Construction method, detection method and application of fingerprint spectrum of heart-nourishing Damwamixib preparation

Abstract

The invention discloses a construction method, a detection method and application of a fingerprint spectrum of a heart-nourishing Damwamixib preparation. The construction method comprises the following steps: detecting a test solution by using high performance liquid chromatography to generate a fingerprint spectrum; a mobile phase A is a 0.05-0.15% formic acid solution, and a mobile phase B is acetonitrile. The detection method comprises the following steps: carrying out ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry detection on a test solution; a mobile phase A is a 0.05-0.15% formic acid solution, and a mobile phase B is acetonitrile. According to the invention, the fingerprint characteristic spectrum of the heart-nourishing Damwaemixib preparation is constructed, 30 common peaks are identified, the authenticity of the sample is effectively identified, the consistency of each batch is judged, and the quality stability of the product is effectively controlled. By applying ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry, 22 components can be effectively separated and quantitatively detected at the same time, and a scientific basis is provided for establishing a quality control system.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for constructing a fingerprint of a Yangxin Dawaei Miceke preparation, a compound detection method and application. BACKGROUND

[0002] Yangxin Dawaei Miceke is derived from a classic compound preparation of Uyghur medicine, composed of 26 kinds of medicine such as Potentilla kleiniana flower, musk, sandalwood, eaglewood, safflower, rose, cassia, and box fruit tree, etc. It was first recorded in the Uyghur medicine classic ancient book "Medicine Essence", and has the effect of regulating the function of viscera. It can not only invigorate the spleen and stomach, and refresh the mind, but also can be used for treating chest tightness and pain, palpitation, weak spleen and stomach, decreased visual acuity, and fatigue.

[0003] However, with the deepening of research, the original standard obviously cannot meet the needs of modern medicine. How to construct an innovative methodology system for the quality standard improvement of traditional medicine, and realize the effective control of the quality of Yangxin Dawaei Miceke preparation, and ensure its safe and effective clinical efficacy, is a problem that must be considered by the technical personnel in the field.

[0004] Traditional Chinese medicine fingerprint refers to the chromatogram obtained by treating a certain traditional Chinese medicine, using a certain analysis method, which can indicate the characteristics of the traditional Chinese medicine. Under the premise that the effective components of the preparation are not completely clear, it is urgent to develop the chromatographic fingerprint of traditional Chinese medicine and establish a high-quality, stable and reliable quality control method. However, there is no research report on the fingerprint of Yangxin Dawaei Miceke preparation in the current literature, and the existing literature only determines the content of 1-2 components in the preparation. Therefore, in order to better monitor the content of each component in Yangxin Dawaei Miceke preparation and systematically clarify its pharmacodynamic material basis, it is urgent to establish a more comprehensive, simple and convenient quality control method for Yangxin Dawaei Miceke preparation. SUMMARY

[0005] The purpose of this invention is to provide a method for constructing a fingerprint spectrum of Yangxin Dawaimishik preparation, a compound detection method, and its application, so as to improve the problems of imperfect quality standards and inaccurate quality control of existing Yangxin Dawaimishik honey ointment.

[0006] Generally, the number of medicinal ingredients in existing traditional Chinese medicine preparations with clearly defined characteristic components is relatively simple, making fingerprint spectroscopy research and quantitative analysis relatively easy to control. However, Yangxin Dawa Yimishike honey ointment or preparations consist of 26 medicinal ingredients, including ox tongue flower, musk, sandalwood, agarwood, saffron, rose, cinnamon, and boxberry vine. The prescription is large, and even a single medicinal material contains hundreds or thousands of components. With 26 medicinal materials, the chemical composition is even more numerous and complex. The chemical components contained in traditional Chinese medicine are complex and difficult to control. Many components have low content and lack legal reference substances. Separation requires high sensitivity, making it even more difficult to identify the specific compounds. Moreover, the physicochemical properties (polarity, hydrophobicity, etc.) of the 22 components vary greatly. It is difficult to find a single chromatographic condition (column, mobile phase, gradient program) that allows all 22 peaks to reach baseline separation. In addition, co-eluting compounds can interfere with each other, causing ion suppression or enhancement effects at the ion source, significantly affecting the accuracy and precision of quantification, making it difficult to determine which compound the fragment ion signal specifically originates from. Therefore, the quality control research of Yangxin Dawayimisik honey ointment or preparations is quite difficult.

[0007] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0008] This invention provides a method for constructing a fingerprint spectrum of a Dawaimisik preparation, which includes the following steps: detecting the test solution using high performance liquid chromatography and generating a fingerprint spectrum from the detection results;

[0009] The test solution is a solution containing Yangxin Dawaimisik preparation;

[0010] In the high-performance liquid chromatography, the detection wavelength is 254 nm; the mobile phase A is a 0.05%-0.15% (v / v) formic acid solution, and the mobile phase B is acetonitrile; the gradient elution conditions for the mobile phase A and the mobile phase B are as follows:

[0011]

[0012] The percentages in the table represent the volume percentage of each component in the total volume of mobile phase A and mobile phase B, respectively.

[0013] In this invention, the solvent used in the test solution is preferably an alcohol or an aqueous alcohol solution.

[0014] The alcohol in the alcohol or the aqueous alcohol solution may be methanol or ethanol. The volume percentage of the aqueous alcohol solution is preferably 30-70%, more preferably 40-60%, for example 45%, 50% or 55%.

[0015] In this invention, the extract concentration (raw drug content) in the test solution is preferably 8-12 mg / mL, more preferably 10 mg / mL.

[0016] In this invention, the preparation method of the test solution preferably includes the following steps: ultrasonically extracting the mixture of the Yangxin Dawaimisik preparation and the solvent, allowing it to stand, taking the supernatant, centrifuging, diluting, and filtering to obtain the test solution.

[0017] The preferred mass-to-volume ratio of the heart-nourishing davaimisik preparation to the solvent is (8-12) g: 100 mL, for example, 10 g: 100 mL.

[0018] The power of the ultrasonic extraction can be 250 W.

[0019] The frequency of the ultrasonic extraction can be 40 kHz.

[0020] The ultrasonic extraction time can be 0.5-1.5 hours, for example, 1 hour.

[0021] The settling time can be 20-30 hours, for example, 24 hours.

[0022] In a preferred embodiment, the test solution is prepared by the following steps: a mixture of 10 g of Yangxin Dawa Yimisik preparation and 100 mL of 50% methanol aqueous solution is extracted by ultrasonic extraction for 1 hour (250 W, 40 kHz), allowed to stand for 24 hours, 1 mL of the supernatant is placed in a 10 mL volumetric flask, and the volume is adjusted to the mark with 50% methanol aqueous solution to make the crude drug concentration 10 mg / mL, and filtered through a 0.22 μm microporous membrane.

