Fingerprint establishment method and quality monitoring method of traditional Chinese medicine composition containing tangerine peel

Abstract

The present invention relates to the field of quality monitoring of traditional Chinese medicine, and specifically, to a method for establishing a fingerprint of a traditional Chinese medicine composition containing dried tangerine peel and a quality monitoring method. The method includes: heating each gram of the drug reagent to be tested with 20-30mL of alcohol solvent for 20-40 minutes, then supplementing with alcohol solvent, then filtering to form a filter residue and dissolving it in water, then extracting and collecting the extract with methyl chloride and ester solvents in turn, spin-drying the extract and adding alcohol solvent to dilute and fix the volume; using acetonitrile as mobile phase A and 0.05%-0.3% phosphoric acid solution as mobile phase B, gradient elution; respectively accurately drawing 5-20 μL of the test solution and the reference solution, injecting into a liquid chromatograph, measuring, recording a chromatogram, and obtaining. The method overcomes the shortcomings of the prior art that the one-sided detection cannot fully reflect the quality attributes of the product, and can comprehensively and systematically detect the method of Shenshu granules.

Description

Technical Field

[0001] The invention relates to the field of quality monitoring of traditional Chinese medicines, and in particular to a method for establishing a fingerprint spectrum of a traditional Chinese medicine composition containing tangerine peel and a method for quality monitoring. Background Art

[0002] In the Shenshu Powder formula, tangerine peel is used as the main ingredient to regulate qi and strengthen the spleen, dry dampness and resolve phlegm, and unblock the three burners; Atractylodes lancea is pungent and strong, can raise yang, ward off evil, move the spleen to dry dampness, and relieve depression; Magnolia officinalis is bitter and warm, can remove dampness, disperse fullness, digest food, and thicken the intestines. The two herbs are used together as the ministers, which can not only help tangerine peel to regulate qi and strengthen the spleen, dry dampness and resolve phlegm, but also promote qi and harmonize the stomach, remove dampness and strengthen the spleen, unblock the qi in the middle burner and eliminate all symptoms; Patchouli and Acorus calamus are added as adjuvants, because their pungent fragrance can open the orifices, correct the body, ward off evil and benefit the stomach; Licorice is used as an adjuvant to harmonize the middle and regulate the spleen and earth, tonify the spleen and replenish qi, and harmonize all the herbs. Ginger is pungent and warm, and can penetrate evil. Jujube is sweet and mild in nature, and they are both adjuvant drugs. "Yi Zong Jin Jian·Deleted and Supplemented Famous Physicians' Prescriptions" says: "Ginger and jujube complement each other, and licorice can harmonize the yang surface and yin interior qi, blood and camp. They can coexist without conflict. It is the combination of hardness and softness to achieve harmony." The combination of these drugs has the effects of drying dampness and clearing turbidity, and expelling filth with fragrance. It is a good remedy for treating epidemics.

[0003] Traditional Chinese medicine fingerprint is a comprehensive and quantifiable identification method. Traditional Chinese medicine and its preparations are complex multi-component systems. Therefore, their quality should be evaluated by appropriate detection methods that can provide rich identification information. The establishment of traditional Chinese medicine fingerprint will be able to more comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicine and its preparations, and then provide an overall description and evaluation of the quality of the medicine.

[0004] The current product quality standards only use high performance liquid chromatography to determine the content of hesperidin in tangerine peel for effective ingredient detection, and at the same time, other medicinal ingredients are exclusively identified. However, since Taiwushenshu Granules contain eight Chinese medicines with complex ingredients, the qualitative and quantitative analysis of individual ingredients is still difficult to fully reflect the comprehensive information of the medicine. Although CN111407877A has constructed a detection method for a Chinese medicine composition for treating cold and dampness, especially for treating new coronavirus pneumonia, this method still has the disadvantage of a single and one-sided detection and identification index, which is easy to cause fluctuations in the accuracy and reliability of quality control, which is not conducive to comprehensive monitoring of the quality of Taiwushenshu Granules. Summary of the invention

[0005] The present invention provides a method for establishing a fingerprint spectrum of a traditional Chinese medicine composition containing dried tangerine peel and a method for monitoring its quality, which overcomes the shortcoming of the prior art that the one-sided detection cannot comprehensively reflect the quality attributes of the product, and can comprehensively and systematically detect the Shenshu granules, especially the method for establishing the fingerprint spectrum of the Shenshu granules, which is of great significance for its comprehensive quality detection and overall quality control.

