Fingerprint spectrum of qi-regulating and blood-activating preparation and establishment method thereof

Abstract

The invention belongs to the technical field of medicine detection, and particularly relates to a qi-regulating and blood-activating preparation fingerprint spectrum and an establishment method, the qi-regulating and blood-activating preparation is prepared from fructus cinnamoii, borneol, ligusticum wallichii and allium macrostemon, the fingerprint spectrum is determined by adopting a UPLC-MS method, and compared with a common liquid chromatography method, the fingerprint spectrum has the advantages of being higher in separation degree, more sensitive in detection, higher in accuracy and the like. The separation time is shorter, and the contained chemical components are clearer; and meanwhile, establishment of a perfect quality control system of the product is facilitated, and the medicine quality and the clinical curative effect are guaranteed.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug detection, and particularly relates to a fingerprint spectrum of a qi-regulating and blood-activating preparation and a method for establishing the same. Background Art

[0002] The qi-regulating and blood-activating dropping pills are a pure traditional Chinese medicine preparation composed of volatile oil of Litsea lancifolia, Allium macrostemon, Ligusticum wallichii, and borneol, which uses Litsea lancifolia, a drug commonly used in the Miao nationality in Guizhou Province to treat chest pain, as the monarch drug, and is rationally formulated according to the medication habits of the Miao nationality and traditional Chinese medicine theory. It has the effects of aromatic-warming and qi-regulating, and relieving pain. It is mainly used to treat chest pain due to yang deficiency and cold coagulation (angina pectoris of coronary heart disease), manifested as chest tightness, shortness of breath, palpitation, fear of cold, and even pain radiating to the back, severe pain when feeling cold, and difficulty in breathing. It has achieved good results in clinical prevention and treatment of chest pain due to yang deficiency and cold coagulation.

[0003] In the prior art, Patent Document 1 discloses a "quality control method for dropping pill preparations for treating chest pain" (CN101708274A), which mainly uses thin-layer chromatography to qualitatively identify Litsea lancifolia and Ligusticum wallichii in the qi-regulating and blood-activating dropping pills, and uses gas chromatography to determine the contents of cineole and borneol. Subsequently, Patent Document 2 discloses a "detection method for a Migao Xinle preparation" (CN103105447A), which mainly adds the determination of the content of ferulic acid on the basis of Patent Document 1.

[0004] At present, the fingerprint spectrum of traditional Chinese medicine is an effective mode for quality control of traditional Chinese medicine. However, up to now, no fingerprint spectrum study has been carried out on the qi-regulating and blood-activating dropping pills, especially the rapid separation and accurate identification of the chemical components contained in the qi-regulating and blood-activating dropping pills by UPLC-MS. In order to comprehensively analyze the chemical components contained in the qi-regulating and blood-activating dropping pills and comprehensively and effectively control its quality, the applicant has conducted an in-depth study on the fingerprint spectrum of the qi-regulating and blood-activating dropping pills. Summary of the Invention

[0005] The present invention provides a method for establishing a fingerprint spectrum of a qi-regulating and blood-activating preparation in view of the imperfect detection method in the prior art of the qi-regulating and blood-activating preparation, comprising the following steps:

[0006] (1) Preparation of the test solution: Take the qi-regulating and blood-activating preparation, add methanol, dissolve it by ultrasonic treatment, filter, and obtain the test solution;

[0007] (2) Preparation of the reference solution: Take an appropriate amount of one or more reference substances such as senkyunolide A, senkyunolide G, senkyunolide H, (-)-camphor, senkyunolide I, ferulic acid, ligustilide, linoleic acid, cinnamic acid, and butenylphthalide, add methanol to make a solution with a concentration of 0.01 - 0.1 mg / ml, shake well, and set aside;

[0008] (3) Chromatographic conditions: The chromatographic column is packed with octadecylsilyl silica gel. Acetonitrile is used as mobile phase A, and an aqueous solution of 0.5% - 2% ammonium formate or ammonium acetate is used as mobile phase B for gradient elution. The elution gradient is as follows: 0 - 5 min, 5% - 15% A; 5 - 10 min, 15% - 35% A; 10 - 20 min, 35% - 55% A; 20 - 40 min, 55% - 85% A; 40 - 50 min, 85% - 95% A. The flow rate is 0.4 - 1.0 mL / min;

[0009] (4) Mass spectrometry conditions: Positive and negative ion analysis modes are adopted; the ionization method is electrospray ionization; accurate mass number calibration is performed using a standard tuning solution before injection analysis; the detection range of the first - stage mass spectrometry scan is 100 - 1700 m / z; nitrogen is used as the solvent removal drying gas; the temperature is 300 - 350 °C; the flow rate is 5 - 7 L / min; the sheath gas temperature is 320 - 380 °C; the capillary voltage is 3.0 - 4.0 kV; the Fragment voltage is 150 - 200 V;

[0010] (5) Establishment of the fingerprint of the regulating qi and activating blood circulation preparation: Using the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints of the Pharmacopoeia Commission of the People's Republic of China, the liquid chromatograms of the test solution and the reference solution are respectively imported with data, corrected at multiple points and matched with data to obtain the fingerprint of the regulating qi and activating blood circulation preparation.