[0023] In this invention, the chromatographic column used in the high-performance liquid chromatography (HPLC) detection is preferably an ACQUITY UPLC BEH Shield RP18. The column specifications are preferably 1.7 μm and 2.1 × 100 mm.

[0024] In this invention, the volume percentage of the mobile phase A in the high-performance liquid chromatography detection is preferably 0.08-0.12%, for example, 0.1%.

[0025] In this invention, the injection volume in the high-performance liquid chromatography detection can be conventional in the art, preferably 1~4μL, for example 2μL.

[0026] In this invention, the flow rate of the test solution in the high-performance liquid chromatography detection is preferably 0.1~0.5 mL / min, more preferably 0.2 mL / min.

[0027] In this invention, preferably, the chromatogram is recorded within 73 minutes during the high-performance liquid chromatography detection.

[0028] In a preferred embodiment of the present invention, the method for constructing the fingerprint spectrum of the Yangxin Dawaimisik preparation includes the following steps:

[0029] a. Prepare several solutions of the test sample described above;

[0030] b. The test solution was analyzed by high-performance liquid chromatography as described above;

[0031] Based on the similarity evaluation system of chromatographic fingerprint of traditional Chinese medicine, a test solution is selected as a reference, a characteristic chromatogram is constructed, and a corresponding standard fingerprint chromatogram is generated.

[0032] The fingerprint spectrum contains 30 common peaks, which constitute the fingerprint characteristics of the Yangxin Dawaimisik preparation. This fingerprint spectrum serves as the standard fingerprint spectrum for the Yangxin Dawaimisik preparation. The fingerprint spectrum of the Yangxin Dawaimisik preparation is a single high-performance liquid chromatogram, as shown below. Figure 2 As shown. According to Figure 2 Based on the retention time of the common peaks, 30 common peaks can be effectively detected within 73 minutes.

[0033] In a preferred embodiment, the fingerprint spectrum includes 30 common characteristic peaks. Taking the test solution S1 sample as a reference, the retention time, relative retention time, peak area, and relative peak area of ​​the common characteristic peaks are as follows:

[0034] .

[0035] This invention also provides a method for detecting compounds in Yangxin Dawaimisik preparations, which includes the following steps: performing ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-QQQ-LIT-MS / MS) on the test sample solution to detect the analyte;

[0036] The test solution is a solution containing Yangxin Dawaimisik preparation; the analyte includes one or more of the following: cryptochlorogenic acid, epicatechin, p-coumaric acid, catechin, quercetin, caffeic acid, rutin, rosmarinic acid, gallic acid, luteolin, ellagic acid, kaempferol, kaempferol-3-O-rutin, narcissin, isoquercitrin, silagetin, chlorogenic acid, neochlorogenic acid, quinic acid, astragaloside, and hyperoside.

[0037] In the high-performance liquid chromatography, mobile phase A is a 0.05%-0.15% (v / v) formic acid solution, and mobile phase B is acetonitrile; the gradient elution conditions for mobile phase A and mobile phase B are as follows:

[0038] .

[0039] In this invention, the ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry detection column is preferably a Waters ACQUITY UPLC HSS T3. The column specifications are preferably 2.1 mm × 100 mm, 1.8 μm.

[0040] In this invention, in the ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry detection, the volume percentage of the mobile phase A is preferably 0.08-0.12%, for example 0.1%.

[0041] In this invention, in the ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry detection, the injection volume in ultra-high performance liquid chromatography detection can be conventional in the art, preferably 1~4μL, for example 2μL.

[0042] In this invention, in the ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry detection, the column temperature in the ultra-high performance liquid chromatography detection is preferably 30~40℃, more preferably 35℃.

[0043] In this invention, during high-performance liquid chromatography detection, the flow rate of the test solution is preferably 0.15~0.35 mL / min, more preferably 0.25 mL / min.

[0044] In this invention, the preferred mass spectrometry detection conditions in the ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry detection are:

[0045] The ion source was an electrospray ionization (ESI) source; the scanning modes were: multiple reaction detection (MRM) mode and rapid switching mode between positive and negative ions; the ion source temperature was 450℃; the ionization voltages were 5500 V and -4500 V; the curtain gas, ion source spray gas GasⅠ, and auxiliary heating gas GasⅡ were all N2, with pressures of 35, 55, and 55 psi, respectively; the collision gas was set to medium.

[0046] In this invention, during the ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry detection, the m / z, declustering voltage (DP), and collision energy (CE) of the analyte are detailed in the table below:

[0047] .

[0048] In this invention, the solvent used in the test solution is preferably an alcohol or an aqueous alcohol solution.

[0049] The alcohol in the alcohol or the aqueous alcohol solution may be methanol or ethanol. The volume percentage of the aqueous alcohol solution is preferably 40-60%, for example, 45%, 50% or 55%.

[0050] In this invention, the extract concentration (raw drug content) in the test solution is preferably 8-12 mg / mL, more preferably 10 mg / mL.

[0051] In this invention, the preparation method of the test solution preferably includes the following steps: ultrasonically extracting the mixture of the Yangxin Dawaimisik preparation and the solvent, allowing it to stand, taking the supernatant, centrifuging, diluting, and filtering to obtain the test solution.

[0052] The preferred mass-to-volume ratio of the heart-nourishing davaimisik preparation to the solvent is (8-12) g: 50 mL, for example, 10 g: 50 mL.

[0053] The power of the ultrasonic extraction can be 250 W.

[0054] The frequency of the ultrasonic extraction can be 40 kHz.

[0055] The ultrasonic extraction time can be 0.5-1.5 hours, for example, 1 hour.

[0056] The settling time can be 20-30 hours, for example, 24 hours.

[0057] In a preferred embodiment, the test solution is prepared by the following steps: a mixture of 10 g of Yangxin Dawaimisik preparation and 50 mL of 50% ethanol aqueous solution is ultrasonically treated (power 250 W, frequency 40 kHz) for 60 min, allowed to stand for 24 h, 1 mL of the supernatant is placed in a 10 mL volumetric flask, and 50% ethanol aqueous solution is added to make up to the mark to obtain an extract concentration (crude drug amount) of 10.0 mg / mL, which is then filtered through a 0.22 μm microporous membrane.