[0006] The present invention is achieved in that:

[0007] In a first aspect, an embodiment of the present invention provides a method for establishing a fingerprint of a Chinese medicine composition containing tangerine peel, comprising: Step 1: Preparation of a test solution:

[0008] Each gram of the drug reagent to be tested is heated with 20-30 mL of alcohol solvent for 20-40 minutes, and then supplemented with alcohol solvent, and then filtered to form a filter residue and dissolved with water, and then extracted with chloroform and ester solvent in sequence and collected the extract, the extract is dried and diluted with alcohol solvent to a fixed volume;

[0009] Step 2: HPLC:

[0010] Octadecylsilane bonded silica gel is used as filler, acetonitrile is used as mobile phase A, 0.05%-0.3% phosphoric acid solution is used as mobile phase B, and gradient elution is performed; the flow rate is 0.9-1.2 mL / min, the column temperature is 28-32°C, the detection wavelength is 263-273 nm, and the theoretical plate number calculated based on the hesperidin peak should be no less than 2000;

[0011] Step 3: Detection:

[0012] Accurately pipette 5-20 μL of the test solution and the reference solution respectively, inject into the liquid chromatograph, measure, and record the chromatogram to obtain the product.

[0013] Further, in a preferred embodiment of the present invention, the alcohol solvent comprises a monohydric alcohol, preferably methanol, preferably 70-100% methanol.

[0014] Further, in a preferred embodiment of the present invention, each gram of the drug reagent to be tested is heated with 20-30mL70-100% methanol for 20-40 minutes, cooled, weighed, and then supplemented with methanol, and then filtered to form a filter residue and dissolved in water, and then extracted with dichloromethane, the water layer is extracted with ethyl acetate and the ethyl acetate portion is collected, then spin-dried and methanol is added to dilute the volume, and filtered.

[0015] Further, in a preferred embodiment of the present invention, the test solution is prepared by mixing each gram of the drug reagent to be tested with 20-30 mL of 70% ethanol, heating and reflux for 30 minutes, cooling, weighing, and then supplementing with methanol, and then filtering to form a filter residue and dissolving 28-32 mL of water, extracting with dichloromethane twice, extracting the water layer with ethyl acetate three times, each time 28-32 mL, and collecting the ethyl acetate portion, then spin-drying and adding methanol to dilute to volume, and filtering.

[0016] Further, in a preferred embodiment of the present invention, the elution procedure is as follows:

[0017] Time (min) Mobile phase A (%) Mobile phase B (%) 0 5 95 20 20 80 30 20 80 45 35 65 55 90 10 60 90 10 .

[0018] Furthermore, in a preferred embodiment of the present invention, mobile phase B is 0.1% phosphoric acid.

[0019] Furthermore, in a preferred embodiment of the present invention, the specification of the chromatographic column is 250 mm×4.6 mm, and the silica gel particle size is 5 μm.

[0020] Furthermore, in a preferred embodiment of the present invention, it includes: using traditional Chinese medicine chromatographic fingerprint software to calculate the similarity between the chromatogram of the test solution and the fingerprint of the control melt, and the calculated similarity is greater than 0.9.

[0021] Furthermore, in a preferred embodiment of the present invention, the Chinese medicine chromatographic fingerprint software is "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition".

[0022] In a second aspect, an embodiment of the present invention provides a method for monitoring the quality of a Chinese medicinal composition containing tangerine peel, which includes a method for establishing a fingerprint of the Chinese medicinal composition containing tangerine peel.

[0023] The beneficial effect of the present invention is that the present invention can comprehensively and systematically detect the Shenshu granules through the above method, especially the method of establishing the fingerprint spectrum of the Shenshu granules, which is of great significance for its comprehensive quality detection and overall quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 is a test result diagram provided by Example 1 of the present invention;

[0026] Figure 2 It is a test result diagram of peak attribution and peak identification in the verification example of the present invention;

[0027] Figure 3 It is a test result diagram of the specificity experiment in the verification example of the present invention;

[0028] Figure 4 It is a test result diagram of the overall investigation in the verification example of the present invention;

[0029] Figure 5 It is a test result diagram of the instrument precision test in the verification example of the present invention;

[0030] Figure 6is a graph showing the test results of the stability test in the verification example of the present invention;

[0031] Figure 7 It is an overlay diagram of the repeatability results in the verification example of the present invention;

[0032] Figure 8 It is a superimposed graph of the results of three different flow rates in the durability test in the verification example of the present invention;

[0033] Fig. 9 This is a graph showing the relative retention time results of three different flow rates in a durability test in a verification example of the present invention;

[0034] Fig.10 It is a superimposed diagram of the results of different column temperatures in the durability test in the verification example of the present invention;

