Fingerprint spectrum of liqi huoxue preparation and establishment method thereof

Abstract

The application belongs to the technical field of medicine detection, and particularly relates to a kind of fingerprint spectrum of qi-regulating and blood-activating preparation and a method for establishing, the qi-regulating and blood-activating preparation is made of Litsea rotundifolia, moxa tablet, chuanxiong, and baiyunqing, the fingerprint spectrum is determined by UPLC-MS method, compared with ordinary liquid chromatography, has the advantages of higher resolution, more sensitive detection, shorter separation time, and more explicit chemical components; at the same time, it is helpful to establish a perfect quality control system of product, and is helpful to guarantee the quality of medicine and clinical efficacy.

Description

Technical Field

[0001] This invention belongs to the field of drug detection technology, specifically relating to a fingerprint spectrum of a qi-regulating and blood-activating preparation and its establishment method. Background Technology

[0002] Qi-regulating and Blood-activating Dripping Pills are a traditional Chinese medicine preparation made from Litsea cubeba, a herb commonly used by the Miao people of Guizhou Province to treat chest pain and angina pectoris. Based on Miao medicinal practices and traditional Chinese medicine theory, the formula is composed of Litsea cubeba volatile oil, Allium macrostemon, Ligusticum chuanxiong, and Artemisia argyi. It possesses aromatic and warming properties, regulates qi, and relieves pain. It is primarily used to treat chest pain and angina pectoris (coronary heart disease angina) caused by cold stagnation and lack of warmth, with symptoms including chest tightness, shortness of breath, palpitations, aversion to cold, and even severe chest pain radiating to the back, exacerbated by cold, and difficulty breathing. Clinically, it has shown good results in the prevention and treatment of chest pain and angina pectoris caused by cold stagnation.

[0003] In the prior art, Patent Document 1 discloses a "Quality Control Method for a Droplet Preparation for Treating Chest Pain and Heart Pain" (CN101708274A), which mainly uses thin-layer chromatography to qualitatively identify Ligusticum chuanxiong and Ligusticum striatum in the Qi-regulating and Blood-activating Droplet Preparation, and uses gas chromatography to determine the content of eucalyptol and borneol. Subsequently, Patent Document 2 disclosed a "Detection Method for a Mixed Ginseng and Heart-Nourishing Preparation" (CN103105447A), which mainly adds the determination of ferulic acid content based on Patent Document 1.

[0004] Currently, fingerprinting is an effective quality control method for traditional Chinese medicine (TCM). However, to date, no fingerprinting study has been conducted on Liqi Huoxue Dripping Pills, especially for the rapid separation and accurate identification of its chemical components using UPLC-MS. To comprehensively analyze the chemical components of Liqi Huoxue Dripping Pills and effectively control its quality, the applicant has conducted in-depth research on its fingerprinting. Summary of the Invention

[0005] This invention addresses the shortcomings of existing detection methods for qi-regulating and blood-activating preparations by providing a method for establishing fingerprint spectra of such preparations, comprising the following steps:

[0006] (1) Preparation of test solution: Take the qi-regulating and blood-activating preparation, add methanol, sonicate to dissolve, filter, and the test solution is obtained.

[0007] (2) Preparation of reference solution: Take an appropriate amount of one or more of the following reference standards: ligustrazine A, ligustrazine G, ligustrazine H, L-camphor, ligustrazine I, ferulic acid, ligustilide, linoleic acid, cinnamic acid, and butenylphthalide, add methanol to prepare a solution of 0.01-0.1 mg / ml, shake well, and set aside.