[0058] In this invention, the preferred linear regression equation and linear range for the analyte are as follows: where X is the concentration in µg / mL and Y is the peak area:

[0059] Quinic acid: y = 3115.4889 x - 170237.3212, linear range 0.05-5 μg / mL, R 2 It is 0.9990;

[0060] Gallic acid: y = 75,350.1372x + 20,453,831.7218, linear range 0.12-6 μg / mL, R 2 It is 0.9946;

[0061] Neochlorogenic acid: y = 73294.2964x + 404815.7394, linear range 0.001-1 μg / mL, R 2 It is 0.9994;

[0062] Catechins: y = 25086.8449 x - 711.4828, linear range 0.00008-0.04 μg / mL, R 2 It is 0.9994;

[0063] Chlorogenic acid: y = 51027.1256x + 2290245.1194, linear range 0.04-4 μg / mL, R 2 It is 0.9975;

[0064] Caffeic acid: y = 178,124.0604x + 2,193,843.4736, linear range 0.004-0.8 μg / mL, R 2 It is 0.9984;

[0065] Cryptochlorogenic acid: y = 75,264.8823x + 233,959.4559, linear range 0.0008-0.8 μg / mL, R 2 It is 0.9993;

[0066] Epicatechin: y = 12938.6798 x + 14090.8505, linear range 0.0005-0.1 μg / mL, R 2 It is 0.9979;

[0067] p-Coumaric acid: y = 100,026.4170x + 331,961.6823, linear range 0.0012-0.6 μg / mL, R 2 It is 0.9997;

[0068] Ellagic acid: y = 9,704.1020x + 246,568.4084, linear range 0.05-5 μg / mL, R 2 It is 0.9987;

[0069] Hyperoside: y = 18170.5665x + 20795.4240, linear range 0.0008-0.16 μg / mL, R 2It is 0.9992;

[0070] Rutin: y = 13,124.4407x + 101,448.8140, linear range 0.002-2 μg / mL, R 2 It is 0.9993;

[0071] Isoquercetin: y = 103386.1338x + 438435.7990, linear range 0.0015-0.3 μg / mL, R 2 It is 0.9981;

[0072] Kaempferol-3-O-rutin: y = 20,789.6469 x - 6,129.6574, linear range 0.0002-0.2 μg / mL, R 2 It is 0.9998;

[0073] Astragaloside: y = 3588.7468x + 14091.4662, linear range 0.001-0.4 μg / mL, R 2 It is 0.9974;

[0074] Quercetin: y = 89997.5557x + 59638.4806, linear range 0.0002-0.15 μg / mL, R 2 It is 0.9991;

[0075] Narcisin: y = 15214.9910x + 43710.9840, linear range 0.0008-0.16 μg / mL, R 2 It is 0.9975;

[0076] Rosmarinic acid: y = 110892.2160x + 1060434.6524, linear range 0.002-1.5 μg / mL, R 2 It is 0.9981;

[0077] Quercetin: y = 136719.1259 x + 204606.8507, linear range 0.0005-0.1 μg / mL, R 2 It is 0.9975;

[0078] Luteolin: y = 239605.5882 x + 223692.0938, linear range 0.0006-0.06 μg / mL, R 2 It is 0.9994;

[0079] Silver calcinate: y = 40377.9259x + 60639.6685, linear range 0.001-0.1 μg / mL, R 2 It is 0.9990;

[0080] Kaempferol: y = 19033.5098x + 18002.9745, linear range 0.00008-0.08 μg / mL, R 2 It is 0.9976.

[0081] In some preferred embodiments, the ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry detection includes the following steps:

[0082] a. Prepare a series of reference solutions of different concentrations for the analyte;

[0083] The above detection method was used to analyze and detect the reference solution and the test solution respectively; the peak area of ​​the analyte in the reference solution and the peak area of ​​the test solution were measured.

[0084] b. Based on the peak area and corresponding concentration of the analyte in the reference solution, perform linear regression to obtain the standard curve equation;

[0085] c. For the same analyte, the peak area measured by the test solution is substituted into its corresponding standard curve equation to obtain the concentration of the analyte.

[0086] In step a, the reference solution can be prepared using a conventional solvent that can dissolve it, such as methanol.

[0087] In step a, the concentration of the reference solution can be referenced from the concentration used when preparing a standard curve equation of concentration versus peak area in the art.

[0088] In step b, the standard curve equation can be obtained by referring to conventional calculation methods in the art. For example, the standard curve equation can be obtained by performing linear regression with the concentration of the analyte in the reference solution as the abscissa and the logarithm of the peak area corresponding to the concentration of the analyte as the ordinate.

[0089] In this invention, the UPLC-QQQ-LIT-MS / MS is a combined analytical technique that integrates ultra-high performance liquid chromatography (UPLC), triple quadrupole mass spectrometry (QQQ), and linear ion trap mass spectrometry (LIT).

[0090] The present invention also provides the application of the fingerprint spectrum obtained by the method for constructing the fingerprint spectrum of the Yangxin Dawaimishik preparation and / or the detection method of the compound in the quality control of the Yangxin Dawaimishik preparation.

[0091] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0092] The reagents and raw materials used in this invention are all commercially available.

[0093] The positive and progressive effects of this invention are as follows:

[0094] This invention constructed a fingerprint characteristic spectrum for Yangxin Dawaimexik preparation and identified 30 common peaks in the spectrum. The characteristic features of the fingerprint spectrum can effectively identify the authenticity of samples, determine the consistency of batches of products, effectively control product quality, and ensure relative stability of product quality. Similarity analysis of the fingerprint spectrum results show that the production process of Yangxin Dawaimexik preparation has good stability.

[0095] Based on the above research, UPLC-QQQ-LIT-MS / MS technology was used to determine the content of multiple indicators in the Yangxin Dawaimisik preparation, and a method for determining the content of 22 components was established for the first time. This method can simultaneously and effectively separate and quantitatively detect 22 components. Among the 22 components are several components with cardiovascular and cerebrovascular protective effects, including gallic acid, epicatechin, chlorogenic acid, and rosmarinic acid. This clarifies the pharmacodynamic material basis of the preparation and provides a scientific basis for establishing a quality control system.

[0096] The method of this invention is applicable not only to Yangxin Dawaimishik honey ointment, but also to the quality control method of other Yangxin Dawaimishik preparations such as pills and tablets that retain the original preparation intermediates. Attached Figure Description

[0097] Figure 1 Fingerprint chromatograms of 14 batches of Yangxin Dawa Yimishike Honey Ointment.

[0098] Figure 2 A comparison image of the Dawaimishik Honey Paste for nourishing the heart.

[0099] Figure 3 The high-performance liquid chromatogram of sample S1, Yangxin Dawa Yimishike Honey Ointment.

[0100] Figure 4 High-performance liquid chromatogram of Yangxin Dawa Yimishike Honey Ointment.

[0101] Figure 5 The high-performance liquid chromatogram of Yangxin Dawa Yimishike Honey Ointment in Comparative Example 1 is shown.