[0035] Fig.11 This is a graph showing the relative retention time results of different column temperatures in a durability test in a verification example of the present invention;

[0036] Fig.12 It is an overlay diagram of the results of different chromatographic columns in the durability test in the verification example of the present invention;

[0037] Fig.13 is a graph showing the relative retention time results of different chromatographic columns in a durability test in a verification example of the present invention;

[0038] Fig.14 It is a superimposed graph of the results of two instruments in the durability test in the verification example of the present invention;

[0039] Fig.15 It is a graph showing the relative retention time results of two instruments in the durability test in the verification example of the present invention;

[0040] Fig.16 It is a common pattern diagram of 12 batches of characteristic spectra in the verification example of the present invention;

[0041] Fig.17 It is a control characteristic spectrum generated from the common pattern diagram of 12 batches of characteristic spectrum in the verification example of the present invention;

[0042] Figure 18-19 This is a graph showing the similarity detection results of different batches of Shenshu granules in the verification example of the present invention.

[0043] Fig. 20 This is a test result diagram of Comparative Example 1 of the present invention;

[0044] Fig.21 This is a graph showing the test results of Comparative Example 2 of the present invention. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0046] The following is a detailed description of a method for establishing a fingerprint of a Chinese medicine composition containing dried tangerine peel and a method for monitoring its quality, provided in an embodiment of the present invention.

[0047] The embodiment of the present invention provides a method for establishing a fingerprint of a Chinese medicine composition containing dried tangerine peel, comprising:

[0048] Step 1: Prepare the test solution:

[0049] Each gram of the drug reagent to be tested is heated with 20-30 mL of alcohol solvent for 20-40 minutes, and then supplemented with alcohol solvent, and then filtered to form a filter residue and dissolved with water, and then extracted with chloroform and ester solvent in sequence and collected the extract, the extract is dried and diluted with alcohol solvent to a fixed volume;

[0050] Specifically, each gram of the drug reagent to be tested is mixed with 20-30 mL (e.g., 20 mL, 21 mL, 22 mL, 23 mL, 24 mL, 25 mL, 26 mL, 27 mL, 28 mL, 29 mL, and 30 mL, etc., any value between 20-30 mL) of 70-100% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, and 100%, etc., any value between 70-100%) of methanol, weighed, and heated for 20-40 minutes (e.g., After 20 minutes, 25 minutes, 30 minutes, 35 minutes and 40 minutes (any value between 20-40 minutes), cool, weigh, and then use methanol to make up the lost weight, shake well, filter, and dissolve the filter residue in 30 ml of water, then shake and extract with dichloromethane 3 times, discard the dichloromethane liquid, take the water layer and shake and extract with ethyl acetate 3 times, 30 ml each time, combine and collect the ethyl acetate part, then spin dry and add methanol to dissolve and transfer to a volumetric flask, then dilute to volume with methanol, mix and filter.

[0051] Step 2: HPLC:

[0052] Octadecylsilane bonded silica gel is used as a filler, acetonitrile is used as a mobile phase A, and 0.05%-0.3% phosphoric acid solution is used as a mobile phase B, for example, any value between 0.05% phosphoric acid, 0.1% phosphoric acid, 0.15% phosphoric acid, 0.2% phosphoric acid, 0.25% phosphoric acid and 0.3% phosphoric acid, etc.; gradient elution is performed; the flow rate is 0.9-1.2 mL / min, for example, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min and 1.2 mL / min, etc. 0.9-1.2 m L / min; the column temperature is 28-32°C, for example, 28°C, 29°C, 30°C, 31°C, 32°C and any value between 28-30°C such as 28-32°C; the detection wavelength is 263-273nm, for example, 263nm, 264nm, 265nm, 266nm, 267nm, 268nm, 269nm, 270nm, 271nm, 272nm and any value between 263-273nm such as 273nm; the number of theoretical plates calculated based on the hesperidin peak should not be less than 2000.

[0053] Step 3: Detection:

[0054] Accurately pipette 5-20 μL of the test solution and the reference solution respectively, inject into the liquid chromatograph, measure, and record the chromatogram to obtain the product.

[0055] Further, the elution procedure is as follows:

[0056] Time (min) Mobile phase A (%) Mobile phase B (%) 0 5 95 20 20 80 30 20 80 45 35 65 55 90 10 60 90 10

[0057] The specifications of the chromatographic column are 250 mm × 4.6 mm, and the silica gel particle size is 5 μm.