[0008] (3) Chromatographic conditions: the chromatographic column is filled with octadecylsilane-bonded silica gel, acetonitrile is used as mobile phase A, and 0.5%-2% ammonium formate or ammonium acetate aqueous solution is used as mobile phase B for gradient elution, and the elution gradient is: 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 used; the ionization mode is electrospray ionization; before sample analysis, standard tuning liquid is used for accurate mass number correction; the detection range of primary mass spectrometry scanning detection is: 100-1700 m / z; nitrogen is used as a solvent 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 Liqi Huoxue preparation fingerprint: using the national pharmacopoeia commission traditional Chinese medicine chromatographic fingerprint similarity evaluation system, the liquid chromatography of the test solution and the reference solution is respectively subjected to data import, multi-point correction and data matching, and the Liqi Huoxue preparation fingerprint is obtained.

[0011] Specifically, the Liqi Huoxue preparation fingerprint is divided into a fingerprint under a positive ion mode and a fingerprint under a negative ion mode.

[0012] Specifically, the fingerprint under the positive ion mode has 26 common chromatographic peaks, and No. 29 peak, Yangchuanmou lactone A, is used as a reference peak S.

[0013] Specifically, the fingerprint under the negative ion mode has 26 common chromatographic peaks, and No. 9 peak, Yangchuanmou lactone G, is used as a 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 1% ammonium formate aqueous solution is used as mobile phase B for gradient elution, and the elution gradient is: 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: using positive and negative ion analysis mode; ionization mode is electrospray ionization (ESI); before sample analysis, using Agilent standard tuning liquid (G1969-85000) for accurate mass number correction; primary mass spectrometry scan detection detection range: 100-1700 m / z; using nitrogen as a solvent drying gas; temperature is 325℃; flow rate is 6.8L·min -1 ; sheath gas temperature is 350℃; capillary voltage is 4.0kV; Fragment voltage is 150V; secondary fragment voltage 20V, 30V, 40V, the first three strong are selected for induced collision dissociation (CID) to obtain the secondary mass spectrometry data on the basis of primary scan.

[0017] In some embodiments, the qi-regulating and blood-activating preparation is a qi-regulating and blood-activating drop pill, and the detection method of the fingerprint spectrum thereof comprises the following steps:

[0018] (1) Preparation of test sample solution: crush the qi-regulating and blood-activating drop pill, take 2.0g of the powder, add 20ml of methanol, ultrasonic for 20min, take the supernatant and filter, to obtain the test sample solution;

[0019] (2) Preparation of reference material solution: take appropriate amount of control samples of chuangxiongol A, chuangxiongol G, ferulic acid, chuangxiongol H, levorotatory camphor and linoleic acid, and prepare a 0.02mg / ml solution with methanol, shake well and reserve;

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

[0021] (4) Mass spectrometry conditions: using positive and negative ion analysis mode; ionization mode is electrospray ionization; before sample analysis, using Agilent standard tuning liquid for accurate mass number correction; primary mass spectrometry scan detection detection range: 100-1700 m / z; using nitrogen as a solvent drying gas; temperature is 325℃; flow rate is 6.8L / min; sheath gas temperature is 350℃; capillary voltage is 4.0kV; Fragment voltage is 150V, secondary fragment voltage 20V, 30V, 40V;

[0022] (5) Determination method: 2 μl of the reference solution and the sample solution were precisely taken respectively and injected into the liquid chromatograph, determination was carried out according to the chromatographic conditions and mass spectrometric conditions, and the total ion flow chromatogram was recorded;

[0023] The positive ion mode fingerprint spectrum of the sample should present the chromatographic peak with the same retention time as the reference substance ligustilide A, and the negative ion mode fingerprint spectrum of the sample should present the chromatographic peak with the same retention time as the reference substance ligustilide G; the similarity of the sample fingerprint spectrum and the control fingerprint spectrum should not be less than 0.90 according to the traditional Chinese medicine chromatographic fingerprint similarity evaluation system.