[0102] Figure 6 The high-performance liquid chromatogram of the Yangxin Dawa Yimishike Honey Ointment in Comparative Example 2 is shown.

[0103] Figure 7The high-performance liquid chromatograms of Comparative Example 5 and Example 4, namely, the Yangxin Dawa Yimishik Honey Ointment. Figure 7 Part A is Example 4. Figure 7 Part B is Comparative Example 5.

[0104] Figure 8 The high-performance liquid chromatogram of Comparative Example 6, Yangxin Dawa Yimishike Honey Ointment.

[0105] Figure 9 The high-performance liquid chromatogram of Comparative Example 7, Yangxin Dawa Yimishike Honey Ointment. Detailed Implementation

[0106] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0107] Instruments: UHPLC-Q-Orbitrap-HRMS (Ultra-High Performance Liquid Chromatography-Quadrupole-Electrostatic Orbiter High Resolution Mass Spectrometer) was purchased from Thermo Fisher Scientific, USA, Germany; SK7210 HP ultrasonic cleaner was purchased from Shanghai Kedao Ultrasonic Instrument Co., Ltd., China; EYELA cooling water circulation system was purchased from Shanghai Ailang Instrument Co., Ltd.; BUCHI Recirculating Chiller F-305 rotary evaporator was purchased from Shanghai Ailang Instrument Co., Ltd.; Allegra 25R high-speed centrifuge was purchased from Beckman Coulter GmbH, Germany; Qtrap 6500+ triple quadrupole linear ion trap mass spectrometer was purchased from AB Sciex, USA; eppendorf centrifuge was purchased from Eppendorf Biotechnology, Germany; SQP 0.01% balance was purchased from Sartorius Scientific Instruments Beijing Co., Ltd., Germany; AE224C analytical balance was purchased from Mettler Toledo, USA; KQ-2508 ultrasonic cleaner was purchased from Jiangsu Kunshan Ultrasonic Instrument Co., Ltd.

[0108] Reference standards: p-coumaric acid (batch number WP24020412), rutin (batch number WP24010302), kaempferol-3-O-rutin (batch number WP24080702), rosmarinic acid (batch number wkq23020112), and luteolin (batch number wkq23021710) were all purchased from Sichuan Weikeqi Biotechnology Co., Ltd. Neochlorogenic acid (batch number WP24060712), caffeic acid (batch number WP24070112), quinic acid (batch number 15073002), epicatechin (batch number 15112504), ellagic acid (batch number 20090806), astragaloside (batch number 19121510), cynomol (batch number 21011602), kaempferol (batch number 20062810), and catechin (batch number 15092804) were all purchased from Shanghai Chunyou Biotechnology Co., Ltd. Hypericin (batch number 111521-201809), isoquercitrin (batch number 111809-201804), quercetin (batch number 111538-201606), narcissin (batch number 111997-201501), and quercetin (batch number 100081-200406) were all purchased from the China National Institutes for Food and Drug Control. Gallic acid (batch number 149-91-7) was purchased from Beijing Century Aoke Biotechnology Co., Ltd. Chlorogenic acid (batch number 20140617) was purchased from Luoyang Qifeng Biopharmaceutical Co., Ltd. Cryptochlorogenic acid (batch number GSB11-3795-2020) was purchased from the Xinjiang Institute of Physics and Chemistry, Chinese Academy of Sciences.

[0109] Reagents: Acetonitrile and formic acid were of mass spectrometry grade (Thermo Fisher Scientific Inc., USA); ultrapure water (Watsons, China); anhydrous ethanol was of analytical grade (Tianjin Huihang Chemical Technology Co., Ltd., China); all other reagents were of analytical grade.

[0110] Sample: Yangxin Dawaimishik Honey Ointment was purchased from Xinjiang Uygur Pharmaceutical Co., Ltd. A total of 14 batches were produced, serial numbers S1 to S14, and batch numbers 2206079, 2206080, 2206086, 2206087, 2206094, 2206095, 2206102, 2206103, 2207044, 2207045, 2207052, 2207053, 2208003, and 2208004.

[0111] Example 1: Fingerprint Map Construction and Similarity Evaluation

[0112] 1.1 Research Methods: Chromatographic conditions: ACQUITY UPLC BEH Shield RP18 (1.7 μm 2.1×100 mm) column, mobile phase was 0.1% formic acid solution (v / v) (A)-acetonitrile (B), gradient elution as shown in the table below; flow rate was 0.2 mL / min; injection volume was 2 μL, and detection wavelength was 254 nm.

[0113] Table 1 Chromatographic conditions for fingerprint spectroscopy

[0114]

[0115] Preparation of test solution: Weigh 10 g of sample and place it in a stoppered conical flask. Add 100 mL of 50% methanol aqueous solution and extract by sonication for 1 hour (power 250 W, frequency 40 kHz). Let stand for 24 hours, cool, take 1 mL of supernatant and place it in a 10 mL volumetric flask. Dilute to the mark with 50% methanol aqueous solution to make the crude drug concentration 10 mg / mL. Filter with a 0.22 μm microporous membrane for later use.

[0116] 1.2 Results and Discussion

[0117] For 14 batches of Yangxin Dawa Yimisik Honey Ointment, test solutions were prepared according to the preparation method of the test solution in Part 1.1, and analyzed according to the UPLC chromatographic conditions in Part 1.1. Using the 2012 version of the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System, sample S1 was selected as a reference to construct a characteristic chromatogram (see...). Figure 1 ), and generate the corresponding standard fingerprint spectrum, automatically matching 42 common peaks, and identifying 30 of them (see Figure 2 ).

[0118] Figure 1 Fingerprint chromatograms of 14 batches of Yangxin Dawa Yimishike Honey Ointment. Figure 1 In the diagram, S1-S14 correspond to 14 batches of samples, and R is the generated control fingerprint spectrum.

[0119] Figure 2 A comparison chart (i.e., a common peak spectrum) of Yangxin Dawa Yimishike Honey Paste.

[0120] Figure 3 The figure shows the high-performance liquid chromatogram (HPLC) of sample S1, Yangxin Dawa Yimisik Honey Ointment. As can be seen from the figure, under these elution conditions, the chromatographic peaks are sharp, narrow, and without tailing; the baseline is stable with low noise; and the chromatographic separation is clear, indicating the optimal elution gradient. At a wavelength of 254 nm, the chromatographic peak information is abundant, the signal is strongest, and interference is minimal.

[0121] The specific data is shown in the table below.