[0058] Furthermore, the chromatogram of the test solution and the fingerprint of the control solution were similarly calculated using the Chinese medicine chromatogram fingerprint software, and the similarity was calculated to be greater than 0.9. The Chinese medicine chromatogram fingerprint software is the "Chinese Medicine Chromatography Fingerprint Similarity Evaluation System 2012 Edition".

[0059] In a second aspect, an embodiment of the present invention provides a method for monitoring the quality of a Chinese medicinal composition containing tangerine peel, which includes a method for establishing a fingerprint of the Chinese medicinal composition containing tangerine peel.

[0060] The following is a detailed description of the fingerprint establishment method and quality monitoring method of a Chinese medicine composition containing tangerine peel provided by the present invention in conjunction with specific embodiments.

[0061] Example 1

[0062] This embodiment provides a method for establishing a fingerprint of a Chinese medicine composition containing dried tangerine peel, wherein:

[0063] 1) Reagents

[0064] Dichloromethane 20201118 Fuchen (Tianjin Chemical Reagent Co., Ltd.)

[0065] Ethyl acetate 20210818 Fuchen (Tianjin Chemical Reagent Co., Ltd.)

[0066] Phosphoric acid 20180420 Fuchen (Tianjin Chemical Reagent Co., Ltd.)

[0067] Methanol V3TG1H Honeywell

[0068] Acetonitrile V6KA1H Honeywell

[0069] 2) Standard products

[0070] Hesperidin 110721-201818 China Food and Drug Administration 96.2%

[0071] Magnolia officinalis 110729-201714 China Food and Drug Administration 100.0%

[0072] Licorice glycoside 110610-201908 China Food and Drug Administration 95.0%

[0073] Honokiol 110730-201915 China Food and Drug Administration 99.8%

[0074] Ammonium glycyrrhizinate 110731-201619 China Food and Drug Administration 97.7%

[0075] 6-Gingerol 111833-201806 China Food and Drug Administration 99.9%

[0076] 3) Chromatographic column

[0077] WelchromC18(II),4.6*250mm,5μm,SN:60209900803

[0078] 4) Divine Art Granules with sample batch number 220301

[0079] 5) Preparation of reference and test solutions:

[0080] Preparation of test solution: Take about 2g of sample powder, weigh accurately, put it in a stoppered conical flask, accurately add 50ml of 70% methanol, weigh the weight, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, let cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, add 30ml of water to the residue to dissolve, shake and extract with dichloromethane twice, 30ml each time, discard the dichloromethane solution, continue to shake and extract with ethyl acetate 3 times, 30ml each time, combine the ethyl acetate, evaporate to dryness, add methanol to dissolve and transfer to a 10ml volumetric flask, dilute to the scale with methanol, mix and filter.

[0081] Preparation of reference solution: Hesperidin: Accurately weigh 5.06 mg of hesperidin reference substance and place it in a 100 ml volumetric flask, add 30 ml of methanol to dissolve it, dilute to the scale with methanol, mix well, and obtain.

[0082] Magnolia officinalis: Accurately weigh 7.98 mg of magnolia officinalis reference substance and place it in a 200 ml volumetric flask, add 30 ml of methanol to dissolve it, dilute to the scale with methanol, mix well, and obtain.

[0083] Honokiol: Accurately weigh 6.10 mg of honokiol reference substance and place it in a 25 ml volumetric flask, add 15 ml of methanol to dissolve it, dilute it to the scale with methanol, mix it, accurately pipette 1.0 ml of the above solution into a 10 ml volumetric flask, dilute it to the scale with methanol, mix it, and obtain.

[0084] Licorice glycoside: Accurately weigh 5.01 mg of licorice glycoside reference substance and place it in a 25 ml volumetric flask. Add 15 ml of methanol to dissolve it. Dilute it to the mark with methanol and mix well. Accurately pipette 1.0 ml of the above solution into a 10 ml volumetric flask, dilute it to the mark with methanol and mix well.

[0085] Ammonium glycyrrhizinate: Accurately weigh 5.14 mg of ammonium glycyrrhizinate reference substance and place it in a 25 ml volumetric flask. Add 15 ml of methanol to dissolve it. Dilute to the mark with methanol and mix well.

[0086] Accurately pipette 5-20 μL of the test solution and the reference solution respectively, inject into the liquid chromatograph, measure, and record the chromatogram, wherein the chromatographic conditions include: octadecylsilane bonded silica gel as the filler, acetonitrile as the mobile phase A, the mobile phase B is 0.1% phosphoric acid, the flow rate is 1.0 mL / min, the column temperature is 30°C, and the detection wavelength is 268 nm.