[0024] In some embodiments, the Liqi Huoxue Dropping Pills are prepared from the following raw medicinal materials by weight: Litsea cubeba 450 parts, moxa tablet 9 parts, Chuanxiong 360 parts, and Baiyun 36 parts. The preparation method is as follows: Litsea cubeba is extracted by steam distillation to obtain essential oil; Chuanxiong is crushed into coarse powder, extracted with ethanol solution, and purified to obtain Chuanxiong extract; Baiyun is crushed into coarse powder, extracted with ethanol by reflux to obtain Baiyun extract; the Chuanxiong extract and the Baiyun extract are mixed, heated slightly, and uniformly mixed; the essential oil of Litsea cubeba and the moxa tablet are uniformly mixed; and the mixture is dropped to obtain the Liqi Huoxue Dropping Pills.

[0025] Beneficial effects:

[0026] 1. The UPLC-MS is used to establish the fingerprint spectrum of the Liqi Huoxue preparation, combined with various chemical pattern recognition methods such as cluster analysis, and the main chemical components of Liqi Huoxue are identified, which can more comprehensively evaluate the product quality of the Liqi Huoxue preparation, guarantee the clinical efficacy of the product, and fill the blank of the detection method of the Liqi Huoxue preparation fingerprint spectrum.

[0027] 2. Compared with the ordinary liquid chromatography, the technical scheme has the advantages of higher separation degree, more sensitive detection, and shorter separation time. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Fingerprint spectrum of Liqi Huoxue Dropping Pills (positive ion mode)

[0029] Figure 2 Fingerprint spectrum of Liqi Huoxue Dropping Pills (negative ion mode)

[0030] Figure 3 Total ion flow diagram of the sample under positive ion mode (A) and negative ion mode (B)

[0031] Figure 4Total ion chromatogram of the reference substance in positive ion mode (C) and negative ion mode (D), wherein peak No. 5 is ferulic acid; peak No. 9 is senkyunolide G; peak No. 19 is senkyunolide I; peak No. 21 is ligustilide; peak No. 25 is senkyunolide H; peak No. 29 is butylidenephthalide; peak No. 43 is cinnamic acid; peak No. 45 is levorotatory camphor; and peak No. 50 is linoleic acid

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

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

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

[0035] Figure 8 PLS-DA VIP value diagram of Liqi Huoxue Dropping Pills in positive ion mode (A) and negative ion mode (B) DETAILED DESCRIPTION

[0036] In order to make the technical solutions and technical effects of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application are described clearly and completely. The embodiments described below are part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by those skilled in the art on the basis of the embodiments in the present application without creative labor belong to the scope of protection of the present application.

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

[0038] The UPLC-MS is used for detecting the fingerprint spectrum of the Liqi Huoxue Dropping Pills, wherein the test conditions of the high performance liquid chromatography are as follows: the chromatographic column is Agilent ZORBAX Eclipse Plus C18 (3.0mmx100 mm.1.8 μm), the mobile phase A is acetonitrile, the mobile phase B is 1% ammonium formate aqueous solution, and gradient elution is carried out; 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 mass spectrometry conditions are as follows: the positive and negative ion analysis modes are used; the ionization mode is electrospray ionization (ESI); the Agilent standard tuning liquid (G1969-85000) is used for accurate mass number correction before sample injection analysis; the first mass spectrum scanning detection range is 100-1700 m / z; nitrogen is used as the desolvent drying gas; the temperature is 325 DEG C; the flow rate is 6.8 L·min -1 ; the sheath gas temperature is 350 DEG C; the capillary voltage is 4.0 kV; the Fragment voltage is 150 V; the secondary fragment voltage is 20 V, 30 V and 40 V.

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

[0040] In some embodiments of the present application, the injection amount is 2 μL

[0041] The technical solutions provided by the present application are described in detail below in combination with specific examples.

[0042] In the examples, each raw reagent material can be commercially available, and the experimental method without specific conditions is a conventional method and conventional condition known in the field, or is according to the conditions recommended by the instrument manufacturer.

[0043] The instruments and reagents used in the following examples and comparative examples are as follows:

[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 (3.0mmx100mm, 1.8 μm) chromatographic column (Agilent Technology Company, USA); ultrasonic instrument (Shanghai Keduo Ultrasonic Instrument Co., Ltd.), S-23 high-speed bench centrifuge (Hunan Xiangyi Instrument Co., Ltd.); AUW120D one ten-thousandth electronic analytical balance (Japan SHIMADZU Company).

