Quality detection method of Juanbi decoction

Abstract

The application provides a quality detection method of Juanbi decoction, and belongs to the technical field of medicine detection. The quality detection method is to prepare a test sample solution of Juanbi decoction, and then to detect the quality of the Juanbi decoction by using high performance liquid chromatography tandem mass spectrometry with acetonitrile (A)-0.04-0.06wt% formic acid aqueous solution (B) as a mobile phase. The method of liquid mass connection is used to qualitatively analyze the chemical components of the substance reference of Juanbi decoction, to provide a scientific basis for the substance basis research of the substance reference of Juanbi decoction, and to clarify the main chemical components.

Description

Technical Field

[0001] This invention relates to drug testing technology, and more particularly to a method for quality testing of Juanbi Decoction. Background Technology

[0002] Juanbi Decoction is composed of Notopterygium root, Angelica pubescens root, Gentiana macrophylla root, Piper kadsura stem, Ligusticum chuanxiong rhizome, Cinnamon bark, Vaccaria root (processed with vinegar), Aucklandia root, Angelica sinensis root, Morus alba twig, and Glycyrrhiza uralensis root (processed). It is indicated for stroke with body aches and pains, stiffness in the neck and back, cold and numb hands and feet, heaviness in the lower back and knees, and difficulty in movement. Juanbi Decoction is the 79th of the 100 classic prescriptions in the "Catalogue of Famous Ancient Prescriptions (First Batch)". Classic prescriptions have proven clinical efficacy. Developing these prescriptions into convenient and easy-to-take Chinese medicine preparations using modern science and technology not only preserves traditional Chinese medicine but also better promotes their clinical application.

[0003] High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) offers high efficiency and speed, providing accurate molecular structure information and enabling the acquisition of first-order and multi-order mass spectrometry data for individual compounds. This provides a valuable technical means for the multi-index component analysis of complex traditional Chinese medicine systems. Juanbi Decoction is composed of 11 traditional Chinese medicines, resulting in an extremely complex composition. Currently, it is impossible to comprehensively monitor the quality of Juanbi Decoction, let alone perform qualitative analysis of its chemical components. Therefore, it is necessary to develop a quality detection method for Juanbi Decoction to more comprehensively assess its quality. Summary of the Invention

[0004] To address the above problems, this invention provides a method for quality testing of Juanbi Decoction.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for quality testing of a traditional Chinese medicine formula called Juanbi Decoction includes the following steps:

[0007] S1. Prepare the test solution using Juanbi Decoction;

[0008] S2. The quality of Juanbi Decoction was determined by high performance liquid chromatography-tandem mass spectrometry using the test solution.

[0009] The mobile phase A for the high-performance liquid chromatography detection is acetonitrile, and the mobile phase B is an aqueous solution of formic acid with a concentration of 0.04-0.06 wt%.

[0010] The elution method for the high-performance liquid chromatography detection is gradient elution;

[0011] The elution conditions for the gradient elution are as follows:

[0012] 0–12 min, 1% → 11% mobile phase A, 99% → 89% mobile phase B;

[0013] 12–14 min, 11% → 13% mobile phase A, 89% → 87% mobile phase B;

[0014] 14–15 min, 13% → 14% mobile phase A, 87% → 86% mobile phase B;

[0015] 15–18.5 min, 14% → 16% mobile phase A, 86% → 84% mobile phase B;

[0016] 18.5–21.5 min, 16% mobile phase A, 84% mobile phase B;

[0017] 21.5–25 min, 16% → 19% mobile phase A, 84% → 81% mobile phase B;

[0018] 25–26 min, 19% → 26% mobile phase A, 81% → 74% mobile phase B;

[0019] 26–32 min, 26% mobile phase A, 74% mobile phase B;

[0020] 32–33.5 min, 26% → 44% mobile phase A, 74% → 56% mobile phase B;

[0021] 33.5–45 min, 44% → 46% mobile phase A, 56% → 54% mobile phase B;

[0022] 45–46 min, 46% → 66% mobile phase A, 54% → 34% mobile phase B;

[0023] 46–55 min, 66% mobile phase A, 34% mobile phase B.

[0024] Furthermore, the detection wavelength of the high-performance liquid chromatography is 290–330 nm.

[0025] Furthermore, the flow rate for the high-performance liquid chromatography detection is 0.38–0.45 mL / min, and the column temperature is 38–45 °C.

[0026] Furthermore, the chromatographic column used for the high-performance liquid chromatography detection is packed with octadecylsilane-bonded silica gel.

[0027] Furthermore, the mass spectrometry detection is performed in both positive ion mode and negative ion mode.

[0028] Furthermore, the mass spectrometry detection conditions are as follows: drying gas temperature 345–355℃, drying gas flow rate 8–12 L / min, nebulizing gas pressure 34–36 psi, sheath gas temperature 345–355℃, sheath gas flow rate 11–13 L / min, capillary voltage in positive ion mode 3400–3600 V, and capillary voltage in negative ion mode 3400–3600 V.

[0029] Furthermore, during the mass spectrometry detection process, the first-stage mass spectrometry uses MS mode with a mass scan range of 100–1200 m / z, and the second-stage mass spectrometry uses Auto MSMS mode.

[0030] Furthermore, the test solution is prepared by adding solid Juanbi Decoction to a methanol-water solution, sonicating, filtering, and then obtaining the solution.

[0031] The concentration of the methanol aqueous solution is 70–85 vol%.

[0032] The solid form of Juanbi Decoction is Juanbi Decoction dry paste powder, and the weight-volume ratio of Juanbi Decoction to methanol aqueous solution is 0.9-1.1g:10mL.

