Method for detecting chemical components of heart-nourishing Damwai-mimic honey paste

Abstract

The invention provides a method for detecting the chemical components of heart-nourishing Damwai-Mimike honey cream, which comprises the following steps: carrying out gradient elution on the heart-nourishing Damwai-Mimike honey cream by adopting a UPLC (Ultra Performance Liquid Chromatography) method, and then testing by adopting a Q-TOF (Quantitative Time of Flight) method, and the chromatographic conditions adopted in the UPLC method are as follows: a mobile phase A is 0.1% formic acid aqueous solution, a mobile phase B is 0.1% formic acid aqueous solution, and a mobile phase C is 0.1% formic acid aqueous solution; the percentage is the volume ratio of the fingernail acid to the formic acid aqueous solution, the mobile phase A is acetonitrile, and the percentage is the volume ratio of the fingernail acid to the formic acid aqueous solution, the mobile phase B is acetonitrile, and gradient elution is carried out. And an experimental basis is provided for quality control and basic research on pharmacodynamic substances of the heart-nourishing Damwai-memicok honey cream.

Description

Technical Field

[0001] This invention relates to a method for detecting the chemical components of Yangxin Dawa Yimishike Honey Paste, belonging to the field of traditional Chinese medicine detection technology. Background Technology

[0002] The Yangxin Dawaimishik Honey Ointment is recorded in the Uyghur classical medical text, *Karbadin Kader*. It is composed of twenty-six medicinal ingredients, including saffron, musk, sandalwood, cinnamon, pearl, and frankincense, resulting in a complex chemical composition. Research on the chemical components of traditional Chinese medicine is an important means of studying the material basis of Chinese medicine and a prerequisite and foundation for in-depth explanation of its mechanisms of action.

[0003] Traditional solvent extraction methods, such as reflux extraction, decoction extraction, water extraction with alcohol precipitation, alcohol extraction with water precipitation, and acid-base extraction, have drawbacks including affecting efficacy, complex procedures, time and labor consumption, low purity, loss of organic reagents, and high labor costs. Liquid chromatography-mass spectrometry (LC-MS) technology not only improves the sensitivity and accuracy of detection but also enables the simultaneous detection of multiple components, significantly improving detection efficiency and quality. It plays a crucial role in the qualitative identification of traditional Chinese medicine compound preparations.

[0004] Currently, publicly available technical data, such as CN103908621A and "Li Zhen, Song Zhiming. Exploration of the Application of Nursing Intervention in the Treatment of Coronary Heart Disease and Angina Pectoris with Uyghur Medicine Yangxin Dawaimishike, Chinese Journal of Ethnic Medicine," indicate that Yangxin Dawaimishike honey ointment has good clinical efficacy and is used to treat chest pain, palpitations, stomach weakness, visual impairment, and neurasthenia. However, due to its complex chemical composition, it is difficult to elucidate the material basis for its true efficacy. With the deepening research on ethnic medicine, clarifying the main chemical components of Yangxin Dawaimishike honey ointment is essential for promoting its clinical application research. Currently, there are no reported methods for qualitative and quantitative analysis of the chemical components of Yangxin Dawaimishike honey ointment. Summary of the Invention

[0005] The technical problem this invention aims to solve is to overcome the limitation of existing technologies in rapidly and accurately analyzing and identifying the chemical components of Yangxin Dawaimishike Honey Ointment, and to provide a method for detecting the chemical components of Yangxin Dawaimishike Honey Ointment. This invention provides a comprehensive, rapid, and accurate analysis and identification of the chemical components of Yangxin Dawaimishike Honey Ointment, offering experimental evidence for quality control and pharmacodynamic research of the ointment.

[0006] This invention provides a method for detecting the chemical components of Yangxin Dawa Yimisik Honey Paste, which includes the following steps:

[0007] The UPLC method was used to perform gradient elution of Yangxin Dawayimisik honey paste, followed by Q-TOF testing.