[0045] Drugs and reagents: chromatographic grade methanol, mass spectrometry grade acetonitrile (Merck Millipore, Germany); distilled water (Shenzhen Watson Distilled Water Co., Ltd.); mass spectrometry grade ammonium formate (Fisher, USA). Reference substances: senkyunolide A (batch number: 21080506), senkyunolide G (batch number 21072411), senkyunolide H (batch number: 21012602), levorotatory camphor (20201012), senkyunolide I (batch number: 21012506), cinnamic acid (batch number: 21061603) were purchased from Chengdu Pu Feide Biotechnology Co., Ltd., ferulic acid (batch number: 919C021), ligustrazine (batch number: 903C021) were purchased from Beijing Solabio Technology Co., Ltd., linoleic acid (batch number: HR2178W10) was purchased from Baoji Chen Guang, butenyl phthalide (Shanghai Shidand Biotechnology Co., Ltd., batch number: 18443).

[0046] Preparation method of Liqi Huoxue Dropping Pills

[0047] Take 450g of Chinese medicine Plagiostachys collniana, 9g of moxa, 360g of Szechuan lovage rhizome, and 36g of Chinese chive, and make them according to the following method: extract essential oil from Plagiostachys collniana by steam distillation, and grind Szechuan lovage rhizome into coarse powder, then extract twice by refluxing with 10 times the amount of 95% ethanol, 6 times the amount of ethanol for 2 hours for the first time, and refluxing for 3 hours, 4 times the amount of ethanol for 2 hours for the second time, combine the two extraction solutions, recover ethanol under reduced pressure to a relative density of about 1.1, take the middle layer liquid, and concentrate under reduced pressure to a relative density of about 1.2 (55-60°C), adjust the pH value to 9 with ammonia water, then extract three times with 6 times the amount of chloroform, combine the extraction solutions, recover chloroform under reduced pressure, and concentrate to a relative density of 1.01-1.05 (55-60°C) to obtain Szechuan lovage rhizome extract, grind Chinese chive into coarse powder, immerse in 6 times the amount of 95% ethanol, and extract twice by refluxing for 1 hour each time, concentrate under reduced pressure to a relative density of 1.12-1.16 (55-60°C) to obtain Chinese chive extract, mix Szechuan lovage rhizome extract and Chinese chive extract, heat slightly to mix evenly, add melted polyethylene glycol, mix well, add essential oil of Plagiostachys collniana and moxa, mix evenly, and drop to form dropping pills.

[0048] Fingerprint detection method of Liqi Huoxue Dropping Pills

[0049] 2.1. Preparation of test solution: grind Liqi Huoxue Dropping Pills, take 2.0g of powder, add 20ml of methanol, and dissolve by ultrasonic for 20min, then take the supernatant after standing, centrifuge at 3000r·min -1 for 5min. After centrifugation, filter the supernatant with a 0.22μm microporous filter to obtain the test solution.

[0050] 2.2. Preparation of reference solution: Take 4.0 mg of each of ligustilide A, ligustilide H, ligustilide G, ferulic acid, levorotatory camphor, linoleic acid, and cinnamic acid reference substances, accurately weigh, and transfer into 25 ml volumetric flasks, respectively. Add an appropriate amount of methanol to ultrasonically dissolve, stand for 10 min, and then dilute to volume. Filter with a microporous filter membrane to obtain the stock solution of the reference substances. Mix 100 μl of each of the stock solutions of the reference substances to obtain the reference solution.

[0051] 2.3. Chromatographic conditions: The chromatographic column 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, and the elution gradient is: 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 spectrometric conditions: Positive and negative ion analysis modes are used; the ionization mode is electrospray ionization (ESI); before sample analysis, Agilent standard tuning solution (G1969-85000) is used for accurate mass number correction; the detection range of primary mass spectrometric scanning detection is: 100-1700 m / z; nitrogen is used as the solvent 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 secondary fragment voltage is 20 V, 30 V, and 40 V.

