak 8 is the peak of cinnamic acid, peak 9 is the peak of psoralen, peak 10 is the peak of isopsoralen, and peak 12 is the peak of limonin. Figures 18 to 24 Peak 1 is the psoralen peak, and peak 2 is the isopsoralen peak; Figures 25 to 32 The middle peak 1 is all cinnamic acid peak; Figures 33 to 39 From left to right in the figure are the test solution, limonin reference solution, cinnamaldehyde reference solution, and psoralen reference solution. DETAILED DESCRIPTION

[0053] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0054] Example 1 Preparation of Wumeizhili Granules

[0055] The preparation method of Wumeizhili granules is as follows: take the medicinal pieces constituting Wumeizhili granules, decoct them separately with water, filter, concentrate the filtrate to a clear paste, add or not add maltodextrin, mix well, and granulate.

[0056] The decoction pieces that make up Wumei Zhili Granules were taken (Wumei Zhili Granules are composed of the following decoction pieces: 60g of black plum, 20g of cassia twig, 5g of cinnamon bark, 20g of dried ginger, 3g of Zanthoxylum bungeanum, 3g of asarum, 4g of Evodia rutaecarpa, 12g of processed aconite root, 10g of psoralea corylifolia, 12g of Schisandra chinensis, 30g of Codonopsis pilosula, 12g of stir-fried angelica root, and 30g of stir-fried barley sprouts). Three batches of Wumei Zhili Granules were prepared according to the above method. The specific production data are shown in Table 1.

[0057] Table 1

[0058]

[0059] Example 2 Quality Inspection of Wumeizhili Granules

[0060] 1. Characteristic spectrum identification

[0061] 1.1 Detection method

[0062] 1.1.1 Preparation of reference substance solutions: Using methanol as solvent, prepare 15 μg / mL psoralen reference substance solution, 15 μg / mL isopsoralen reference substance solution, 15 μg / mL coumarin reference substance solution, 10 μg / mL hyperoside reference substance solution, 20 μg / mL codonopsis pilosula reference substance solution, 30 μg / mL limonin, and 30 μg / mL cinnamic acid reference substance solution.

[0063] 1.1.2 Preparation of test solution: Take an appropriate amount of Wumeizhili granules, grind them into powder, take 1g, weigh it accurately, put it into a stoppered conical flask, add 20mL of methanol accurately, weigh it, and treat it with ultrasound (power 250W, frequency 50kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0064] 1.1.3 Determination: Accurately pipette 2 μL of reference solution and test solution, inject into high performance liquid chromatograph, record the chromatogram, and calculate the relative retention time.

[0065] Liquid chromatography conditions were as follows: column: Sepax GP-C18, 2.1×100 mm, 1.8 μm; detection wavelength: 210 nm; flow rate: 0.2 mL / min; column temperature: 30°C; injection volume: 2 μL; mobile phase: acetonitrile as mobile phase A, 0.05 wt% phosphoric acid as mobile phase B, gradient elution as shown in Table 2:

[0066] Table 2

[0067]