[0008] The chromatographic conditions used in the UPLC method are as follows:

[0009] Mobile phase A is a 0.1% formic acid aqueous solution, where the percentage is the volume ratio of formic acid to the formic acid aqueous solution. Mobile phase B is acetonitrile, and gradient elution is used. The parameters of the gradient elution are as follows:

[0010] time The percentage of mobile phase B in the total volume of the mobile phase. 0min~5min 5％～25％ 5 min ~ 15 min 25％～30％ 15–45 min 30％～99％

[0011] The mass spectrometry conditions used in the Q-TOF method are as follows:

[0012] An electrospray ionization source is used, with positive ion detection mode and / or negative ion detection mode. The ion source temperature is 120℃, the desolvation gas temperature is 450℃, and the desolvation gas flow rate is 800L / h. In the positive ion detection mode, the capillary voltage is +3.0KV, and in the negative ion detection mode, the capillary voltage is -2.5KV.

[0013] In this invention, the gradient elution in the UPLC method is a linear gradient change.

[0014] In this invention, the chromatographic column used in the UPLC method is preferably a C18 column, such as a Waters Xtimate BEHC18 column.

[0015] The Waters Xtimate BEH C18 chromatographic column preferably has an inner diameter of 4.6 mm, a column length of 250 mm, and a packing particle size of 5 μm.

[0016] In this invention, the flow rate of the mobile phase in the UPLC method is preferably 0.4 mL / min.

[0017] In this invention, the injection temperature in the UPLC method is preferably 10°C.

[0018] In this invention, the injection volume in the UPLC method is preferably 2 μL.

[0019] In this invention, the column temperature of the chromatographic column in the UPLC method is preferably 40°C.

[0020] In this invention, the detection wavelength in the UPLC method is preferably 190–500 nm.

[0021] In this invention, during the UPLC method, the Yangxin Dawaimishik honey paste is preferably loaded in the form of a Yangxin Dawaimishik honey paste solution.

[0022] The preferred method for preparing the Yangxin Dawaimishik honey ointment solution includes: mixing the Yangxin Dawaimishik honey ointment with an aqueous solution of methanol.

[0023] The volume percentage of methanol in an aqueous solution can be 40% to 60%, for example 50%. Volume percentage refers to the percentage of the volume of methanol in the aqueous solution.

[0024] The preferred method for the mixing process is ultrasonic mixing.

[0025] The mixing process can take 20 to 50 minutes, for example, 30 minutes.

[0026] The mixing process may further include a purification step. Preferably, the purification method employs a filter membrane, and the filter membrane preferably has a pore size of 0.22 μm.

[0027] In this invention, the chromatograph used in the UPLC method can be a Model 2998 ultra-high performance liquid chromatograph.

[0028] In this invention, the separation results can be obtained after gradient elution, and the Q-TOF method is then used to test the separation results.

[0029] In this invention, the desolvation gas in the Q-TOF method is conventional in the art, such as nitrogen.

[0030] In this invention, the carrier gas in the Q-TOF method is conventional in the art, such as nitrogen.

[0031] In this invention, the orifice voltage in the Q-TOF method can be 40V. The orifice gas flow rate can be 50L / h.

[0032] In this invention, the collision-induced dissociation gas used in the Q-TOF method is conventional in the art, such as argon. The purity of the argon gas can be 99.95%.

[0033] In this invention, the low collision energy in the Q-TOF method can be 6 eV, and the high collision energy can be 12–45 eV.

[0034] In this invention, the mass spectrometer used in the Q-TOF method can be the Xevo G2-S Q-TOF high-resolution time-of-flight mass spectrometer.

[0035] In this invention, the method for detecting chemical components in the Yangxin Dawa Yimishike Honey Ointment may further include a data processing step.

[0036] Preferably, the data processing steps include:

[0037] S1: Process the test results under the positive ion detection mode and / or the test results under the negative ion detection mode to obtain the results under the positive ion mode and / or the results under the negative ion mode.