[0011] Specifically, the fingerprint of the regulating qi and activating blood circulation preparation is divided into fingerprints in the positive - ion mode and the negative - ion mode.

[0012] Specifically, the fingerprint in the positive - ion mode has a total of 26 chromatographic peaks, and the ligustilide A at the 29th peak is used as the reference peak S.

[0013] Specifically, the fingerprint in the negative - ion mode has a total of 26 chromatographic peaks, and the ligustilide G at the 9th peak is used as the reference peak S.

[0014] Preferably, the chromatographic column is Agilent ZORBAX Eclipse Plus C18 (3.0 mm x 100 mm, 1.8 μm).

[0015] Preferably, the mobile phase is: Acetonitrile is used as mobile phase A, and an aqueous solution of 1% ammonium formate is used as mobile phase B for gradient elution. The elution gradient is as follows: 0 - 5 min, 5% - 15% A; 5 - 10 min, 15% - 35% A; 10 - 20 min, 35% - 55% A; 20 - 40 min, 55% - 85% A; 40 - 50 min, 85% - 95% A.

[0016] Preferably, the mass spectrometry conditions are as follows: positive and negative ion analysis modes are adopted; the ionization method is electrospray ionization (ESI); accurate mass number calibration is performed using an Agilent standard tuning solution (G1969-85000) before sample injection and analysis; the detection range of the first-stage mass spectrometry scan is 100-1700 m / z; nitrogen is used as the solvent removal and drying gas; the temperature is 325 °C; the flow rate is 6.8 L·min -1 ; the sheath gas temperature is 350 °C; the capillary voltage is 4.0 kV; the Fragment voltage is 150 V; the second-stage fragment voltages are 20 V, 30 V, and 40 V. Based on the first-stage scan, the top three intensities are selected for collision-induced dissociation (CID) to obtain their second-stage mass spectrometry data.

[0017] In some embodiments, the regulating qi and activating blood circulation preparation is regulating qi and activating blood circulation dropping pills, and the detection method of its fingerprint includes the following steps:

[0018] (1) Preparation of the test solution: Crush the regulating qi and activating blood circulation dropping pills, weigh 2.0 g of the powder, add 20 ml of methanol, sonicate for 20 min, take the supernatant and filter to obtain the test solution;

[0019] (2) Preparation of the reference solution: Take appropriate amounts of ferulic acid, senkyunolide A, senkyunolide G, senkyunolide H, (-)-borneol, linoleic acid, and cinnamic acid reference standards, dissolve them in methanol to make a solution of 0.02 mg / ml, shake well, and set aside;

[0020] (3) Chromatographic conditions: The chromatographic column used is an Agilent ZORBAX Eclipse Plus C18 column. Acetonitrile is used as mobile phase A, and 1% ammonium formate aqueous solution is used as mobile phase B for gradient elution. The elution gradient is as follows: 0-5 min, 5%-15% A; 5-10 min, 15%-35% A; 10-20 min, 35%-55% A; 20-40 min, 55%-85% A; 40-50 min, 85%-95% A; the flow rate is 0.4 mL / min;

[0021] (4) Mass spectrometry conditions: Positive and negative ion analysis modes are adopted; the ionization method is electrospray ionization; accurate mass number calibration is performed using an Agilent standard tuning solution before sample injection and analysis; the detection range of the first-stage mass spectrometry scan is 100-1700 m / z; nitrogen is used as the solvent removal and drying gas; the temperature is 325 °C; the flow rate is 6.8 L / min; the sheath gas temperature is 350 °C; the capillary voltage is 4.0 kV; the Fragment voltage is 150 V, and the second-stage fragment voltages are 20 V, 30 V, and 40 V;

[0022] (5) Assay: Precisely pipette 2 μl each of the reference substance solution and the test substance solution, inject them into the liquid chromatograph, and perform the determination according to the chromatographic conditions and mass spectrometry conditions, and record the total ion current chromatogram;

[0023] In the fingerprint of the test substance, the chromatographic peak with the same retention time as ligustilide A in the reference substance should appear in the positive ion mode, and the chromatographic peak with the same retention time as ligustilide G in the reference substance should appear in the negative ion mode; calculated by the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints, the similarity between the fingerprint of the test substance and the control fingerprint should not be lower than 0.90.