[0068] 1.2 Validation of characteristic pattern detection methodology

[0069] 1.2.1 Positioning and System Suitability: Using methanol as the solvent, prepare 15 μg / mL psoralen reference solution, 15 μg / mL isopsoralen reference solution, 15 μg / mL coumarin reference solution, 10 μg / mL hyperoside reference solution, 20 μg / mL codonopsis pilosula reference solution, 30 μg / mL limonin, and 30 μg / mL cinnamic acid reference solution. Accurately weigh 1.0 g of Wumeizhili Granules powder (Batch No. WMZL-K-250317-02) and place it in a stoppered conical flask. Accurately add 20 mL of methanol and weigh the solution. Ultrasonicate (power 250 W, frequency 50 kHz) for 30 minutes. Allow to cool and weigh again. Make up the loss with methanol. Shake well and filter. The filtrate is used as the test solution. Accurately draw 2 μL of reference solution and test solution respectively, inject into high performance liquid chromatograph, measure, record chromatogram, relevant chromatogram see Figures 1 to 8 It can be seen that the retention time of hyperoside in the chromatogram of the reference solution is 15.363min, the theoretical plate number is 16675, and the tailing factor is 1.03; the retention time of coumarin is 21.060min, the theoretical plate number is 16143, and the tailing factor is 1.01; the retention time of codonopsis pilosula is 27.053min, the theoretical plate number is 95515, and the tailing factor is 1.00; the retention time of cinnamic acid is 35.317min, the theoretical plate number is 16143, and the tailing factor is 1.01; min, the theoretical plate number was 29473, and the tailing factor was 1.07; the retention time of psoralen was 39.407 min, the theoretical plate number was 21064, and the tailing factor was 1.01; the retention time of isopsoralen was 43.243 min, the theoretical plate number was 20628, and the tailing factor was 1.01; the retention time of limonin was 64.940 min, the theoretical plate number was 304215, and the tailing factor was 0.96. The retention time of hyperoside in the chromatogram of the test solution is 15.393min, the theoretical plate number is 29059, and the tailing factor is 1.09; the retention time of coumarin is 21.053min, the theoretical plate number is 17731, and the tailing factor is 0.90; the retention time of codonopsis pilosula is 27.000min, the theoretical plate number is 144108, and the tailing factor is 1.04; the retention time of cinnamic acid is 35.320mi n, the number of theoretical plates was 30904, and the tailing factor was 0.99; the retention time of psoralen was 39.377 min, the number of theoretical plates was 17035, and the tailing factor was 1.01; the retention time of isopsoralen was 43.257 min, the number of theoretical plates was 21805, and the tailing factor was 0.97; the retention time of limonin was 64.940 min, the number of theoretical plates was 361979, and the tailing factor was 0.98. This indicates that the system has good applicability.

[0070] 1.2.2 Durability test

[0071] 1.2.2.1 Column temperature durability:

[0072] According to the specified chromatographic conditions, the column temperature was changed by ±5°C, and Wumei Zhili Granules (batch number: WMZL-K-250317-02) was taken. The test solution was prepared according to the method under "1.1.2". 2 μL of the test solution was accurately aspirated and injected into the high performance liquid chromatograph. The retention time of the common peak was recorded. The peak corresponding to the peak of the hyperoside reference substance was taken as the S1 peak. The relative retention time and RSD value of the characteristic peaks of peak 1, peak 2, peak 3, and peak 6 with the S1 peak were calculated. The peak corresponding to the peak of the limonin reference substance was taken as the S2 peak. The relative retention time and RSD value of the characteristic peaks of peak 11, peak 13, and peak 14 with the S2 peak were calculated. The results are shown in Table 3. The relevant chromatograms are shown in Figures 9 and 10 .

[0073] Table 3

[0074]

[0075] The results showed that the relative retention time RSD of peaks 1, 2, and 6 was greater than 5%, so the column temperature was set at 30°C.

[0076] 1.2.2.2 Flow rate durability

[0077] According to the specified chromatographic conditions, the flow rate was changed by ±0.02 mL / min, Wumei Zhili Granules (batch number: WMZL-K-250317-02) were taken, and the test solution was prepared according to the method under "1.1.2". 2 μL of the test solution was accurately aspirated and injected into the high performance liquid chromatograph. The retention time of the common peak was recorded. The peak corresponding to the peak of the hyperoside reference substance was taken as the S1 peak. The relative retention time and RSD value of the characteristic peaks of peak 1, peak 2, peak 3, and peak 6 with the S1 peak were calculated. The peak corresponding to the peak of the limonin reference substance was taken as the S2 peak. The relative retention time and RSD value of the characteristic peaks of peak 11, peak 13, and peak 14 with the S2 peak were calculated. The results are shown in Table 4. The relevant chromatograms are shown in Figure 11-12 .

[0078] Table 4

[0079]

[0080] 1.2.2.3 Wavelength durability

[0081] According to the prescribed chromatographic conditions, the wavelength was changed by ±5nm, and Wumei Zhili Granules (batch number: WMZL-K-250317-02) was taken. The test solution was prepared according to the method under "1.1.2". 2μL of the test solution was accurately aspirated and injected into the high performance liquid chromatograph. The retention time of the common peak was recorded. The peak corresponding to the peak of the hyperoside reference substance was the S1 peak. The relative retention time and RSD value of the characteristic peaks of peak 1, peak 2, peak 3, and peak 6 with the S1 peak were calculated. The peak corresponding to the peak of the limonin reference substance was the S2 peak. The relative retention time and RSD value of the characteristic peaks of peak 11, peak 13, and peak 14 with the S2 peak were calculated. The results are shown in Table 5. The relevant chromatograms are shown in Figure 13-14 .