[0038] S2: Compare the results under the positive ion mode and / or the results under the negative ion mode with the database respectively to determine the chemical composition of the Yangxin Dawa Yimishike Honey Ointment.

[0039] The database is preferably a collection of information about Yangxin Dawa Yimishike Honey Ointment from one or more databases, including the SciFinder database, TCMID database, TCM@Taiwan database, TCMSP database, and CNKI database.

[0040] The results in the positive ion mode and / or negative ion mode were processed using ChemFinder for Office 22.0.0, Compound Discover 2.0, and Xcalibur 3.2 software, respectively, to obtain the retention time, molecular formula, mass-to-charge ratio, and primary and secondary mass spectrometry fragment information of the compounds.

[0041] The positive and progressive effects of this invention are as follows:

[0042] This invention employs a combination of UPLC and Q-TOF methods to comprehensively, rapidly, and accurately analyze and identify the chemical components of Yangxin Dawaimishike Honey Ointment, providing experimental basis for the quality control and pharmacodynamic material basis research of Yangxin Dawaimishike Honey Ointment. Attached Figure Description

[0043] Figure 1 This is the base chromatogram (BPC) of the Yangxin Dawa Yimishike Honey Paste in positive ion mode, as shown in Example 1.

[0044] Figure 2 This is the base peak diagram (BPC) of the Yangxin Dawa Yimishike Honey Paste in negative ion mode in Example 1. Detailed Implementation

[0045] 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.

[0046] Constructing a database of the chemistry of Yangxin Dawa Yimishik Honey Ointment:

[0047] The chemical composition information of Compound Yangxin Dawaimishike Honey Ointment was compiled from the SciFinder, TCMID, TCM@Taiwan, TCMSP, and CNKI databases and digitized (its two-dimensional structural formula *.mol) to form a database of the chemical composition of Yangxin Dawaimishike Honey Ointment.

[0048] Instruments and software:

[0049] The instrument used was a Model 2998 ultra-high performance liquid chromatograph and a Xevo G2-S Q-TOF high-resolution time-of-flight mass spectrometer (Waters Corporation, USA), along with Masslynx and UNIFI software.

[0050] Reagents and materials:

[0051] Mass spectrometry grade methanol, mass spectrometry grade acetonitrile, and mass spectrometry grade formic acid were all purchased from Thermo Fisher Scientific Ltd., and Wahaha purified water was purchased from Hangzhou Wahaha Group Co., Ltd.

[0052] Yangxin Dawaimishike Honey Ointment originates from Xinjiang Uygur Pharmaceutical Co., Ltd., Z65020175.

[0053] Example 1

[0054] Step 1: Weigh 1.0g of Yangxin Dawa Yimishike honey paste and place it in a 50mL stoppered Erlenmeyer flask. Add 25mL of 50% methanol-water solution, sonicate for 30min, let cool, add methanol to make up the weight, mix well, and filter through a 0.22μm microporous membrane to obtain the test solution.

[0055] Step 2: The test solution is injected into a 2998 ultra-high performance liquid chromatograph and a Xevo G2-SQ-TOF high-resolution time-of-flight mass spectrometer for analysis.

[0056] The chromatographic conditions are as follows:

[0057] The chromatographic column was a Waters Xtimate BEH C18 column (4.6 x 250 mm, 5 μm). Mobile phase A was 0.1% formic acid in water (v / v), and mobile phase B was acetonitrile. Gradient elution was used with an elution program of 0–45 min. The volume percentage of mobile phase B was 5–99%. The gradient elution parameters were as follows:

[0058] time The percentage of mobile phase B in the total volume of the mobile phase. 0min~5min 5％～25％ 5 min ~ 15 min 25％～30％ 15–45 min 30％～99％

[0059] Gradient elution is a linear gradient change.