[0024] In some embodiments, the regulating qi and activating blood circulation dropping pills are prepared from the following raw materials in parts by weight: 450 parts of Litsea lancifolia, 9 parts of borneol, 360 parts of Ligusticum chuanxiong, and 36 parts of Allium macrostemon. The preparation method is as follows: extract the essential oil from Litsea lancifolia by steam distillation; crush Ligusticum chuanxiong into coarse powder, extract and purify it with ethanol solution to obtain Ligusticum chuanxiong extract; crush Allium macrostemon into coarse powder, extract it by ethanol reflux to obtain Allium macrostemon extract, mix the Ligusticum chuanxiong extract and Allium macrostemon extract, heat slightly and mix evenly, add it to molten polyethylene glycol, mix well, add the obtained essential oil of Litsea lancifolia and borneol, mix evenly, and drop into dropping pills to obtain.

[0025] Beneficial effects:

[0026] 1. The present invention uses UPLC-MS to establish the fingerprint of the regulating qi and activating blood circulation preparation, combines various chemical pattern recognition methods such as cluster analysis, and identifies the main chemical components of regulating qi and activating blood circulation, which can more comprehensively evaluate the product quality of the regulating qi and activating blood circulation preparation, ensure the clinical efficacy of the product, and fill the blank of the fingerprint detection method for the regulating qi and activating blood circulation preparation.

[0027] 2. Compared with the ordinary liquid chromatography method, the technical solution of the present invention has the advantages of higher resolution, more sensitive detection, and shorter separation time. Description of the drawings

[0028] Figure 1 Fingerprint of regulating qi and activating blood circulation dropping pills (positive ion mode)

[0029] Figure 2 Fingerprint of regulating qi and activating blood circulation dropping pills (negative ion mode)

[0030] Figure 3 Total ion current chromatograms of the sample in positive ion mode (A) and negative ion mode (B)

[0031] Figure 4Total ion chromatograms of the reference substance in positive ion mode (C) and negative ion mode (D), where peak 5 is ferulic acid; peak 9 is senkyunolide G; peak 19 is senkyunolide I; peak 21 is ligustilide; peak 25 is senkyunolide H; peak 29 is senkyunolide A; peak 37 is butenylphthalide; peak 43 is cinnamic acid; peak 45 is (-)-camphor; peak 50 is linoleic acid

[0032] Figure 5 Cluster analysis diagrams of 15 batches of Liqi Huoxue Pills in positive ion mode (A) and negative ion mode (B)

[0033] Figure 6 PCA score diagrams of Liqi Huoxue Pills in positive ion mode (A) and negative ion mode (B)

[0034] Figure 7 PLS-DA score diagrams of Liqi Huoxue Pills in positive ion mode (A) and negative ion mode (B)

[0035] Figure 8 PLS-DA VIP value diagrams of Liqi Huoxue Pills in positive ion mode (A) and negative ion mode (B) Specific implementation manners

[0036] To make the technical solutions and technical effects of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described below. The embodiments described below are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art without creative efforts in combination with the embodiments of the present invention belong to the scope of protection of the present invention.

[0037] In a specific embodiment of the present application, the present application provides a fingerprint detection method for Liqi Huoxue Pills, which includes the following steps:

[0038] The fingerprint of Liqi Huoxue Dropping Pills was detected by UPLC-MS. Among them, the test conditions of high performance liquid chromatography were as follows: the chromatographic column was Agilent ZORBAX Eclipse Plus C18 (3.0 mm x 100 mm, 1.8 μm), acetonitrile was used as mobile phase A, and 1% ammonium formate aqueous solution was used as mobile phase B for gradient elution; the elution gradient was: 0 - 5 min, 5% - 15% A; 5 - 10 min, 15% - 35% A; 10 - 20 min, 35% - 55% A; 20 - 40 min, 55% - 85% A; 40 - 50 min, 85% - 95% A. The mass spectrometry conditions were as follows: positive and negative ion analysis modes were adopted; the ionization method was electrospray ionization (ESI); accurate mass number correction was carried out using Agilent standard tuning solution (G1969-85000) before injection analysis; the detection range of primary mass spectrometry scan was: 100 - 1700 m / z; nitrogen was used as the solvent removal drying gas; the temperature was 325 °C; the flow rate was 6.8 L·min -1 ; the sheath gas temperature was 350 °C; the capillary voltage was 4.0 kV; the Fragment voltage was 150 V; the secondary fragment voltages were 20 V, 30 V, 40 V.

[0039] In some embodiments of the present application, the flow rate of the mobile phase was 0.4 ml / min;

[0040] In some embodiments of the present application, the injection volume was 2 μL

[0041] The technical solutions provided by the present invention will be described in detail below in conjunction with specific embodiments.