[0053] 2.5. Determination method: 2 μl of the reference solution and the test sample solution, respectively, are accurately pipetted and injected into the liquid chromatograph, and determination is performed according to the high performance liquid chromatography conditions and the mass spectrometry conditions, and the chromatogram and mass spectrum are recorded.

[0054] The test sample fingerprint should exhibit chromatographic peaks with the same retention time as the reference chromatographic peaks. According to the Chinese medicine chromatographic fingerprint similarity evaluation system, the similarity of the test sample fingerprint to the control fingerprint should not be lower than 0.90.

[0055] Example 3. Establishment of the Liqi Huoxue Dropping Pill Fingerprint

[0056] 3.1. Preparation of the test sample solution:

[0057] The Liqi Huoxue Dropping Pill is crushed, 2.0 g of the powder is weighed, 20 ml of methanol is added, ultrasonic dissolution is performed for 20 min, and after standing, the supernatant is taken, and the solution is centrifuged at 3000 r·min -1Centrifuge for 5 min. After centrifugation, the supernatant was filtered with a 0.22 μm microporous filter to obtain the test solution.

[0058] 3.2. Preparation of the control solution:

[0059] Take 4.0 mg of each of the ten control samples of ligustilide A, ligustilide H, ligustilide G, ligustilide I, ferulic acid, levorotatory camphor, ligustilide, linoleic acid, cinnamic acid, and butenyl phthalide, precisely weigh, and transfer to a 25 ml volumetric flask. Add an appropriate amount of methanol to ultrasonically dissolve, stand for 10 min, and then dilute to the mark. Filter with a microporous filter to obtain the stock solution of the ten control samples. Take 100 μl of each of the control stock solutions, mix, and obtain the control solution.

[0060] 3.3. Chromatographic conditions:

[0061] The chromatographic column is Agilent ZORBAX Eclipse Plus C18 (3.0 mm x 100 mm.1.8 μm), with acetonitrile as mobile phase A and 1% ammonium formate aqueous solution as mobile phase B, gradient elution, elution gradient: 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] The positive and negative ion analysis modes are used; the ionization mode is electrospray ionization (ESI); before sample analysis, the Agilent standard tuning liquid (G1969-85000) is used for accurate mass number correction; the detection range of the primary mass spectrum scanning detection is 100-1700 m / z; nitrogen is used as the solvent drying gas; the temperature is 325℃; the flow rate is 6.8 L·min -1 ; the sheath gas temperature is 350℃; the capillary voltage is 4.0 kV; the Fragment voltage is 150 V; the secondary fragment voltage is 20 V, 30 V, and 40 V.

[0064] 3.5. Establishment of the fingerprint and similarity evaluation

[0065] Take 15 batches of Liqi Huoxue Dropping Pills respectively, prepare test sample solution according to the method under item "3.1", and determine under the chromatographic conditions in items "3.3 and 3.4", record the chromatogram, and import the data into "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version)", set the time window as 0.1, take the chromatogram of sample S1 as the reference chromatogram for the positive ion flow chart and the negative ion flow chart, perform chromatographic peak matching by the average method, and the fingerprint chromatograms of the 15 batches of samples are shown in Figure 1 , 2 , and the similarity is shown in Table 1. By comparison with the total ion flow chart of the control (see Figure 3 , Figure 4 ), 10 components including senkyunolide A, senkyunolide H, levorotatory camphor, senkyunolide G, senkyunolide I, ferulic acid, ligustilide, linoleic acid, cinnamic acid and butenyl phthalide are identified.