[0122] Table 2

[0123]

[0124] The similarity was evaluated using a software system for evaluating the similarity of chromatographic fingerprints of traditional Chinese medicine. The results showed that the similarity of 14 batches of Yangxin Dawaimishik preparations ranged from 0.873 to 0.988, specifically 0.975, 0.982, 0.988, 0.980, 0.987, 0.970, 0.979, 0.985, 0.986, 0.942, 0.873, 0.920, 0.877, and 0.893, with minimal differences between batches. The experimental results indicate that the medicinal materials used in Yangxin Dawaimishik preparations are of consistent origin and the processing technology is stable.

[0125] Example 2: Investigation of the extraction solvent for the test sample

[0126] Except for the following conditions, the operation and conditions of Example 2 are the same as those of Example 1.

[0127] In the preparation method of the test solution, a 30% methanol aqueous solution is used as the solvent.

[0128] Example 3: Investigation of the extraction solvent for the test sample

[0129] Except for the following conditions, the operation and conditions of Example 3 are the same as those of Example 1.

[0130] In the preparation method of the test solution, a 70% methanol aqueous solution is used as the solvent.

[0131] Figure 4 The figure shows the high-performance liquid chromatogram of Yangxin Dawaimishik honey paste. As can be seen from the figure, when using 50% methanol aqueous solution (Example 1), 70% methanol aqueous solution (Example 3), and 30% methanol aqueous solution (Example 2) as extraction solvents, the sample solution extracted with 50% methanol aqueous solution showed more chromatographic peaks and a higher response value. Therefore, the optimal extraction solvent is 50% methanol aqueous solution.

[0132] Comparative Example 1: Elution Gradient Investigation

[0133] Except for the elution gradient conditions described below, all other operations are the same as in Example 1.

[0134] Table 3

[0135]

[0136] Figure 5 The figure shows the high-performance liquid chromatogram of Yangxin Dawa Yimishike Honey Ointment in Comparative Example 1. As shown in the figure, the chromatogram of Comparative Example 1 has a longer peak elution time, and the retention time of the same components fluctuates more than that of Example 1. The baseline is unstable and there is baseline drift and noise interference.

[0137] Comparative Example 2: Elution Gradient Investigation

[0138] Except for the elution gradient conditions described below, all other operations are the same as in Example 1.

[0139] Table 4

[0140]

[0141] Figure 6 The figure shows the high-performance liquid chromatogram of Yangxin Dawa Yimisik Honey Ointment in Comparative Example 2. As shown in the figure, the chromatogram of Comparative Example 2 shows some peaks with tailing, broadening, baseline drift, and high overall noise. Some chromatographic peaks have high overlap, indicating poor separation.

[0142] Comparative Examples 3-4: Investigation of Detection Wavelengths

[0143] Except for the following differences, all other operations are the same as in Example 1.

[0144] In Comparative Example 3, the detection wavelength was 210 nm. In Comparative Example 4, the detection wavelength was 360 nm.

[0145] According to the high performance liquid chromatogram of Yangxin Dawa Yimishike Honey Ointment in Comparative Example 3, at a wavelength of 210 nm, the peak elution time is too long, the response signal is weak, the separation is poor, and there are impurities, making it difficult to achieve complete separation of the chromatographic peaks.

[0146] According to the high performance liquid chromatogram of Yangxin Dawa Yimishike Honey Ointment in Comparative Example 4, at a wavelength of 360 nm, the number of visible chromatographic peaks is significantly reduced, the interference of impurity peaks is more significant, and the baseline noise is relatively large. This indicates that the detection specificity is poor and the signal-to-noise ratio is low at this wavelength, which is not conducive to the accurate identification and quantification of target compounds.

[0147] Therefore, the optimal detection wavelength for high-performance liquid chromatography (HPLC) is 254 nm during fingerprint pattern construction.

[0148] Example 4: Quantitative analysis of 22 components

[0149] 4.1 Research Methods

[0150] Chromatographic conditions: A Waters ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) was used; the mobile phase was 0.1% formic acid solution (A)-acetonitrile (B); the flow rate was 0.25 mL / min; the column temperature was 35℃; the injection volume was 2 uL; the detection wavelength was 254 nm; and the gradient elution program was shown in the table below.

[0151] Table 5 chromatographic conditions for quantitative analysis

[0152]

[0153] Mass spectrometry conditions: The ion source was an electrospray ionization (ESI) source, with multiple reaction detection (MRM) mode and a rapid switching mode between positive and negative ions; the ion source temperature was 450℃; the ionization voltages were 5500 V and -4500 V; the curtain gas, ion source spray gas GasⅠ, and auxiliary heating gas GasⅡ were all N2, with pressures of 35, 55, and 55 psi, respectively; the collision gas was set to medium. The m / z, declustering voltage (DP), and collision energy (CE) of the parent and daughter ions for the 22 components are detailed in the table below.

[0154] Table 6. Optimization results of mass spectrometry conditions for 22 chemical components

[0155]

[0156] Preparation of reference solutions: Accurately weigh 5.0 mg of each of the 22 reference standards, including cryptochlorogenic acid, epicatechin, p-coumaric acid, quercetin, rosmarinic acid, ellagic acid, and kaempferol, and place them in separate 10-100 mL volumetric flasks. Add methanol to make up to the volume and shake well to obtain single reference stock solutions of 50 μg / mL ellagic acid and 500 μg / mL of the other reference standards. All solutions were stored at 4℃ and filtered through a 0.22 μm microporous membrane before analysis.

[0157] Preparation of mixed reference solutions: Accurately measure the above-mentioned reference standard filtrate and place it in a 10 mL volumetric flask. Dilute to the mark with methanol and dilute according to a certain ratio to prepare mixed reference solutions of different concentrations.

[0158] Preparation of the test solution: Weigh 10.0 g of Yangxin Dawa Yimishike honey paste sample, add 50 mL of 50% ethanol aqueous solution, and extract by ultrasonication (power 250 W, frequency 40 kHz) for 60 min. Let stand for 24 h, take 1 mL of supernatant and put it into a 10 mL volumetric flask, add 50% ethanol aqueous solution to make up to the mark, and the extract concentration (crude drug amount) is 10.0 mg / mL. Filter with a 0.22 μm microporous membrane before analysis.