[0082] Table 5

[0083]

[0084] 1.3 Sample testing

[0085] Three batches of Wumei Zhili Granules (Batch Nos.: WMZL-K-250317-01, WMZL-K-250317-02, and WMZL-K-250317-03) were tested according to Section 1.1. The relative retention times of the characteristic spectra of Wumei Zhili Granules are shown in Table 6. Figure 15 This is the chromatogram obtained by determining the characteristic spectrum of Wumei Zhili Granules with batch number WMZL-K-250317-02.

[0086] Table 6

[0087]

[0088] 2. Determination of psoralen and isopsoralen content

[0089] 2.1 Determination method

[0090] 2.1.1 Preparation of reference solution: Accurately weigh the psoralen and isopsoralen reference substances and add methanol to prepare 15 μg / mL psoralen reference solution and 15 μg / mL isopsoralen reference solution.

[0091] 2.1.2 Preparation of test solution: Take an appropriate amount of Wumeizhili granules, grind them into powder, take 1g, weigh it accurately, put it into a stoppered conical flask, add 20mL of methanol accurately, weigh it, and treat it with ultrasound (power 250W, frequency 50kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0092] 2.1.3 Determination: Accurately pipette 2 μL of reference solution and test solution, inject into high performance liquid chromatograph, record the peak area, and calculate the content.

[0093] Chromatographic conditions were as follows: column: Sepax GP-C18, 2.1×100 mm, 1.8 μm; detection wavelength: 244 nm; flow rate: 0.2 mL / min; column temperature: 30°C; injection volume: 2 μL; mobile phase: acetonitrile as mobile phase A, 0.05 wt% phosphoric acid as mobile phase B, gradient elution according to the requirements in Table 7:

[0094] Table 7

[0095]

[0096] 2.2 Methodological validation

[0097] 2.2.1 Positioning and system applicability

[0098] Use methanol as solvent to prepare 15μg / mL psoralen reference solution and 15μg / mL isopsoralen reference solution. Accurately weigh an appropriate amount of Wumeizhili Granules (batch number: WMZL-K-250317-02), grind it into powder, take 1g, put it in a stoppered conical flask, accurately add 20mL of methanol, weigh the weight, ultrasonically treat (power 250W, frequency 50kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate as the test solution. Accurately aspirate 2μL of the reference solution and the test solution respectively, inject them into the high performance liquid chromatograph, and measure, such as Figures 16 to 18 As shown. In the chromatogram of the reference solution, the retention time of psoralen is 39.403 min, the number of theoretical plates is 21071, and the tailing factor is 1.02; the retention time of isopsoralen is 43.243 min, the number of theoretical plates is 20594, and the tailing factor is 1.02. In the chromatogram of the test solution, the retention time of psoralen is 39.410 min, the number of theoretical plates is 20274, and the tailing factor is 1.03; the retention time of isopsoralen is 43.257 min, the number of theoretical plates is 20724, and the tailing factor is 1.01. This indicates that the system suitability is good.

[0099] 2.2.2 Durability test

[0100] 2.2.2.1 Column temperature durability

[0101] According to the specified chromatographic conditions, change the column temperature by ±5°C, take Wumei Zhili Granules (batch number: WMZL-K-250317-02), prepare the test solution according to the method under "2.1.2", and determine the content of psoralen and isopsoralen. The results are shown in Table 8. The relevant chromatograms are shown in Figures 19 and 20 .

[0102] Table 8

[0103]

[0104] 2.2.2.2 Flow rate durability

[0105] According to the specified chromatographic conditions, change the flow rate by ±0.02mL / min, take Wumei Zhili Granules (batch number: WMZL-K-250317-02), prepare the test solution according to the method under "2.1.2", and determine the content of psoralen and isopsoralen. The results are shown in Table 9. The relevant chromatogram is shown in Figure 21-22 .

[0106] Table 9

[0107]

[0108] 2.2.2.3 Wavelength durability

[0109] According to the specified chromatographic conditions, change the wavelength ± 5nm, take Wumei Zhili Granules (batch number: WMZL-K-250317-02), prepare the test solution according to the method under "2.1.2", and determine the content of psoralen and isopsoralen. The results are shown in Table 10. The relevant chromatogram is shown in Figure 23-24 .