[0066] From Figure 1 , Figure 2 , the fingerprint chromatograms of Liqi Huoxue Dropping Pills in positive and negative ion modes can be seen that the chemical components of the 15 batches of Liqi Huoxue Dropping Pills are relatively stable in general. As can be seen from Table 1, the similarity of the 15 batches of Liqi Huoxue Dropping Pills is all ≥0.978, indicating that the quality of each batch of the 15 batches of Liqi Huoxue Dropping Pills is relatively stable.

[0067] Table 1 Similarity of the peaks of 15 batches of samples to the control peaks

[0068]

[0069] Example 4 Methodological verification of fingerprint chromatogram

[0070] 4.1 Precision test Take Liqi Huoxue Dropping Pills, prepare test sample solution according to the method under item "3.1", and continuously inject 6 times under the chromatographic mass spectrometric conditions in items "3.3 and 3.4", and take senkyunolide A (peak 29) and senkyunolide G (peak 9) as the reference peaks (S), and 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 RSD of the relative retention time and relative peak area of each common peak is less than 3.0%, indicating that the precision of the instrument is good.

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

[0072] 4.3 Repeatability test Take 6 portions of the same batch of Liqi Huoxue Dropping Pills, prepare test solution according to the method of item 3.1, and inject under the chromatographic conditions of items 3.3 and 3.4, take Yangchuanxiong lactone A (peak 29) and Yangchuanxiong lactone G (peak 9) as reference peaks (S), and calculate the relative retention time and relative peak area of each chromatographic peak (as above). The results show that the RSD of each common peak is less than 3.0%, indicating that the method has good repeatability.

[0073] Example 5 Establishment of chemical pattern recognition analysis method of Liqi Huoxue Dropping Pills

[0074] 5.1 Cluster analysis

[0075] Introduce the peak area of common peaks in the fingerprint chromatograms of Liqi Huoxue Dropping Pills under positive and negative ion modes into SPSS software, and perform cluster analysis by intergroup connection 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, 15 batches of samples under positive ion mode can be divided into 6 categories. The first category is S13, S14, S15, S10, S11 and S11 samples, the second category is S7, S8, S9 and S6 samples, the third category is S1 sample, the fourth category is S1 sample, the fifth category is S3 and S4 samples, and the sixth category is S5 sample. Under negative ion mode, 15 batches of samples can be divided into 6 categories. The first category is S7, S8, S9, S10, S11, S12 and S14 samples, the second category is S13 and S15 samples, the third category is S5 and S6 samples, the fourth category is S3 and S4 samples, the fifth category is S2 sample, and the sixth category is S1 sample.

[0077] It indicates that the overall quality of 15 batches of Liqi Huoxue Dropping Pills is basically stable, and the common components of each batch of preparation may differ due to different sources of raw materials, preparation processes, etc.

[0078] 5.2 Principal component analysis (PCA)

[0079] Take the peak area of common peaks in the fingerprint chromatograms of Liqi Huoxue Dropping Pills under positive and negative ion modes as variables, introduce them into SPSS software, and perform PCA after standardizing the data. Take the characteristic value > 1 as the standard, extract 5 principal components under positive ion mode, and the cumulative variance contribution rate is 89.203%. Extract 2 principal components under negative ion mode, and the cumulative variance contribution rate is 89.383%. It can objectively reflect most of the information of Liqi Huoxue Dropping Pills, see Tables 2 and 3. Draw the PCA score chart using SIMCA-P software, see Figure 6 .

[0080] As can be seen from Figure 6It was found that 15 batches of samples could be classified into 6 groups in positive and negative ion modes, which was consistent with the results of cluster analysis.

[0081] Table 2 PCA characteristic values and variance contribution rates of Liqihuoxue Pill samples (positive ion mode)

[0082]

[0083] Table 3 PCA characteristic values and variance contribution rates of Liqihuoxue Pill samples (negative ion mode)

[0084]

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

[0086] The peak areas of common peaks in the fingerprint spectra of Liqihuoxue Pill in positive and negative ion modes were introduced into SIMCA software for PLS-DA, and the results are shown in Figure 7 .