[0159] 4.2 Results and Discussion

[0160] The established UHPLC-MS / MS method successfully achieved simultaneous quantitative analysis of 22 active ingredients in Yangxin Dawa Yimishike Honey Ointment. Method validation results showed that its linearity, sensitivity, precision, stability, and repeatability all met the requirements of the Chinese Pharmacopoeia and ICH guidelines, as detailed below:

[0161] (1) Examination of linear relationships

[0162] Accurately pipette mixed reference solutions of different concentrations and determine them according to the chromatographic conditions in section "4.1". Record the peak areas and plot a standard curve with reference concentration X on the x-axis and reference peak area Y on the y-axis. Perform linear regression to obtain the regression equation, linear range, and correlation coefficient (R²). 2 The results showed that all 22 active ingredients exhibited good linear relationships across various concentration ranges. The linear regression equations, linear ranges, and correlation coefficients for the 22 reference standards are as follows:

[0163] Quinic acid: y = 3115.4889 x - 170237.3212, linear range 0.05-5 μg / mL, R 2 It is 0.9990;

[0164] Gallic acid: y = 75,350.1372x + 20,453,831.7218, linear range 0.12-6 μg / mL, R 2 It is 0.9946;

[0165] Neochlorogenic acid: y = 73294.2964x + 404815.7394, linear range 0.001-1 μg / mL, R 2 It is 0.9994;

[0166] Catechins: y = 25086.8449 x - 711.4828, linear range 0.00008-0.04 μg / mL, R 2 It is 0.9994;

[0167] Chlorogenic acid: y = 51027.1256x + 2290245.1194, linear range 0.04-4 μg / mL, R 2 It is 0.9975;

[0168] Caffeic acid: y = 178,124.0604x + 2,193,843.4736, linear range 0.004-0.8 μg / mL, R 2 It is 0.9984;

[0169] Cryptochlorogenic acid: y = 75,264.8823x + 233,959.4559, linear range 0.0008-0.8 μg / mL, R 2 It is 0.9993;

[0170] Epicatechin: y = 12938.6798 x + 14090.8505, linear range 0.0005-0.1 μg / mL, R 2 It is 0.9979;

[0171] p-Coumaric acid: y = 100,026.4170x + 331,961.6823, linear range 0.0012-0.6 μg / mL, R 2 It is 0.9997;

[0172] Ellagic acid: y = 9,704.1020x + 246,568.4084, linear range 0.05-5 μg / mL, R 2 It is 0.9987;

[0173] Hyperoside: y = 18170.5665x + 20795.4240, linear range 0.0008-0.16 μg / mL, R 2 It is 0.9992;

[0174] Rutin: y = 13,124.4407x + 101,448.8140, linear range 0.002-2 μg / mL, R 2 It is 0.9993;

[0175] Isoquercetin: y = 103386.1338x + 438435.7990, linear range 0.0015-0.3 μg / mL, R 2 It is 0.9981;

[0176] Kaempferol-3-O-rutin: y = 20,789.6469 x - 6,129.6574, linear range 0.0002-0.2 μg / mL, R 2 It is 0.9998;

[0177] Astragaloside: y = 3588.7468x + 14091.4662, linear range 0.001-0.4 μg / mL, R 2 It is 0.9974;

[0178] Quercetin: y = 89997.5557x + 59638.4806, linear range 0.0002-0.15 μg / mL, R 2 It is 0.9991;

[0179] Narcisin: y = 15214.9910x + 43710.9840, linear range 0.0008-0.16 μg / mL, R 2 It is 0.9975;

[0180] Rosmarinic acid: y = 110892.2160x + 1060434.6524, linear range 0.002-1.5 μg / mL, R 2 It is 0.9981;

[0181] Quercetin: y = 136719.1259 x + 204606.8507, linear range 0.0005-0.1 μg / mL, R 2 It is 0.9975;

[0182] Luteolin: y = 239605.5882 x + 223692.0938, linear range 0.0006-0.06 μg / mL, R 2 It is 0.9994;

[0183] Silver calcinate: y = 40377.9259x + 60639.6685, linear range 0.001-0.1 μg / mL, R 2 It is 0.9990;

[0184] Kaempferol: y = 19033.5098x + 18002.9745, linear range 0.00008-0.08 μg / mL, R 2 It is 0.9976.

[0185] (2) Determination of the content of 22 compounds in 10 batches of Yangxin Dawa Yimi Xike honey paste samples

[0186] Prepare test solutions for batches S1-S10 according to the preparation method of test solution under section "4.1".

[0187] Each test solution was injected and analyzed according to the chromatographic and mass spectrometric conditions described in section "4.1" to obtain a chromatogram of the sample. The peak areas corresponding to 22 compounds in each chromatogram were detected, and the contents of each compound in the 10 batches of samples were calculated using the standard curve as shown in the table below.

[0188] Table 7. Determination of the content of 22 compounds in 10 batches of Yangxin Dawa Yimishike Honey Paste samples (unit: μg / g)

[0189]

[0190] (3) Precision, stability and repeatability tests

[0191] 1) Precision test: Accurately pipette the mixed reference solution under section “4.1”, inject it continuously 6 times under the chromatographic conditions under section “4.1”, measure the peak area, and calculate its RSD value.

[0192] 2) Repeatability test: Take the same batch of Yangxin preparation sample (S1), prepare 6 test solutions in parallel according to the test solution preparation method under section “4.1”, inject the samples according to the chromatographic and mass spectrometric conditions under section “4.1”, record the peak area of ​​the analyte, obtain the concentration, and calculate the RSD value.

[0193] 3) Stability Study: Samples (S1) from the same batch of Yangxin preparation were used to prepare test solutions according to the method described in section "4.1". Peak areas were measured under the chromatographic conditions described in section "4.1" at time intervals of 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, and 48 h, and the RSD values ​​were calculated. The results of precision, stability, and repeatability tests are as follows.

[0194] Table 8. Results of precision, stability, and repeatability experiments for the quantitative methods.

[0195]

[0196] (4) Recovery test

[0197] Nine 5.0 g portions of the Yangxin sample were accurately weighed and divided into three groups. Three portions of each reference solution were added to each of the three groups of samples at ratios of high (1:1.2), medium (1:1), and low (1:0.8). The test solutions were prepared according to the method described in section "4.1". Peak areas were measured, and sample content (based on the standard curve), recovery rate, and RSD were calculated. The results are shown below.

[0198] Table 9. Recovery results of 22 compounds

[0199]

[0200]

[0201]

[0202]

[0203]

[0204] Comparative Example 5: Investigation of the chromatographic column

[0205] Comparative Example 5 differs from Example 4 only in the chromatographic column used; the column was an ACQUITY UPLCBEH Shield RP18 (1.7 μm 2.1 × 100 mm). All other operations and conditions were the same as in Example 4.

[0206] Compared with Example 4, Comparative Example 5 had difficulty separating the components and had a low degree of separation, while Example 4 had a better degree of separation of the product.

[0207] Figure 7 The high-performance liquid chromatograms of Comparative Example 5 and Example 4, namely, the Yangxin Dawa Yimishik Honey Ointment. Figure 7 Part A is Example 4. Figure 7 Part B is Comparative Example 5. It can be seen that Comparative Example 5 is difficult to separate the components, with narrow peak spacing, few peaks, peak tailing, and low resolution. In contrast, Example 4 has better resolution of the product, with more chromatographic peaks, sharp and symmetrical peaks, and high efficiency of the chromatographic column in separating the components.