[0042] In the embodiments, all the original reagent materials were commercially available. The experimental methods without specific conditions were conventional methods and conventional conditions well-known in the art, or the conditions recommended by the instrument manufacturer.

[0043] The instruments and reagents used in the following examples and comparative examples were:

[0044] Instruments: Agilent 1290 UPLC-6540 0-TOF chromatography-mass spectrometry combined instrument (configured with MassHunter mass spectrometry workstation and Mass analysis mass spectrometry data processing system), Agilent ZORBAX Eclipse Plus C18 (3.0 mm x 100 mm, 1.8 μm) chromatographic column (Agilent Technologies, USA); ultrasonic instrument (Shanghai Kedao Ultrasonic Instrument Co., Ltd.), S-23 high-speed tabletop centrifuge (Hunan Xiangyi Instrument Co., Ltd.); AUW120D one-ten-thousandth electronic analytical balance (SHIMADZU Corporation, Japan).

[0045] Drugs and reagents: Chromatographic grade methanol, mass spectrometry grade acetonitrile (Merck Millipore, Germany); distilled water (Watsons Distilled Water Co., Ltd., Shenzhen); mass spectrometry grade ammonium formate (Fisher, USA). Reference substances: Senkyunolide A (batch number: 21080506), Senkyunolide G (batch number 21072411), Senkyunolide H (batch number: 21012602), (-)-Camphor (20201012), Senkyunolide I (batch number: 21012506), Cinnamic acid (batch number: 21061603) were all purchased from Chengdu Pufeide Biotechnology Co., Ltd., Ferulic acid (batch number: 919C021), Ligustilide (batch number: 903C021) were all purchased from Beijing Solarbio Science & Technology Co., Ltd., Linoleic acid (batch number: HR2178W10) was purchased from Baoji Chenguang, Butylidenephthalide (Shanghai Standard Biotech Co., Ltd., batch number: 18443).

[0046] Example 1 Preparation method of Liqi Huoxue Dropping Pills

[0047] Weigh 450 g of Litsea lancifolia, 9 g of Borneol, 360 g of Ligusticum chuanxiong, and 36 g of Allium macrostemon, and prepare according to the following method: Extract the essential oil from Litsea lancifolia by steam distillation method, crush Ligusticum chuanxiong into coarse powder, and then extract it twice by reflux under reduced pressure with 10 times the amount of 95% ethanol. For the first time, soak it with 6 times the amount of ethanol for 2 hours and reflux for 3 hours. For the second time, use 4 times the amount of ethanol and reflux for 2 hours. Combine the two extraction solutions, recover ethanol under reduced pressure until the relative density is about 1.1, take the middle layer solution, concentrate it under reduced pressure until the relative density is about 1.2 (55 - 60 °C), adjust the pH value to 9 with ammonia water, and then extract it three times with 6 times the amount of chloroform. Combine the extraction solutions, recover chloroform under reduced pressure and concentrate it until the relative density is 1.01 - 1.05 (55 - 60 °C) to obtain Ligusticum chuanxiong extract. Crush Allium macrostemon into coarse powder, soak it with 6 times the amount of 95% ethanol and reflux for extraction twice, each time for 1 hour, and concentrate it under reduced pressure until the relative density is 1.12 - 1.16 (55 - 60 °C) to obtain Allium macrostemon extract. Mix the Ligusticum chuanxiong extract and Allium macrostemon extract, heat slightly to mix evenly, add it to the molten polyethylene glycol, mix well, add the obtained essential oil of Litsea lancifolia and Borneol, mix well, and drop it into dropping pills to obtain the product.

[0048] Example 2 Fingerprint detection method of Liqi Huoxue Dropping Pills

[0049] 2.1. Preparation of test solution: Crush Liqi Huoxue Dropping Pills, weigh 2.0 g of the powder, add 20 ml of methanol, dissolve it by ultrasonic wave for 20 min, let it stand, take the supernatant, and centrifuge at 3000 r·min -1 for 5 min. After centrifugation, take the supernatant and filter it through a 0.22 μm microporous filter membrane to obtain the test solution.

[0050] 2.2. Preparation of the reference solution: Weigh accurately 4.0 mg each of senkyunolide A, senkyunolide H, senkyunolide G, ferulic acid, (-)-camphor, linoleic acid, and cinnamic acid reference substances, and transfer them separately to 25-ml volumetric flasks. Add an appropriate amount of methanol, dissolve by ultrasonic treatment, allow to stand for 10 min, and make up the volume. Filter through a microporous membrane filter to obtain the stock solutions of the reference substances. Measure 100 μl each of the stock solutions of the reference substances, mix them to obtain the reference solution.