[0087] The elution procedure is as follows:

[0088] Time (min) Mobile phase A (%) Mobile phase B (%) 0 5 95 20 20 80 30 20 80 45 35 65 55 90 10 60 90 10

[0089] Test results are shown in Figure 1 .

[0090] After testing, it was determined that Peak 7 was hesperidin, Peak 5 was liquiritin, and Peak 11 was ammonium glycyrrhizate; no obvious chromatographic peaks were found at the elution positions of magnolol, honokiol, and 6-gingerol.

[0091] Comparative Example 1

[0092] 1. Experimental instruments

[0093] Analytical Balance BSA-224S Sartorius Scientific Instruments (Beijing) Co., Ltd.

[0094] Pure water machine FBZ-2002-up-p Qingdao Fulham Technology Co., Ltd.

[0095] Agilent 1260 HPLC

[0096] CNC Ultrasonic Cleaner KQ-500DEKunshan Ultrasonic Instrument Co., Ltd.

[0097] 2. Experimental Reagents

[0098] Methanol V3JG1H Honeywell

[0099] Acetonitrile VBVA1H Honeywell

[0100] Phosphoric acid 20180420 Tianjin Fuchen Chemical Reagent Co., Ltd.

[0101] 3. Preparation of test samples

[0102] Mix and grind the product, take about 0.3g (220301) and weigh accurately, put it in a stoppered conical bottle, accurately add 50ml of methanol, weigh, ultrasonic (500W, 40kHz) for 30 minutes, let it cool, add methanol to make up the weight, shake well and filter, and take the filtrate. This sample must be sealed and refrigerated when not in use.

[0103] 4. Chromatographic conditions

[0104] Mobile phase: 0.1% phosphoric acid water-acetonitrile, chromatographic column: Diamonsil C18(2)4.6*250mm 5μm, column temperature: 30℃, injection volume: 10μL, wavelength 268nm, running time: 90min.

[0105]

[0106] Results Fig. 20 ,according to Fig. 20 It can be seen that the chromatographic peaks are relatively dense, there are many impurity peaks, and the peak shape is poor.

[0107] Comparative Example 2

[0108] 1. Experimental instruments

[0109] Analytical Balance BSA-224S Sartorius Scientific Instruments (Beijing) Co., Ltd.

[0110] Pure water machine FBZ-2002-up-p Qingdao Fulham Technology Co., Ltd.

[0111] Agilent 1260 HPLC

[0112] CNC Ultrasonic Cleaner KQ-500DEKunshan Ultrasonic Instrument Co., Ltd.

[0113] 2. Experimental Reagents

[0114] Methanol V3JG1H Honeywell

[0115] Acetonitrile VBVA1H Honeywell

[0116] Phosphoric acid 20180420 Tianjin Fuchen Chemical Reagent Co., Ltd.

[0117] 3. Preparation of test samples

[0118] Mix and grind the product, take about 0.3g (220301) and weigh accurately, put it in a stoppered conical bottle, accurately add 50ml of methanol, weigh, ultrasonic (500W, 40kHz) for 30 minutes, let it cool, add methanol to make up the weight, shake well and filter, and take the filtrate. This sample must be sealed and refrigerated when not in use.

[0119] 4. Chromatographic conditions

[0120] Mobile phase: 0.1% phosphoric acid water-acetonitrile, chromatographic column: Diamonsil C18(2)4.6*250mm 5μm, column temperature: 30℃, injection volume: 10μL, wavelength 268nm, running time: 60min.

[0121]

[0122] Results Fig.21 ,according to Fig.21 It can be seen that the peak resolution has improved, but the peak shape and resolution are still poor.

[0123] Verification Example

[0124] Fingerprint establishment

[0125] (1) Peak attribution and peak identification

[0126] Preparation of test sample solution: Prepare the test sample solution of single-flavor decoction pieces according to the finished product preparation method:

[0127] Medicinal taste Sample weight (g) Amount of water added (mL) Tangerine peel 0.25 50 Atractylodes macrocephala 0.15 50 Acorus 0.15 50 Magnolia officinalis 0.2 50 Patchouli 0.2 50 Licorice 0.2 50 Jujube 0.25 50 Ginger 0.15 50

[0128] The test solution used was the test solution of Example 1.

[0129] The detected spectrum can be found in Figure 2 .choose Figure 2 Peak 7 (with a large peak area, moderate and stable peak appearance time) was identified as hesperidin through previous comparisons, so it was selected as the reference peak. Figure 2 The peaks from top to bottom are patchouli, atractylodes, jujube, calamus, ginger, magnolia bark, liquorice, tangerine peel and the test sample.