[0060] The flow rate was 0.4 mL / min; the injector temperature was 10℃; the injection volume was 2 μL; the column temperature was 40℃; and the UV detection wavelength range was 190 nm-500 nm.

[0061] Mass spectrometry conditions are:

[0062] An electrospray ionization (ESI) source was used, with both positive and negative ion detection modes. The scanning mode was full scan, and the scanning range was 50-1500 Da. Ion source parameters: ion source temperature 120℃, carrier gas and desolvation gas were nitrogen, cone gas flow rate was 50 L / h, desolvation gas temperature was 450℃, desolvation gas flow rate was 800 L / h, and cone voltage was 40 V; in positive and negative ion detection modes, the capillary voltages were +3.0 KV and -2.5 KV, respectively; the collision-induced dissociation gas was argon (99.95%); the low collision energy was 6 eV, and the high collision energy range was 12-45 eV.

[0063] Step 3 involves processing the test results obtained in Step 2, including:

[0064] S1: The test results include the test results in positive ion mode and the test results in negative ion mode. The test results in positive ion mode and the test results in negative ion mode are processed separately to obtain the results in positive ion mode and the results in negative ion mode.

[0065] The results in the positive ion mode and / or negative ion mode were processed using ChemFinder for Office 22.0.0, Compound Discover 2.0, and Xcalibur 3.2 software, respectively, to obtain the retention time, molecular formula, mass-to-charge ratio, and primary and secondary mass spectrometry fragment information of the compounds.

[0066] S2: Compare the results under positive ion mode and negative ion mode with the database respectively to determine the chemical composition of Yangxin Dawaimishike Honey Cream;

[0067] If the results are consistent with the data information in the database (i.e., retention time, molecular formula, mass-to-charge ratio, and strength, etc.), it indicates that the Yangxin Dawa Yimishike Honey Ointment contains this compound.

[0068] Analysis of results from Example 1:

[0069] Figure 1 and Figure 2 The figures shown are the base peak diagrams (BPC) in positive ion mode and negative ion mode of the Yangxin Dawa Yimisik Honey Paste in Example 1.

[0070] The results of the analysis and identification of the compounds in positive ion mode are shown in Table 1, and the results of the analysis and identification in negative ion mode are shown in Table 2.

[0071] Table 1

[0072]

[0073]

[0074]

[0075]

[0076] Table 2

[0077]

[0078]

[0079]

[0080]

[0081] It can be seen that 101 compounds were preliminarily identified in the Yangxin Dawa Yimishik Honey Ointment of Example 1, including 7 silkworm cocoons, 5 sandalwoods, 15 cinnamons, 9 box fruit vines, 15 roses, 7 large-leaved blood-tonifying herbs, 4 lavender, 7 musks, 31 saffrons, 30 fragrant orchids, 10 amla fruits, and 10 bamboo shavings.

Claims

1. A method for detecting the chemical components of a heart-healthy Dawae Misik honey paste, characterized by, It comprises the following steps: The UPLC method is used for gradient elution of Yangxindawei Micexike honey ointment, and then the Q-TOF method is used for testing; The chromatographic conditions used in the UPLC method are as follows: The mobile phase A is 0.1% formic acid aqueous solution, and the percentage refers to the volume ratio of formic acid to formic acid aqueous solution. The mobile phase B is acetonitrile. Gradient elution is used, and the parameters of the gradient elution are as follows: The mass spectrometry conditions used in the Q-TOF method are as follows: The ion source is electrospray, and the positive ion detection mode and / or the negative ion detection mode are used. The ion source temperature is 120℃, the desolvation gas temperature is 450℃, the desolvation gas flow rate is 800L / h, the capillary voltage is +3.0KV in the positive ion detection mode, and the capillary voltage is -2.5KV in the negative ion detection mode.