[0051] 2.3. Chromatographic conditions: The chromatographic column used is Agilent ZORBAX Eclipse Plus C18 (3.0 mm x 100 mm, 1.8 μm). Acetonitrile is used as mobile phase A, and 1% ammonium formate aqueous solution is used as mobile phase B for gradient elution. The elution gradient is as follows: 0 - 5 min, 5% - 15% A; 5 - 10 min, 15% - 35% A; 10 - 20 min, 35% - 55% A; 20 - 40 min, 55% - 85% A; 40 - 50 min, 85% - 95% A; flow rate: 0.4 mL / min.

[0052] 2.4. Mass spectrometry conditions: Positive and negative ion analysis modes are adopted; the ionization method is electrospray ionization (ESI); accurate mass number calibration is performed using Agilent standard tuning solution (G1969 - 85000) before injection analysis; the detection range of the first-stage mass spectrometry scan is 100 - 1700 m / z; nitrogen is used as the solvent removal drying gas; the temperature is 325 °C; the flow rate is 6.8 L·min -1 ; the sheath gas temperature is 350 °C; the capillary voltage is 4.0 kV; the Fragment voltage is 150 V; the second-stage fragment voltages are 20 V, 30 V, and 40 V.

[0053] 2.5. Determination method: Accurately pipette 2 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, and perform the determination according to the high-performance liquid chromatography conditions and the mass spectrometry conditions, and record the chromatogram and mass spectrum.

[0054] In the fingerprint of the test sample, chromatographic peaks with the same retention time as those of the reference chromatographic peaks should be presented respectively. Calculated by the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints, the similarity between the fingerprint of the test sample and the reference fingerprint should not be lower than 0.90.

[0055] Example 3 Establishment of the fingerprint of Liqi Huoxue Dropping Pills

[0056] 3.1. Preparation of the test solution:

[0057] Pulverize the Liqi Huoxue Dropping Pills, weigh 2.0 g of the powder, add 20 ml of methanol, dissolve by ultrasonic treatment for 20 min, allow to stand, and take the supernatant. Centrifuge at 3000 r·min -1Centrifuge for 5 min. After centrifugation, take the supernatant and filter it through a 0.22 μm microporous membrane to obtain the test solution.

[0058] 3.2. Preparation of reference solution:

[0059] Accurately weigh 4.0 mg each of 10 reference substances including senkyunolide A, senkyunolide H, senkyunolide G, senkyunolide I, ferulic acid, L-camphor, ligustilide, linoleic acid, cinnamic acid, and butenylphthalide, and transfer them separately to 25-ml volumetric flasks. Add an appropriate amount of methanol and dissolve by ultrasonic treatment. Let it stand for 10 min and make up the volume. Filter through a microporous membrane to obtain the stock solutions of the 10 reference substances. Measure 100 μl each of the stock solutions of the reference substances and mix them to obtain the reference solution.

[0060] 3.3. Chromatographic conditions:

[0061] The chromatographic column used is Agilent ZORBAX Eclipse Plus C18 (3.0 mm x 100 mm, 1.8 μm). Acetonitrile is used as mobile phase A and 1% ammonium formate aqueous solution is used as mobile phase B for gradient elution. The elution gradient is as follows: 0 - 5 min, 5% - 15% A; 5 - 10 min, 15% - 35% A; 10 - 20 min, 35% - 55% A; 20 - 40 min, 55% - 85% A; 40 - 50 min, 85% - 95% A; flow rate: 0.4 mL / min.

[0062] 3.4. Mass spectrometry conditions:

[0063] Positive and negative ion analysis modes are adopted; the ionization method is electrospray ionization (ESI); accurate mass number calibration is carried out using Agilent standard tuning solution (G1969 - 85000) before sample injection and analysis; the detection range of the first-stage mass spectrometry scan is 100 - 1700 m / z; nitrogen is used as the desolvation drying gas; the temperature is 325 °C; the flow rate is 6.8 L·min -1 ; the sheath gas temperature is 350 °C; the capillary voltage is 4.0 kV; the Fragment voltage is 150 V; the second-stage fragment voltages are 20 V, 30 V, and 40 V.

[0064] 3.5. Establishment of fingerprint and similarity evaluation

[0065] Fifteen batches of Liqi Huoxue Pills were taken respectively, and the test solution was prepared according to the test solution method under item "3.1". Injection and determination were carried out under the chromatographic conditions under items "3.3, 3.4", the chromatogram was recorded, and the data was imported into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition)". The time window was set to 0.1. The positive ion flow diagram was referenced by the chromatogram of sample S1, and the negative ion flow diagram was referenced by the chromatogram of sample S1. Chromatographic peak matching was performed by the average method, and the fingerprint diagrams of 15 batches of samples are shown in Figure 1 、 2 , and the similarity is shown in Table 1. By comparing with the total ion flow diagram of the reference substance (see Figure 3 、 Figure 4 ), 10 components including senkyunolide A, senkyunolide H, (-)-camphor, senkyunolide G, senkyunolide I, ferulic acid, ligustilide, linoleic acid, cinnamic acid, and butenylphthalide were identified.