[0208] Examination of the elution procedure in Comparative Example 6

[0209] Comparative Example 6 differs from Example 4 only in the elution program, which is as follows: 0-2 min, 0% B; 2-3 min, 0%-2%; 3-22 min, 2%-12%; 22-38 min, 12%-20%; 38-43 min, 20%-25%; 43-53 min, 25%-40%; 53-55 min, 40%-95%; 55-60 min, 95%; 60-61 min, 95%-0%.

[0210] Figure 8 The high-performance liquid chromatogram of Comparative Example 6, Yangxin Dawa Yimishike Honey Ointment.

[0211] Comparative Example 6 showed weak chromatographic peak signal intensity, while Example 4 showed a chromatographic peak signal intensity 2.1 times stronger than that of Comparative Example 6. Furthermore, Comparative Example 6 exhibited a small number of chromatographic peaks, peak tailing, and incomplete separation.

[0212] Comparative Example 7: Examination of the elution procedure

[0213] Comparative Example 7 differs from Example 4 only in the elution program, which is as follows: 0-2 min, 2% B; 2-22 min, 2%-12%; 22-38 min, 12%-20%; 38-43 min, 20%-25%; 43-48 min, 25%-40%; 48-50 min, 40%-95%; 50-55 min, 95%; 55-56 min, 95%-2%.

[0214] Figure 9 The high-performance liquid chromatogram of Comparative Example 7, Yangxin Dawa Yimishike Honey Ointment.

[0215] Compared with Example 4, Comparative Example 7, although the signal intensity was acceptable, had issues such as overlapping adjacent chromatographic peaks, incomplete separation, and large impurity peaks. Overall, it did not have the same separation effect as Example 4.

Claims

1. A method for constructing a fingerprint spectrum of Yangxin Dawaishiyimi preparation, characterized in that, It comprises the following steps: detecting the test sample solution by high performance liquid chromatography, and generating a fingerprint spectrum based on the detection result; The test sample solution is a solution containing the YXDWYMYC preparation; In the high performance liquid chromatography, the detection wavelength is 254 nm; the mobile phase A used is a 0.05%-0.15% formic acid solution by volume percentage, and the mobile phase B is acetonitrile; the gradient elution conditions of the mobile phase A and the mobile phase B are as follows: ； The percentage is the volume percentage of each component in the total volume of the mobile phase A and the mobile phase B.

2. The method for constructing a fingerprint as claimed in claim 1, wherein, The construction method of the fingerprint spectrum meets one or more of the following conditions: (1) In the test sample solution, the solvent used is alcohol or an alcohol-water solution, wherein the alcohol or the alcohol in the alcohol-water solution is preferably methanol or ethanol; the volume percentage of the alcohol-water solution is preferably 40-60%, for example, 45%, 50% or 55%; (2) In the test sample solution, the extract concentration is 8-12 mg / mL, preferably 10 mg / mL; and (3) The preparation method of the test sample solution comprises the following steps: ultrasonic extraction of the mixture of the YXDWYMYC preparation and the solvent, and preparation of the test sample solution by taking the supernatant; The mass-volume ratio of the YXDWYMYC preparation to the solvent is preferably (8-12) g:100 mL, for example, 10 g:100 mL; The power of the ultrasonic extraction is preferably 250 W; The frequency of the ultrasonic extraction is preferably 40 kHz; The ultrasonic extraction time is preferably 0.5-1.5 h, for example, 1 h; Preferably, the test sample solution is prepared by the following steps: mixing 10 g of YXDWYMYC preparation with 100 mL of 50% methanol solution, ultrasonic extraction for 1 hour, ultrasonic power 250 W, ultrasonic frequency 40 kHz, dilution with 50% methanol solution to make the crude drug concentration 10 mg / mL, and filtration with a 0.22 μm microporous filter.

3. The method for constructing a fingerprint as claimed in claim 1, wherein, The construction method of the fingerprint spectrum meets one or more of the following conditions: (1) In the high performance liquid chromatography detection, the chromatographic column is an ACQUITY UPLC BEH Shield RP18; (2) In the high performance liquid chromatography detection, the specification of the chromatographic column is 1.7 μm 2.1×100 mm; (3) In the high performance liquid chromatography detection, the volume percentage of the mobile phase A is 0.08-0.12%, for example, 0.1%; (4) In the high performance liquid chromatography detection, the injection volume is 1-4 μL, for example, 2 μL; (5) In the high performance liquid chromatography detection, the flow rate of the test sample solution is 0.1-0.5 mL / min, preferably 0.2 mL / min; (6) The fingerprint spectrum construction method of the YXDWYMYC preparation comprises the following steps: a. Prepare several test sample solutions; b. The test sample solution is detected according to the high performance liquid chromatography; according to the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, one test sample solution is selected as a reference to construct a characteristic spectrum and generate a corresponding standard fingerprint spectrum; The fingerprint spectrum has 30 common peaks, and the 30 common peaks constitute the fingerprint characteristics of the Yangxin Dawa Yimisike preparation, which is used as the standard fingerprint spectrum of the Yangxin Dawa Yimisike preparation. The fingerprint spectrum of the Yangxin Dawa Yimisike preparation is one high performance liquid chromatogram, as shown in FIG. 2; and, (7) The fingerprint spectrum includes 11 common characteristic peaks, and the retention time, relative retention time, peak area and relative peak area of the common characteristic peaks are as follows, with the sample solution S1 as a reference: 。 4. A method for detecting a compound in a preparation of Nangkawur Miqie, characterized in that, It comprises the following steps: the test sample solution is subjected to ultra-high performance liquid chromatography-triple quadrupole mass spectrometry detection of the to-be-detected components; The test sample solution is a solution containing the Yangxin Dawa Yimisike preparation; and the to-be-detected components include one or more of chlorogenic acid, epicatechin, p-coumaric acid, catechin, quercitrin, quercetin, caffeic acid, rutin, rosmarinic acid, gallic acid, luteolin, ellagic acid, kaempferol, kaempferol-3-O-rutinoside, zephyranthine, isoquercitrin, syringin, chlorogenic acid, neochlorogenic acid, quinic acid, astragalin and hyperoside. In the high performance liquid chromatography, the mobile phase A used is 0.05%-0.15% formic acid solution by volume percentage, and the mobile phase B is acetonitrile; and the gradient elution conditions of the mobile phase A and the mobile phase B are as follows: 。 5. The method of claim 4, wherein the detection is performed by a method selected from the group consisting of mass spectrometry, nuclear magnetic resonance, and chromatography. The detection method meets one or more of the following conditions: (1) In the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry detection, the chromatographic column for ultra-high performance liquid chromatography detection is Waters ACQUITY UPLC HSS T3; (2) In the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry detection, the specification of the chromatographic column for ultra-high performance liquid chromatography detection is 2.1 mm x 100 mm, 1.8 μm; (3) In the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry detection, the volume percentage of the mobile phase A is 0.08-0.12%, for example, 0.1%; (4) In the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry detection, the injection volume in the ultra-high performance liquid chromatography detection is 1-4 μL, for example, 2 μL; (5) In the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry detection, the column temperature in the ultra-high performance liquid chromatography detection is 30-40℃, preferably 35℃; and, (6) In the high performance liquid chromatography detection, the flow rate of the test sample solution is 0.15-0.35 mL / min, preferably 0.25 mL / min.