[0110] Table 10

[0111]

[0112] The results of the above durability tests showed that the durability of the column temperature, flow rate and wavelength of this method met the requirements for content determination.

[0113] 2.3 Sample determination: Three batches of Wumei Zhili Granules (WMZL-K-250317-01, WMZL-K-250317-02, and WMZL-K-250317-03) were taken and their contents were determined according to the law. See Table 11.

[0114] Table 11

[0115]

[0116] 3. Determination of cinnamic acid content

[0117] 3.1 Determination method

[0118] 3.1.1 Preparation of reference solution: Take cinnamic acid reference solution and add methanol to make a 30 μg / mL reference solution.

[0119] 3.1.2 Preparation of test solution: Take an appropriate amount of Wumeizhili granules, grind them into powder, take 1g, place it in a stoppered conical flask, accurately add 20mL of methanol solution, weigh the weight, ultrasonically treat (power 250W, frequency 50kHz) for 30 minutes, let it cool, weigh the weight again, make up the lost weight with methanol, shake well, filter, and take the filtrate.

[0120] 3.1.3 Determination: Accurately pipette 2 μL of each reference solution and test solution, inject into liquid chromatograph, record the peak area, and calculate the content;

[0121] The chromatographic conditions were as follows: chromatographic column: Sepax GP-C18, 2.1×100 mm, 1.8 μm; detection wavelength: 280 nm; flow rate: 0.2 mL / min; column temperature: 30°C; injection volume: 2 μL; mobile phase: acetonitrile as mobile phase A, 0.05 wt% phosphoric acid as mobile phase B; gradient elution procedure was the same as in item “2.1.3”.

[0122] 3.2 Methodological validation

[0123] 3.2.1 Positioning and system applicability

[0124] Use methanol as solvent to prepare 30μg / mL cinnamic acid reference solution. Accurately weigh an appropriate amount of Wumei Zhili Granules (batch number: WMZL-K-250317-02), grind it into powder, take 1g, put it into a stoppered conical flask, accurately add 20mL of methanol, weigh the weight, ultrasonically treat (power 250W, frequency 50kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate as the test solution. Accurately aspirate 2μL of the reference solution and the test solution respectively, inject them into the high performance liquid chromatograph, and measure, such as Figure 25-26 As shown in the chromatogram of the reference solution, the retention time of cinnamic acid is 35.317 min, the number of theoretical plates is 29522, and the tailing factor is 1.06. The retention time of cinnamic acid in the chromatogram of the test solution is 35.337 min, the number of theoretical plates is 29367, and the tailing factor is 1.04. This indicates good system suitability.

[0125] 3.2.2 Durability test

[0126] 3.2.2.1 Column temperature durability

[0127] According to the specified chromatographic conditions, change the column temperature by ±5°C, take Wumei Zhili Granules (batch number: WMZL-K-250317-02), prepare the test solution according to the method under "3.1.2", and determine the content of cinnamic acid. The results are shown in Table 12. The relevant chromatogram is shown in Figure 27-28 .

[0128] Table 12

[0129]

[0130] 3.2.2.2 Flow rate durability

[0131] According to the specified chromatographic conditions, change the flow rate by ±0.02mL / min, take Wumei Zhili Granules (Batch No.: WMZL-K-250317-02), prepare the test solution according to the method under "3.1.2", and determine the content of cinnamic acid. The results are shown in Table 13. The relevant chromatogram is shown in Figures 29 and 30 .

[0132] Table 13

[0133]

[0134] 3.2.2.3 Wavelength durability

[0135] According to the specified chromatographic conditions, change the wavelength ± 5nm, take Wumei Zhili Granules (batch number: WMZL-K-250317-02), prepare the test solution according to the method under "3.1.2", and determine the content of cinnamic acid. The results are shown in Table 14. The relevant chromatogram is shown in Figure 31-32 .

[0136] Table 14

[0137]

[0138] The results of the above durability tests showed that the durability of the column temperature, flow rate and wavelength of this method met the requirements for content determination.