[0066] From Figure 1 、 Figure 2 the fingerprint diagrams of Liqi Huoxue Pills in positive and negative ion modes, it can be seen that the chemical components of 15 batches of Liqi Huoxue Pills are generally stable. Combining with Table 1, it can be known that the similarities of 15 batches of Liqi Huoxue Pills are all ≥0.978, indicating that the quality of each batch of 15 batches of Liqi Huoxue Pills is relatively stable.

[0067] Table 1 Similarity table of peaks of 15 batches of samples and their reference peaks

[0068]

[0069] Example 4 Methodology verification of fingerprint

[0070] 4.1 Precision test Take Liqi Huoxue Pills and prepare the test solution according to the method under item "3.1". Then, inject continuously 6 times according to the chromatographic and mass spectrometric conditions under items "3.3 and 3.4". Using senkyunolide A (peak 29) and senkyunolide G (peak 9) as reference peaks (S) respectively, calculate the relative retention time and relative peak area of each chromatographic peak (26 peaks in positive ion mode and 26 peaks in negative ion mode). The results show that the RSDs of the relative retention time and relative peak area of each common peak are all less than 3.0%, indicating good instrument precision.

[0071] 4.2 Stability test Take Liqi Huoxue Pills and prepare the test solution according to the method under item "3.1". Place it at room temperature for 0, 2, 4, 8, 10, 12, 24 h, inject according to the chromatographic and mass spectrometric conditions under items "3.3 and 3.4". Using senkyunolide A (peak 29) and senkyunolide G (peak 9) as reference peaks (S) respectively, calculate the relative retention time and relative peak area of each chromatographic peak (the same as above). The results show that the RSDs of each common peak are all less than 3.0%, indicating good stability of the solution within 24 h.

[0072] 4.3 Repeatability test Take 6 portions of the same batch of Liqi Huoxue Dropping Pills, prepare the test solution respectively according to the method in item "3.1", inject samples under the chromatographic conditions in items "3.3 and 3.4", take senkyunolide A (peak No. 29) and senkyunolide G (peak No. 9) as the reference peaks (S), calculate the relative retention time and relative peak area of each chromatographic peak (the same as above), and the results show that the RSDs of each common peak are all less than 3.0%, indicating that this method has good repeatability.

[0073] Example 5 Establish a chemical pattern recognition analysis method for Liqi Huoxue Dropping Pills

[0074] 5.1 Cluster analysis

[0075] Import the peak areas of the common peaks in the fingerprint spectra of Liqi Huoxue Dropping Pills in positive and negative ion modes into SPSS software, and perform cluster analysis by between-group linkage and squared Euclidean distance method. The results are shown in Figure 5 .

[0076] As can be seen from Figure 5 , when the classification distance is 3, the 15 batches of samples can be divided into 6 categories in the positive ion mode. The first category is samples S13, S14, S15, S10, S11, S11; the second category is samples S7, S8, S9, S6; the third category is sample S1; the fourth category is sample S1; the fifth category is samples S3, S4; the sixth category is sample S5. In the negative ion mode, they can be divided into 6 categories. The first category is samples S7, S8, S9, S10, S11, S12, S14; the second category is samples S13, S15; the third category is samples S5, S6; the fourth category is samples S3, S4; the fifth category is sample S2; the sixth category is sample S1.

[0077] It shows that the overall quality of the 15 batches of Liqi Huoxue Dropping Pills is basically stable, and the differences in the common components of each batch of preparations may be caused by different raw material sources, preparation processes, etc.

[0078] 5.2 Principal component analysis (PCA)

[0079] Take the peak areas of the common peaks in the fingerprint spectra of Liqi Huoxue Dropping Pills in positive and negative ion modes as variables, import them into SPSS software, perform PCA after data standardization processing. Taking the eigenvalue > 1 as the standard, 5 principal components are extracted in the positive ion mode, and their cumulative variance contribution rate is 89.203%. In the negative ion mode, 2 principal components are extracted, and their cumulative variance contribution rate is 89.383%, which can objectively reflect most of the information of Liqi Huoxue Dropping Pills, as shown in Table 2 and Table 3. Use SIMCA-P software to draw the PCA score plot, as shown in Figure 6 .

[0080] As can be seen from Figure 6It can be seen that the 15 batches of samples can be divided into 6 categories in both positive and negative ion modes, which is consistent with the results of cluster analysis.