2. The method as claimed in claim 1, wherein the chemical constituents of the Naga Dalai Mi Sik Meong Pem are detected. In the UPLC method, the chromatographic column is a C18 chromatographic column; And / or, the flow rate of the mobile phase is 0.4mL / min; And / or, the injection temperature is 10℃; And / or, the injection volume is 2μL; And / or, the column temperature of the chromatographic column is 40℃; And / or, the detection wavelength is 190-500nm; And / or, the gradient elution is linear gradient change.

3. The method as claimed in claim 2, wherein the said chemical constituents of the Naga Dalavoy Mishrik Panchamrit are detected by the method of HPLC. The C18 chromatographic column is a Waters Xtimate BEH C18 chromatographic column. In the Waters Xtimate BEH C18 chromatographic column, the inner diameter of the chromatographic column is preferably 4.6mm, the column length is preferably 250mm, and the particle size of the packing is preferably 5μm.

4. The method as claimed in claim 1, wherein the chemical constituents of the Naga Dalai Mi Sik Meong Pem are detected. The Yangxindawei Micexike honey ointment is loaded in the form of a Yangxindawei Micexike honey ointment solution. The preparation method of the Yangxindawei Micexike honey ointment solution preferably comprises mixing the Yangxindawei Micexike honey ointment with a methanol aqueous solution.

5. The method as claimed in claim 4, wherein the chemical constituents of the Naga Dalai Mi Sik Meong Pem are detected. The volume percentage of methanol in the methanol aqueous solution is 40-60%, for example, 50%. The volume percentage refers to the percentage of the volume of methanol in the volume of the methanol aqueous solution. And / or, the mixing method is ultrasonic mixing; And / or, the mixing time is 20-50min, for example, 30min; And / or, after the mixing treatment, a step of removing impurities is further included. The impurity removal is preferably filter membrane impurity removal, and the filter hole of the filter membrane is preferably 0.22μm.

6. The method as claimed in any one of claims 1 to 5, wherein the chemical components of the Nang Chhai Dawai Mi Sik honey paste are detected. In the Q-TOF method, the desolvation gas is nitrogen; And / or, the carrier gas is nitrogen; And / or, the cone voltage is 40V; And / or, the cone gas flow rate is 50L / h; And / or, the collision-induced dissociation gas is argon, and the purity of the argon can be 99.95%; And / or, the low collision energy is 6eV; And / or, the high collision energy is 12-45eV.

7. The method as claimed in any one of claims 1 to 5, wherein the chemical components of the Nang Chhai Dawai Mi Sik honey paste are detected by the method of claim 6. The chromatograph used in the UPLC method can be a 2998 type ultra-high performance liquid chromatograph; And / or, the mass spectrometer used in the Q-TOF method is an Xevo G2-S Q-TOF type high-resolution time-of-flight mass spectrometer.

8. The method as claimed in any one of claims 1 to 5, wherein the chemical components of the Nang Chai Dawei Mi Cike ointment are detected. The chemical component detection method of the Yangxindawei Micexike honey ointment further comprises a data processing step.

9. The method as claimed in claim 8, wherein the chemical constituents of the Naga Dalai Mi Sik Meong Pung are detected. The data processing step comprises: S1: processing the test results in the positive ion detection mode and / or the test results in the negative ion detection mode to obtain results in the positive ion mode and / or results in the negative ion mode; S2: comparing the results in the positive ion mode and / or the results in the negative ion mode with a database respectively to determine the chemical components in the Yanguan Dawai Michong Gao.

10. The method as claimed in claim 9, wherein the chemical constituents of the Naga Dalai Mi Sik Meong Pung are detected. The database is information of the Yanguan Dawai Michong Gao in one or more of Scifinder database, TCMID database, TCM@Taiwan database, TCMSP database and CNKI database; In step S1, the results in the positive ion mode and / or the results in the negative ion mode are processed by using ChemFinder for Office 22.0.0 software, Compound Discover 2.0 software and Xcalibur 3.2 software respectively to obtain the retention time, molecular formula, mass-to-charge ratio, primary and secondary mass spectrum fragment information of the compound.

Images