6. The detection method of claim 4, wherein, In the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry detection, the mass spectrometry detection conditions are as follows: The ion source is an electrospray ion source; the scanning mode is a mass spectrometry multiple reaction detection mode and a positive and negative ion rapid switching mode; the ion source temperature is 450℃; the ionization voltage is 5500 V and -4500 V, respectively; the gas curtain gas, ion source spray gas Gas I and auxiliary heating gas Gas II are all N2, and the pressures are 35, 55 and 55 psi, respectively; the collision gas is set to medium.

7. The detection method of claim 4, wherein, In the ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry detection, the m / z of the parent ion and daughter ion of the component to be detected, the declustering voltage DP and the collision energy CE are as follows: 。 8. The detection method as described in claim 4, characterized in that, The detection method meets one or more of the following conditions: (1) In the test solution, the solvent used is alcohol or alcohol aqueous solution, wherein the alcohol or alcohol in the alcohol aqueous solution is preferably methanol or ethanol; the volume percentage of the alcohol aqueous solution is preferably 40-60%, for example, 45%, 50% or 55%; (2) In the test solution, the extract concentration is 8-12 mg / mL, preferably 10 mg / mL; (3) The preparation method of the test solution comprises the following steps: ultrasonic extraction of the mixture of the Yangxin Dawa Yimi Sikai preparation and the solvent, and preparation of the test solution by taking the supernatant; Wherein, the mass-volume ratio of the Yangxin Dawa Yimi Sikai preparation and the solvent is preferably (8-12) g: 50 mL, for example, 10 g: 50 mL; The power of the ultrasonic extraction is preferably 250 W; The frequency of the ultrasonic extraction is preferably 40 kHz; The ultrasonic extraction time is preferably 0.5-1.5 h, for example, 1 h; Preferably, the test solution is prepared by the following steps: mixing Yangxin Dawa Yimi Sikai preparation 10 g and 50% ethanol aqueous solution 50 mL, ultrasonic treatment for 60 min, ultrasonic power 250 W, ultrasonic frequency 40 kHz, dilution with 50% ethanol aqueous solution to the extraction concentration of 10.0 mg / mL, 0.22 μm microporous filter membrane filtration.

9. The detection method as described in claim 4, characterized in that, The linear regression equation and linear range of the component to be detected are as follows: wherein X is the concentration µg / mL, and Y is the peak area: Quinic acid: y = 3115.4889 x - 170237.3212, linear range 0.05-5 μg / mL, R 2 was 0.9990; Gallic acid: y = 75,350.1372 x + 20,453,831.7218, linear range 0.12-6 μg / mL, R 2 was 0.9946; Neochlorogenic acid: y = 73294.2964 x + 404815.7394, linear range 0.001-1 μg / mL, R 2 was 0.9994; Catechin: y = 25086.8449 x - 711.4828, linear range 0.00008-0.04 μg / mL, R 2 was 0.9994; Chlorogenic acid: y = 51027.1256 x + 2290245.1194, linear range 0.04-4 μg / mL, R 2 was 0.9975; Caffeic acid: y = 178,124.0604 x + 2,193,843.4736, linear range 0.004-0.8 pg / mL, R 2 was 0.9984; Cryptophycin: y = 75,264.8823 x + 233,959.4559, linear range 0.0008-0.8 μg / mL, R 2 was 0.9993; Epicatechin: y = 12938.6798 x + 14090.8505, linear range 0.0005-0.1 μg / mL, R 2 was 0.9979; Coumaric acid: y = 100,026.4170 x + 331,961.6823, linear range 0.0012-0.6 μg / mL, R 2 was 0.9997; Gallic acid: y = 9,704.1020 x + 246,568.4084, linear range 0.05-5 μg / mL, R 2 was 0.9987; Hyperoside: y = 18170.5665 x + 20795.4240, linear range 0.0008-0.16 μg / mL, R 2 was 0.9992; Rutin: y = 13,124.4407 x + 101,448.8140, linear range 0.002-2 μg / mL, R 2 was 0.9993; Quercitrin: y = 103386.1338 x + 438435.7990, linear range 0.0015-0.3 μg / mL, R 2 was 0.9981; Kaempferol-3-O-rutinoside: y = 20,789.6469 x - 6,129.6574, linear range 0.0002-0.2 μg / mL, R 2 was 0.9998; Tricin: y = 3588.7468 x + 14091.4662, linear range 0.001-0.4 μg / mL, R 2 was 0.9974; Quercitrin: y = 89997.5557 x + 59638.4806, linear range 0.0002-0.15 pg / mL, R 2 was 0.9991 ; Narciclasine: y = 15214.9910 x + 43710.9840, linear range 0.0008-0.16 μg / mL, R 2 was 0.9975; Rosmarinic acid: y = 110892.2160 x + 1060434.6524, linear range 0.002-1.5 μg / mL, R 2 was 0.9981; Quercetin: y = 136719.1259 x + 204606.8507, linear range 0.0005-0.1 μg / mL, R 2 was 0.9975; Luteolin: y = 239605.5882 x + 223692.0938, linear range 0.0006-0.06 μg / mL, R 2 was 0.9994; Silver forge: y = 40377.9259 x + 60639.6685, linear range 0.001-0.1 μg / mL, R 2 was 0.9990; Kaempferol: y = 19033.5098 x + 18002.9745, linear range 0.00008-0.08 μg / mL, R 2 was 0.9976.

10. The application of the fingerprint spectrum of the Yangxin Dawa Yimi Sikai preparation obtained by the construction method of the fingerprint spectrum of the Yangxin Dawa Yimi Sikai preparation according to any one of claims 1-3 and / or the detection method of the compound in the Yangxin Dawa Yimi Sikai preparation according to any one of claims 4-9 in the quality control of the Yangxin Dawa Yimi Sikai preparation.

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