[0081] Table 2 PCA eigenvalue and variance contribution rate of Liqi Huoxue Pills samples (positive ion mode)

[0082]

[0083] Table 3 PCA eigenvalue and variance contribution rate of Liqi Huoxue Dripping Pills samples (negative ion mode)

[0084]

[0085] 5.3 Partial least squares discriminant analysis (PLS-DA)

[0086] Import the peak areas of the common peaks in the fingerprint spectra of Liqi Huoxue Dripping Pills in positive and negative ion modes into SIMCA software for PLS-DA. The results are shown in Figure 7 .

[0087] From Figure 7 it can be seen that the 15 batches of Liqi Huoxue Dripping Pills can be divided into 6 categories, which is consistent with the results of cluster analysis and PCA. Using the variable importance in projection (VIP) method with VIP>1 as the standard, 11 differential markers were obtained in the positive ion mode, which are chromatographic peaks 40, 21, 32, 13, 30, 39, 20, 1, 25 in sequence, indicating that these 9 components are the main components causing the classification differences of Liqi Huoxue Dripping Pills in the positive ion mode; 5 differential markers were obtained in the negative ion mode, which are chromatographic peaks 49, 33, 36, 31, 47 in sequence, indicating that these 5 components are the main components causing the classification differences of Liqi Huoxue Dripping Pills in the negative ion mode (see Figure 8 ). Thus, there are 14 main components that affect the large classification differences of Liqi Huoxue Dripping Pills, and their changes can be focused on during the quality control in the production process.

[0088] 5.4 Qualitative analysis of chemical components of common peaks by UPLC-Q-TOF-MS

[0089] By using the Agilent TCM-DATA traditional Chinese medicine ingredient database to generate molecular formulas and screening according to the principle of error less than 5×10-6, and at the same time retrieving databases such as China National Knowledge Infrastructure (CNKI), PubMed, and Massbank, a chemical component information library of Liqi Huoxue Dripping Pills was established. According to the primary mass spectrum, secondary mass spectrum, comparison with reference substances, and comparison with the self-built traditional Chinese medicine ingredient compound library of the compounds, and combined with the literature, 50 chemical components were determined in Liqi Huoxue Dripping Pills. The results are shown in Table 4.

[0090] Table 4 Identification and analysis of components of common peaks of Liqi Huoxue Dripping Pills by UPLC-Q-TOF / MS

[0091]

[0092]

[0093] *: Confirmed by comparison with reference standards.

[0094] The above embodiments are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for establishing a fingerprint of a preparation for regulating qi and promoting blood circulation, wherein the preparation for regulating qi and promoting blood circulation is made of Litsea cubeba, Artemisia argyi, Rhizoma Chuanxiong and Radix Allii Macrostemonis, and is characterized in that: The steps include: (1) Preparation of test solution: Take the Qi-regulating and blood-activating preparation, add methanol, ultrasonically dissolve, and filter to obtain the test solution; (2) Preparation of reference solution: Take appropriate amount of one or more reference substances selected from ligusticolide A, ligusticolide G, ligusticolide H, L-camphor, ligusticolide I, ferulic acid, ligustilide, linoleic acid, cinnamic acid, and butenylphthalide, add methanol to make a solution of 0.01-0.1 mg / ml, shake well, and set aside; (3) Chromatographic conditions: The column was filled with octadecylsilane bonded silica gel, acetonitrile was used as mobile phase A, and 0.5% to 2% ammonium formate or ammonium acetate aqueous solution was used as mobile phase B. Gradient elution was performed, and the elution gradient was: 0 to 5 min, 5% to 15% A; 5 to 10 min, 15% to 35% A; 10 to 20 min, 35% to 55% A; 20 to 40 min, 55% to 85% A; 40 to 50 min, 85% to 95% A; the flow rate was 0.4 to 1.0 mL / min; (4) Mass spectrometry conditions: positive and negative ion analysis modes were used; the ionization method was electrospray ionization; standard tuning solution was used for accurate mass calibration before sample injection analysis; primary mass spectrometry scanning detection range: 100-1700 m / z; nitrogen was used as the solvent removal and drying gas; the temperature was 300-350 °C; the flow rate was 5-7 L / min; the sheath gas temperature was 320-380 °C; the capillary voltage was 3.0-4.0 kV; and the fragment voltage was 150-200 V; (5) Establishment of fingerprint of Qi-regulating and blood-activating preparations: Using the Chinese Pharmacopoeia Committee's Chinese medicine chromatographic fingerprint similarity evaluation system, the liquid chromatograms of the test solution and the reference solution were subjected to data import, multi-point calibration and data matching to obtain the fingerprint of the Qi-regulating and blood-activating preparations.

2. The method for establishing the fingerprint of the Qi-regulating and blood-activating preparation according to claim 1, characterized in that: The chromatographic column is Agilent ZORBAX Eclipse Plus C18, with a specification of 3.0 mm×100 mm and a particle size of 1.8 μm.