[0130] The relative retention time of each peak and its attribution are shown in the table below.

[0131]

[0132]

[0133] (2) Specificity and integrity

[0134] In order to investigate whether the blank solvent interferes with the spectrum of Taiwushenshu particles, the test solution and blank solvent (dextrin, dissolving solvent) were accurately aspirated and measured according to the chromatographic conditions preliminarily determined. At the same time, the integrity of the sample was investigated and the collection was extended for 30 minutes (using the highest organic phase concentration in the gradient table and isocratic elution).

[0135] Specificity experiment: The results show that the blank solvent has no significant absorption peak at the peak of the test sample, and the specificity is good. See the specific measurement results for details. Figure 3 . Figure 3 The curves of the three species from top to bottom are respectively the dissolving solvent, dextrin, and the test sample.

[0136] Overall inspection: The results show that the test sample has no significant absorption peak at 60min to 90min, and the overall inspection is good. For specific test results, see Figure 4 .

[0137] (3) Instrument precision test

[0138] Take about 2g of this product (batch number: T220301), accurately weigh it, and operate according to the preparation and determination method of the test solution in Example 1. Inject the sample 6 times continuously. The characteristic spectrum of the test sample should show a chromatographic peak with the same retention time as the reference chromatographic peak. Take the S peak as a reference to calculate the relative retention time and relative peak area of ​​the remaining peaks. The specific determination results are shown in Figure 5 and Tables 1 and 2.

[0139] Table 1 Relative retention time results of instrument precision test

[0140]

[0141]

[0142] Table 2 Relative peak area results of instrument precision test

[0143] Serial number S1 S2 S3 S4 S5 S6 Average relative peak area RSD% 1 0.0888 0.0900 0.0899 0.0882 0.0895 0.0887 0.0892 0.81 2 0.0342 0.0346 0.0358 0.0341 0.0343 0.0342 0.0345 1.86 3 0.0252 0.0256 0.0255 0.0253 0.0257 0.0253 0.0254 0.77 4 0.0453 0.0459 0.0460 0.0453 0.0459 0.0454 0.0456 0.73 5 0.5961 0.6053 0.6059 0.5963 0.6050 0.5968 0.6009 0.82 6 0.3479 0.3532 0.3533 0.3479 0.3528 0.3483 0.3506 0.79 7 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.00 8 0.0344 0.0347 0.0348 0.0342 0.0289 0.0343 0.0336 6.82 9 0.1097 0.1119 0.1114 0.1096 0.1118 0.1102 0.1108 0.95 10 0.0299 0.0304 0.0306 0.0301 0.0307 0.0299 0.0303 1.16 11 0.1188 0.1194 0.1196 0.1180 0.1233 0.1179 0.1195 1.66

[0144] Hesperidin peak was used as the reference peak, and the RSD of relative retention time of common chromatographic peaks 1-11 was calculated to be <0.07%, and the RSD of relative peak area was <10%. At the same time, the similarity of each chromatographic fingerprint calculated by the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" software was greater than 0.9, indicating that the instrument had good precision.

[0145] (4) Stability test

[0146] Take about 2g of this product (batch number: T220301), accurately weigh it, and operate according to the preparation and determination method of the test solution in Example 1. Inject and determine at 0, 2, 4, 8, 12, 18, and 24h respectively. The characteristic spectrum of the test product should show a chromatographic peak with the same retention time as the reference chromatographic peak. Take the S peak as a reference to calculate the relative retention time and relative peak area of ​​the remaining peaks. The specific determination results are shown in Figure 6 and Tables 3 and 4.

[0147] Table 3 Relative retention time results of stability test

[0148]

[0149]

[0150] Table 4 Relative peak area results of stability test

[0151] Serial number T0 T2 T4 T8 T12 T18 T24 Average relative peak area RSD% 1 0.0933 0.0932 0.0934 0.0921 0.0920 0.0929 0.0925 0.0928 0.62 2 0.0361 0.0364 0.0364 0.0377 0.0357 0.0374 0.0378 0.0368 2.27 3 0.0272 0.0271 0.0273 0.029 0.0271 0.0271 0.0272 0.0274 2.54 4 0.0483 0.0483 0.0481 0.0458 0.0478 0.0485 0.0485 0.0479 2.00 5 0.6334 0.6325 0.6333 0.6279 0.6260 0.6323 0.633 0.6312 0.47 6 0.3645 0.3644 0.3643 0.3607 0.3602 0.3646 0.3645 0.3633 0.54 7 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.00 8 0.0357 0.0397 0.0395 0.0392 0.0390 0.036 0.0397 0.0384 4.59 9 0.1123 0.1118 0.1121 0.1108 0.1098 0.112 0.1119 0.1115 0.81 10 0.0307 0.0304 0.0306 0.0299 0.0303 0.0303 0.0306 0.0304 0.89 11 0.1186 0.1186 0.1196 0.1189 0.1179 0.1195 0.1194 0.1189 0.52