[0139] 3.3 Sample determination

[0140] Three batches of Wumei Zhili Granules (WMZL-K-250317-01, WMZL-K-250317-02, and WMZL-K-250317-03) were taken and their contents were determined according to the method. See Table 15.

[0141] Table 15

[0142]

[0143] 4. Thin layer identification

[0144] 4.1 Detection method

[0145] 4.1.1 Preparation of test solution: Take an appropriate amount of Wumei Zhili granules, grind them into powder, take 2 g, add 20 mL of ethyl acetate, ultrasonically treat for 30 minutes, filter, and concentrate the filtrate to 2 mL to obtain the test solution.

[0146] 4.1.2 Preparation of reference substance solutions: Take cinnamaldehyde reference substance, limonin reference substance, and psoralen reference substance, and add methanol to prepare cinnamaldehyde reference substance solution, limonin reference substance solution, and psoralen reference substance solution, with a concentration of 1 mg / mL.

[0147] 4.1.3 Determination

[0148] Pipette 10 μL each of the test solution, cinnamaldehyde reference solution, limonin reference solution, and psoralen reference solution, and spot them on the same GF254 thin layer plate. Use toluene-ethyl acetate with a volume ratio of 9:1 as the developing agent, develop, take out, dry, and examine under a 254 nm ultraviolet lamp.

[0149] 4.2 Methodological validation

[0150] 4.2.1 Durability test

[0151] Prepare Wumei Zhili Granules batch number WMZL-K-250317-02 and prepare Wumei Zhili Granules test solution according to the method in section 4.1.1. Prepare negative sample solution with maltodextrin according to the method in section 4.1.1. Prepare cinnamaldehyde reference solution, limonin reference solution, and psoralen reference solution according to the method in section 4.1.2.

[0152] 4.2.1.1 Temperature durability

[0153] 10 μL each of the test solution, negative sample solution, cinnamaldehyde reference solution, limonin reference solution, and psoralen reference solution were taken and spotted on the same GF254 thin layer plate. The plates were developed with toluene-ethyl acetate in a volume ratio of 9:1 at 4°C and 30°C, respectively. The plates were taken out, dried, and examined under a 254nm UV lamp. The thin layer chromatogram at 4°C is shown in Figure 1. Figure 33 , the thin layer chromatogram at 30℃ is shown in Figure 34 The results showed that in the chromatogram of the test sample, a fluorescent spot of the same color appeared at the corresponding position in the chromatogram of the reference sample.

[0154] 4.2.1.2 Humidity durability

[0155] 10 μL of each of the test solution, negative sample solution, cinnamaldehyde reference solution, limonin reference solution, and psoralen reference solution were taken and spotted on the same GF254 thin layer plate. The plates were developed with toluene-ethyl acetate in a volume ratio of 9:1 at 30% and 70% humidity, respectively. The plates were taken out, dried, and examined under a 254 nm UV lamp. The 30% humidity thin layer chromatogram is shown in Figure 2. Figure 35 , the thin layer chromatogram of humidity 70% is shown in Figure 36 The results showed that in the chromatogram of the test sample, a fluorescent spot of the same color appeared at the corresponding position in the chromatogram of the reference sample.

[0156] 4.2.1.3 Thin-layer boards from different manufacturers

[0157] 10 μL of each of the test solution, negative sample solution, cinnamaldehyde reference solution, limonin reference solution, and psoralen reference solution were aspirated and spotted on GF254 thin layer plates from three different manufacturers (Qingdao Ocean Chemical Co., Ltd., Qingdao Shuoyuan Silica Gel Technology Co., Ltd., and Qingdao Xinchanglai Silica Gel Co., Ltd.). The plates were developed using a 9:1 volume ratio of toluene-ethyl acetate. The plates were then taken out, dried, and examined under a 254 nm UV lamp. The corresponding thin layer chromatograms for the thin layer plates purchased from Qingdao Ocean Chemical Co., Ltd. are shown in Figure 2. Figure 37 The corresponding thin layer chromatogram of the thin layer plate purchased from Qingdao Shuoyuan Silica Gel Technology Co., Ltd. is shown in Figure 38 The corresponding thin layer chromatogram of the thin layer plate purchased from Qingdao Shuoyuan Silica Gel Technology Co., Ltd. is shown in Figure 39 The results showed that in the chromatogram of the test sample, a fluorescent spot of the same color appeared at the corresponding position in the chromatogram of the reference sample.