[0087] It was found that 15 batches of Liqihuoxue Pill could be classified into 6 groups, which was consistent with the results of cluster analysis and PCA. Figure 7 According to the variable importance projection (VIP) method, the standard of VIP>1 was used, and 11 differential markers were obtained in positive ion mode, in the order of chromatographic peaks 40, 21, 32, 13, 30, 39, 20, 1, and 25, indicating that these 9 components were the main components causing the classification difference of Liqihuoxue Pill in positive ion mode; 5 differential markers were obtained in negative ion mode, in the order of chromatographic peaks 49, 33, 36, 31, and 47, indicating that these 5 components were the main components causing the classification difference of Liqihuoxue Pill in negative ion mode (see Figure 8 ). Thus, there were 14 main components affecting the large classification difference of Liqihuoxue Pill, and their changes could be focused on in the quality control of the production process.

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

[0089] The molecular formula was generated by using Agilent TCM-DATA Traditional Chinese Medicine Component Database, and the screening was performed according to the principle of error less than 5×10-6, and the databases of China National Knowledge Infrastructure (CNKI), PubMed, and Massbank were searched to establish the chemical component information database of Liqihuoxue Pill. According to the first-order mass spectrum, the second-order mass spectrum, the comparison with reference substances, and the comparison with the self-built traditional Chinese medicine component database, and combined with the literature, 50 chemical components were determined in Liqihuoxue Pill, and the results are shown in Table 4.

[0090] Table 4 Identification and analysis of UPLC-Q-TOF / MS components of common peaks in Liqihuoxue Pill

[0091]

[0092]

[0093] *: confirmed by comparison with a reference substance;

[0094] The above examples are only preferred embodiments of the present application, and it should be noted that for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. A method for establishing a fingerprint of a qi-regulating and blood-activating preparation, the qi-regulating and blood-activating preparation being made of Litsea chenii, moxa, Chuanxiong Rhizoma and Baiyun White, characterized in that, Comprising the following steps: (1) Preparation of test solution: Take Ligui Huoxue preparation, add methanol, ultrasonic dissolution, filter, and obtain the test solution; (2) Preparation of reference solution: Take appropriate amount of Yangchuanmionolide A, Yangchuanmionolide G, Yangchuanmionolide H, levorotatory camphor, Yangchuanmionolide I, ferulic acid, ligusticum lactone, linoleic acid, cinnamic acid, butenyl phthalide reference substance, and prepare 0.01-0.1 mg / ml solution with methanol, shake well, and reserve; (3) Chromatographic conditions: The chromatographic column is filled with octadecylsilane bonded silica gel, the mobile phase A is acetonitrile, and the mobile phase B is 0.5%-2% ammonium formate or ammonium acetate aqueous solution, gradient elution is carried out, the elution gradient is: 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; and the flow rate is 0.4-1.0 mL / min; (4) Mass spectrometry conditions: The positive and negative ion analysis modes are adopted; the ionization mode is electrospray ionization; the standard tuning liquid is used for accurate mass number correction before sample analysis; the primary mass spectrum scanning detection range is: 100-1700 m / z; nitrogen is used as the solvent drying gas; the temperature is 300-350 DEG C; the flow rate is 5-7 L / min; the sheath gas temperature is 320-380 DEG C; the capillary voltage is 3.0-4.0 kV; and the Fragment voltage is 150-200 V; (5) Establishment of Ligui Huoxue preparation fingerprint: The liquid chromatography of the test solution and the reference solution is subjected to data import, multi-point correction and data matching respectively by using the national pharmacopoeia committee traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and the Ligui Huoxue preparation fingerprint is obtained.

2. The method for establishing the fingerprint of Liqi Huoxue preparation according to claim 1, characterized in that, The chromatographic column is Agilent ZORBAX Eclipse Plus C18, the specification is 3.0 mm*100 mm, and the particle size is 1.8 μm.

3. The method for establishing the fingerprint spectrum of Liqi Huoxue 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 Liqi Huoxue preparation according to claim 1, characterized in that, The flow rate is 0.4 mL / min.