[0152] Hesperidin peak was used as the reference peak, and the RSD of the relative retention time of the common chromatographic peaks 1-11 was calculated to be <0.25%, and the RSD of the relative peak area was <10%. At the same time, the similarity of each chromatographic fingerprint calculated by the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" software was greater than 0.9, indicating that the test solution was stable within 48 hours.

[0153] (5) Repeatability test

[0154] Take about 2g (6 portions) of this product (batch number: T220301), weigh accurately, and operate according to the preparation and determination method of the test solution in Example 1. The characteristic spectrum of the test product should show a chromatographic peak with the same retention time as the reference chromatographic peak. Take the S peak as a reference, calculate the relative retention time and relative peak area of ​​the remaining peaks, and the specific determination results are shown in Figure 7 and Tables 5-6.

[0155] Table 5 Relative retention time results of repeatability test

[0156]

[0157]

[0158] Table 6 Relative peak area results of repeatability test

[0159] Serial number S1 S2 S3 S4 S5 S6 Average relative peak area RSD% 1 0.0930 0.0933 0.0937 0.0932 0.0932 0.0930 0.0932 0.27 2 0.0361 0.0362 0.0377 0.0380 0.0365 0.0361 0.0368 2.38 3 0.0272 0.0273 0.0272 0.0272 0.0272 0.0273 0.0272 0.25 4 0.0485 0.0485 0.0485 0.0485 0.0485 0.0485 0.0485 0.05 5 0.6321 0.6329 0.6329 0.6327 0.6333 0.6336 0.6329 0.08 6 0.3647 0.3648 0.3642 0.3642 0.3642 0.3645 0.3644 0.07 7 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.00 8 0.0396 0.0332 0.0334 0.0396 0.0358 0.0359 0.0363 7.83 9 0.1123 0.1120 0.1122 0.1122 0.1125 0.1124 0.1123 0.15 10 0.0308 0.0310 0.0306 0.0307 0.0307 0.0307 0.0307 0.43 11 0.1200 0.1194 0.1195 0.1194 0.1192 0.1195 0.1195 0.24

[0160] Hesperidin peak was used as the reference peak, and the RSD of relative retention time of common chromatographic peaks 1-11 was calculated to be <0.20%, and the RSD of relative peak area was <10%. At the same time, the similarity of each chromatographic fingerprint calculated by the "Similarity Evaluation System of Chinese Medicine Chromatographic Fingerprint 2012 Edition" software was greater than 0.9, indicating that the sample repeatability was good.

[0161] (6) Durability test

[0162] The durability of the chromatographic conditions of 3 different flow rates, 3 different column temperatures, 3 different chromatographic columns, and 2 different manufacturers' instruments was investigated. The characteristic spectrum of the test sample should show a chromatographic peak with the same retention time as the reference chromatographic peak. The relative retention time and relative peak area of ​​the remaining peaks were calculated with the S peak as the reference. The specific measurement results are shown in Figure 8-15 and Tables 7-10.

[0163] Table 7 Durability Relative retention time results at three different flow rates

[0164]

[0165]

[0166] Table 8 Durability Relative retention time results at three different column temperatures

[0167] Serial number 30℃ 28℃ 32℃ Average relative retention time RSD% 1 0.2108 0.2088 0.2110 0.2102 0.58 2 0.3349 0.3363 0.3305 0.3339 0.91 3 0.3894 0.3881 0.3876 0.3884 0.24 4 0.4696 0.4706 0.4642 0.4681 0.74 5 0.7494 0.7448 0.7484 0.7475 0.32 6 0.8598 0.8585 0.8585 0.8589 0.09 7 1.0000 1.0000 1.0000 1.0000 0.00 8 1.1148 1.1046 1.1167 1.1120 0.59 9 1.3066 1.2846 1.3157 1.3023 1.23 10 1.4259 1.3964 1.4413 1.4212 1.61 11 1.4964 1.4633 1.5112 1.4903 1.65

[0168] Table 9 Relative retention time results of three different columns of durability