[0158] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiment. All technical solutions based on the concept of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A quality detection method for Wumeizhili granules, characterized in that: Including characteristic spectrum identification, the specific steps are: (1) Prepare hyperoside reference solution, coumarin reference solution, codonopsis pilosula reference solution, cinnamic acid reference solution, psoralen reference solution, isopsoralen reference solution, and limonin reference solution respectively; (2) Prepare the test solution with Wumei Zhili Granules; (3) Perform liquid chromatography on the seven reference substance solutions and the test solution; The chromatographic conditions of the characteristic spectrum are: Chromatographic column: Sepax GP-C18, 2.1×100 mm, 1.8 μm; mobile phase: acetonitrile as mobile phase A, 0.05 wt% phosphoric acid as mobile phase B; gradient elution; detection wavelength: 210 nm; flow rate: 0.2 ml / min; column temperature: 30°C; injection volume: 2 μL; In the characteristic spectrum identification, the elution procedure is: ； Wumeizhili granules are prepared by the following method: taking the decoction pieces constituting Wumeizhili granules, adding water to decoct, filtering, concentrating the filtrate to a clear paste, adding or not adding maltodextrin, mixing, and granulating; The decoction pieces that make up Wumei Zhili Granules are 60g of black plum, 20g of cassia twig, 5g of cinnamon bark, 20g of dried ginger, 3g of Sichuan pepper, 3g of asarum, 4g of Evodia rutaecarpa, 12g of processed aconite root, 10g of psoralea corylifolia, 12g of Schisandra chinensis, 30g of Codonopsis pilosula, 12g of stir-fried angelica root and 30g of stir-fried barley sprout.

2. The quality detection method of Wumeizhili granules according to claim 1, characterized in that: The quality testing method also includes the determination of psoralen content and isopsoralen content, and the specific steps are as follows: (1) Prepare psoralen reference solution and isopsoralen reference solution respectively; (2) Prepare the test solution with Wumei Zhili Granules; (3) The two reference solutions and the test solution were subjected to liquid chromatography, and the contents of psoralen and isopsoralen were calculated based on the obtained chromatograms.

3. The quality detection method of Wumei Zhili Granules according to claim 2, characterized in that: In the determination of psoralen and isopsoralen content, the chromatographic conditions are: Chromatographic column: Sepax GP-C18, 2.1×100 mm, 1.8 um; mobile phase: acetonitrile as mobile phase A, 0.05 wt% phosphoric acid as mobile phase B; gradient elution; detection wavelength: 244 nm; flow rate: 0.2 ml / min; column temperature: 30°C; injection volume: 2 μL.

4. The quality detection method of Wumeizhili granules according to claim 1, characterized in that: The quality testing method also includes the determination of cinnamic acid content, and the specific steps are as follows: (1) Prepare cinnamic acid reference solution; (2) Prepare the test solution with Wumei Zhili Granules; (3) Perform liquid chromatography on the cinnamic acid reference solution and the test solution, and calculate the cinnamic acid content based on the obtained chromatogram.

5. The quality detection method of Wumeizhili granules according to claim 4, characterized in that: In the determination of cinnamic acid content, the chromatographic conditions are: Chromatographic column: Sepax GP-C18, 2.1×100 mm, 1.8 um; mobile phase: acetonitrile as mobile phase A, 0.05 wt% phosphoric acid as mobile phase B; gradient elution; detection wavelength: 280 nm; flow rate: 0.2 ml / min; column temperature: 30°C; injection volume: 2 μL.

6. The quality detection method of Wumeizhili granules according to claim 1, characterized in that: The quality inspection method also includes thin layer identification, and the specific steps are: (1) Preparation of test solution: Prepare the test solution with Wumei Zhili granules; (2) Preparation of reference solution: prepare cinnamaldehyde reference solution, limonin reference solution, and psoralen reference solution respectively; (3) Inspection: Pipette the test solution, cinnamaldehyde reference solution, limonin reference solution, and psoralen reference solution onto the same thin layer plate and inspect under ultraviolet light.

7. The quality detection method of Wumeizhili granules according to claim 6, characterized in that: Toluene-ethyl acetate was used as the developing solvent in thin layer identification.

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