5. The method for establishing the fingerprint of Liqi Huoxue preparation according to claim 1, characterized in that, The mass spectrometry conditions are that the primary mass spectrum scanning temperature is 325 DEG C, the flow rate is 6.8 L / min; the sheath gas temperature is 350 DEG C; the capillary voltage is 4.0 kV; the Fragment voltage is 150 V; and the secondary fragment voltage is 20 V, 30 V and 40 V. 6.The method for establishing the fingerprint of Liqi Huoxue preparation according to claim 1, characterized in that, The Ligui Huoxue preparation fingerprint is divided into the fingerprints under the positive ion mode and the negative ion mode.

7. The method for establishing the fingerprint spectrum of Liqi Huoxue preparation according to claim 6, characterized in that, The fingerprints under the positive ion mode and the negative ion mode have 50 common chromatographic peaks, and the peak No. 29 Yangchuanmionolide A is used as the reference peak S under the positive ion mode; and the peak No. 9 Yangchuanmionolide G is used as the reference peak S under the negative ion mode.

8. A detection method of a Liqi Huoxue preparation fingerprint, wherein the Liqi Huoxue preparation is Liqi Huoxue Dropping Pills, and the method is characterized by, Comprising the following steps: (1) Preparation of test solution: Take Ligui Huoxue preparation, add methanol, ultrasonic dissolution, filter, and obtain the test solution; (2) Preparation of reference solution: Take appropriate amount of ligustilide A, ligustilide G, ferulic acid, ligustilide H, levorotatory camphor, linoleic acid, and cinnamic acid reference substances, and add methanol to prepare a solution containing 0.02 mg / ml of each, shake well, and reserve; (3) Chromatographic conditions: Agilent ZORBAX Eclipse Plus C18 column, acetonitrile as mobile phase A, 1% ammonium formate aqueous solution as mobile phase B, gradient elution, elution gradient: 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 mode; ionization mode: electrospray ionization; before sample analysis, Agilent standard tuning liquid was used for accurate mass number correction; primary mass spectrometry scanning detection range: 100-1700 m / z; nitrogen was used as a solvent drying gas; temperature: 325 ℃; flow rate: 6.8 L / min; sheath gas temperature: 350 ℃; capillary voltage: 4.0 kV; Fragment voltage: 150 V; secondary fragment voltage: 20V, 30V, 40V; (5) Determination method: precisely take 2 μl of the reference solution and the test sample solution respectively, inject into the liquid chromatograph, and determine according to the chromatographic conditions and mass spectrometry conditions, and record the total ion chromatogram; The positive ion mode fingerprint spectrum of the test sample fingerprint spectrum should present a chromatographic peak with the same retention time as the reference ligustilide A, and the negative ion mode fingerprint spectrum of the test sample fingerprint spectrum should present a chromatographic peak with the same retention time as the reference ligustilide G; according to the Chinese medicine chromatographic fingerprint similarity evaluation system, the similarity of the test sample fingerprint spectrum and the control fingerprint spectrum should not be less than 0.

90.

9. The method according to any one of claims 1 to 8, characterized in that, The qi-regulating and blood-activating preparation is a qi-regulating and blood-activating drop pill, which is prepared from the following raw medicinal materials by weight: Litsea cubeba 450 parts, moxa tablet 9 parts, Szechuan lovage 360 parts, and Chinese onion 36 parts. The preparation method is as follows: Litsea cubeba is extracted by steam distillation to obtain essential oil; Szechuan lovage is crushed into coarse powder, extracted with ethanol solution, and purified to prepare Szechuan lovage extract; Chinese onion is crushed into coarse powder, extracted with ethanol by reflux to prepare Chinese onion extract, the Szechuan lovage extract and the Chinese onion extract are mixed and uniformly heated, the essential oil of Litsea cubeba and the moxa tablet are added and uniformly mixed, and drop pills are prepared.

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