[0169] Serial number Welch Diamonsil Kromasil Average relative retention time RSD% 1 0.2096 0.2097 0.2084 0.2092 0.35 2 0.3326 0.3621 0.3277 0.3408 5.46 3 0.3874 0.3954 0.3979 0.3936 1.39 4 0.4665 0.5020 0.4683 0.4789 4.18 5 0.7463 0.7677 0.7594 0.7578 1.42 6 0.8581 0.8744 0.8640 0.8655 0.95 7 1.0000 1.0000 1.0000 1.0000 0.00 8 1.1113 1.0921 1.1330 1.1121 1.84 9 1.3013 1.2270 1.3522 1.2935 4.87 10 1.4212 1.3399 1.4762 1.4124 4.85 11 1.4888 1.3928 1.5614 1.4810 5.71

[0170] Table 10 Durability Relative retention time results of two instruments

[0171]

[0172]

[0173] (7) Determination of common peaks

[0174] The determined chromatographic method was used to measure 12 batches of samples. 12 batches of samples (200401, 200402, 200403, 200801, 200802, 200803, 220301, 220302, 220303, 220801, 220802, 220803) were selected (the chromatographic peaks with better stability and appropriate response values ​​in the fingerprints were the common peaks. A total of 11 peaks were calibrated as common peaks. The results are shown in Fig.16 .

[0175] (8) Generation of reference feature maps

[0176] The Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software System 2012 Edition issued by the Chinese Pharmacopoeia Committee was used to export the AIA files of 12 batches of HPLC spectra, and the chromatographic peaks were automatically matched to form a common pattern map and generate a reference spectrum. The results are shown in Fig.17 .

[0177] (9) Similarity test results of different batches of magic particles

[0178] See Fig.18 and Fig.19 .

[0179] The fingerprints of 12 batches of trial-production granule samples were similarly calculated using the software "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System, 2012 Edition" recommended by the Pharmacopoeia Committee. The calculation results are shown in Fig.19 , and generate a control map, the results are shown in Fig.18 .Depend on Fig.18 It can be seen that there are 11 common chromatographic peaks, of which peak 7 (S) is hesperidin, peak 5 is liquiritin, and peak 11 is ammonium glycyrrhizinate; compared with the common mode, the similarity values ​​of the 12 batches of particles are all higher than 0.950. Considering comprehensively, it is temporarily stipulated that the fingerprint of the test sample of this product should present chromatographic peaks with the same retention time as the chromatographic peaks of the control fingerprint. According to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint, the similarity between the test sample fingerprint and the control fingerprint shall not be less than 0.9.

[0180] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for establishing a fingerprint of a Chinese medicine composition containing dried tangerine peel, characterized in that: The Chinese medicine composition containing tangerine peel is selected from Shenshu granules; comprising: step 1: preparation of the test solution: Heat each gram of the drug reagent to be tested with 20-30mL 70-100% methanol for 20-40 minutes, cool, weigh, and then add methanol to make up the weight. Then filter to form a filter residue and dissolve it in water, then extract it with dichloromethane, extract the water layer with ethyl acetate and collect the ethyl acetate part, then spin dry and add methanol to dilute to volume, and filter to obtain the product. Step 2: HPLC: Octadecylsilane bonded silica gel is used as filler, acetonitrile is used as mobile phase A, 0.05%-0.3% phosphoric acid solution is used as mobile phase B, and gradient elution is performed; the flow rate is 0.9-1.2 mL / min, the column temperature is 28-32°C, the detection wavelength is 263-273 nm, and the theoretical plate number calculated based on the hesperidin peak should be no less than 2000; The elution procedure is as follows: The specifications of the chromatographic column are 250 mm × 4.6 mm, and the silica particle size is 5 μm; Step 3: Detection: Accurately pipette 5-20 μL of the test solution and the reference solution respectively, inject into the liquid chromatograph, measure, and record the chromatogram to obtain the product.

2. The method for establishing the fingerprint of the Chinese medicine composition containing dried tangerine peel according to claim 1, characterized in that: Mobile phase B was 0.1% phosphoric acid.

3. The method for establishing the fingerprint of the Chinese medicine composition containing dried tangerine peel according to claim 1, characterized in that: include: The chromatogram of the test solution was compared with the fingerprint of the control solution using the traditional Chinese medicine chromatographic fingerprint software, and the similarity was calculated to be greater than 0.

9.

4. The method for establishing the fingerprint of the Chinese medicine composition containing dried tangerine peel according to claim 1, characterized in that: The Chinese medicine chromatographic fingerprint software is "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition".

5. A quality monitoring method for a Chinese medicine composition containing dried tangerine peel, characterized in that: The invention comprises a method for establishing a fingerprint spectrum of the Chinese medicinal composition containing tangerine peel according to any one of claims 1 to 4.

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