3. The method for establishing the fingerprint spectrum of the Qi-regulating and blood-activating preparation according to claim 1, characterized in that: The mobile phase B is 1% ammonium formate aqueous solution.

4. The method for establishing the fingerprint of the Qi-regulating and blood-activating preparation according to claim 1, characterized in that: The flow rate was 0.4 mL / min.

5. The method for establishing the fingerprint of the Qi-regulating and blood-activating preparation according to claim 1, characterized in that: The mass spectrometry conditions are as follows: the primary mass spectrometry scanning temperature is 325°C, the flow rate is 6.8 L / min; the sheath gas temperature is 350°C; the capillary voltage is 4.0 kV; the fragment voltage is 150 V; and the secondary fragment voltages are 20 V, 30 V, and 40 V.

6. The method for establishing the fingerprint spectrum of the Qi-regulating and blood-activating preparation according to claim 1, characterized in that: The fingerprint spectrum of the Qi-regulating and blood-activating preparation is divided into fingerprint spectrum under positive ion mode and fingerprint spectrum under negative ion mode.

7. The method for establishing the fingerprint spectrum of the Qi-regulating and blood-activating preparation according to claim 6, characterized in that: The fingerprint spectrum in the positive ion mode has 26 chromatographic peaks, and peak No. 29, ligusticum chuanxiong lactone A, is used as the reference peak S.

8. The method for establishing the fingerprint spectrum of the Qi-regulating and blood-activating preparation according to claim 6, characterized in that: The fingerprint spectrum in the negative ion mode has 26 chromatographic peaks in total, and peak No. 9, ligusticum chuanxiong lactone G, is used as the reference peak S.

9. A method for detecting fingerprints of a preparation for regulating qi and promoting blood circulation, wherein the preparation for regulating qi and promoting blood circulation is a pill for regulating qi and promoting blood circulation, characterized in that: The steps include: (1) Preparation of test solution: Grind Liqi Huoxue Pills, weigh 2.0 g of powder, add 20 ml of methanol, sonicate for 20 min, and filter the supernatant to obtain the test solution. (2) Preparation of reference solution: Take appropriate amount of ligusticolide A, ligusticolide G, ferulic acid, ligusticolide H, L-camphor, linoleic acid, and cinnamic acid reference substances, add methanol to make a solution containing 0.02 mg / ml of each, shake well, and set aside; (3) Chromatographic conditions: The chromatographic column was Agilent ZORBAX Eclipse Plus C18, with acetonitrile as mobile phase A and 1% ammonium formate aqueous solution as mobile phase B. Gradient elution was performed with the following gradients: 0-5 min, 5%-15% A; 5-10 min, 15%-35% A; 10-20 min, 35%-55% A; 20-40 min, 55%-85% A; 40-50 min, 85%-95% A; flow rate: 0.4 mL / min; (4) Mass spectrometry conditions: positive and negative ion analysis modes were used; the ionization method was electrospray ionization; Agilent standard tuning solution was used for accurate mass calibration before sample injection analysis; the primary mass spectrometry scanning detection range was: 100-1700 m / z; nitrogen was used as the solvent removal and drying gas; the temperature was 325 °C; the flow rate was 6.8 L / min; the sheath gas temperature was 350 °C; the capillary voltage was 4.0 kV; the fragment voltage was 150 V; the secondary fragment voltage was 20 V, 30 V, and 40 V; (5) Determination method: Accurately pipette 2 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, perform the determination according to the chromatographic conditions and mass spectrometry conditions, and record the total ion current chromatogram; The fingerprint of the test sample in the positive ion mode should show a chromatographic peak with the same retention time as the reference substance ligusticum lactone A, and the fingerprint of the test sample in the negative ion mode should show a chromatographic peak with the same retention time as the reference substance ligusticum lactone G; calculated according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprints, the similarity between the test sample fingerprint and the control fingerprint shall not be less than 0.

90.

10. The method according to any one of claims 1 to 9, characterized in that The preparation for regulating qi and activating blood circulation is a Qi-regulating and activating blood circulation pill, which is prepared from the following raw materials in parts by weight: 450 parts of Litsea cubeba, 9 parts of Artemisia argyi tablets, 360 parts of Chuanxiong and 36 parts of Allium macrostemon. The preparation method is as follows: extracting essential oil from Litsea cubeba by steam distillation; crushing Chuanxiong into coarse powder, extracting and purifying with ethanol solution to prepare Chuanxiong extract; crushing Allium macrostemon into coarse powder, extracting with ethanol reflux to prepare Allium macrostemon extract, mixing the Chuanxiong extract and Allium macrostemon extract, mixing evenly with slight heat, adding into molten polyethylene glycol, mixing evenly, adding the obtained Litsea cubeba essential oil and Artemisia argyi tablets, mixing evenly, and dripping into pills.

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