[0033] Furthermore, the test solution is prepared by taking an extract or concentrate of Juanbi Decoction, adding methanol, sonicating, and filtering.

[0034] Furthermore, the volume ratio of Juanbi Decoction extract / Juanbi Decoction concentrate to methanol is 1:3.8–4.2.

[0035] The beneficial effects of the quality testing method for Juanbi Decoction of the present invention are as follows:

[0036] This invention employs liquid chromatography-mass spectrometry (LC-MS) to qualitatively analyze the chemical composition of the material basis of Juanbi Decoction, providing a scientific basis for the study of the material basis of Juanbi Decoction and clarifying its main chemical composition.

[0037] This invention employs liquid chromatography-mass spectrometry (LC-MS) to qualitatively analyze the chemical composition of the reference material of Juanbi Decoction. The analysis is highly efficient and fast, and can obtain accurate molecular structure information of Juanbi Decoction. It can also obtain first-order and multi-order mass spectrometry information of each compound, providing a good technical means for the multi-index component analysis of the complex traditional Chinese medicine system of Juanbi Decoction.

[0038] This invention employs liquid chromatography-mass spectrometry (LC-MS) to qualitatively analyze the chemical composition of the reference material for Juanbi Decoction, providing a scientific basis for the study of the material basis of Juanbi Decoction and elucidating its main chemical composition.

[0039] The quality testing method of this invention can detect almost all the medicinal ingredients in the entire formula of Juanbi Decoction;

[0040] This invention utilizes the different physicochemical properties of the various active ingredients in Juanbi Decoction and changes the chromatographic conditions to achieve the goal of effectively separating the various active ingredients in Juanbi Decoction.

[0041] By selecting specific chromatographic and mass spectrometric conditions, this invention can obtain a better chromatogram of Juanbi Decoction.

[0042] The quality testing method of the present invention also has good feasibility, stability and reproducibility;

[0043] The quality detection method of this invention can detect and clarify the medicinal flavor source and qualitative analysis of 41 peak substances. Among them, 11 coumarin compounds were identified, namely, imperatorin isomer, imperatorin, notopterygium alcohol isomer, and notopterygium alcohol, which are characteristic components of Notopterygium incisum; isoimperatorin, angelicol G / D / B, angelicol A, angelicol C / F / E / H, angelicin, osthol, and dihydroaperitol angelic acid ester, which are characteristic components of Angelica pubescens; 5 iridoid glycosides were identified, namely, loganic acid, gentianin, gentiopicrin, 6'-O-β-D-glucose gentianin, and gentianin, which are characteristic components of Gentiana macrophylla; and 5 phenylpropanoids were identified. Among the compounds identified, chlorogenic acid is a common component of Angelica sinensis, Angelica pubescens, Notopterygium incisum, and Aucklandia lappa; neochlorogenic acid is a common component of Angelica pubescens and Aucklandia lappa; and isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C are specific components of Aucklandia lappa. Five flavonoids were identified: morin A is specific to mulberry twig; glycyrrhizin, apigenin / isoapigenin, isoglycyrrhizin, and gentianin are specific to licorice. Three organic acids were identified: vanillic acid is specific to Angelica sinensis; glycyrrhizic acid is specific to licorice; and ferulic acid is a common component of Angelica sinensis, Angelica pubescens, and Ligusticum chuanxiong. One amino acid was identified: tryptophan is specific to Angelica sinensis. One saponin was identified: adenosine is specific to Angelica sinensis.

[0044] The quality detection method of this invention can obtain the presence and characteristics of common characteristic peaks in the chromatogram of Juanbi Decoction, which can comprehensively monitor the quality of Juanbi Decoction raw materials, semi-finished products and finished products. By comparing the similarity of chromatographic characteristics, the quality of Juanbi Decoction can be evaluated, and its stability and consistency can be examined, which makes up for the deficiencies of the current quality control methods. At the same time, it can also monitor the stability of Juanbi Decoction production process, and ensure its quality is stable, uniform and controllable.

[0045] The quality testing method of this invention has improved the quality control standards of Juanbi Decoction by obtaining its chromatogram, effectively preventing product counterfeiting and ensuring the normal production and circulation order of Juanbi Decoction. Based on this invention, a correlation study can also be conducted between the chromatogram information detected by liquid chromatography-mass spectrometry and the pharmacodynamic information, thereby further clarifying the correlation between the intrinsic chemical components of Juanbi Decoction and the efficacy of the preparation.

[0046] The quality testing method described in this invention can be used for quality evaluation or control throughout the entire process of research, development, production, and clinical application of Juanbi Decoction. Attached Figure Description

[0047] Figure 1 These are the UPLC-UV chromatogram and UPLC-MS total ion chromatogram of Juanbi Decoction in Example 1 of the present invention, wherein from top to bottom are the UPLC-MS total ion chromatogram (positive ion mode TIC chromatogram), the UPLC-MS total ion chromatogram (negative ion mode TIC chromatogram) and the UPLC-UV chromatogram of Juanbi Decoction (chromatogram under UV 310nm conditions).

[0048] Figure 2 This is the TOF-MS first-order mass spectrum of peaks 1 to 41 of the Juanbi Decoction in Example 1 of this invention;

[0049] Figure 3 This is the MS / MS secondary mass spectrum of peaks 1 to 41 of the Juanbi Decoction in Example 1 of this invention. Detailed Implementation

[0050] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0051] Example 1: A method for quality testing of a traditional Chinese medicine formula called Juanbi Decoction

[0052] In this embodiment, the quality of Juanbi Decoction is tested using a reference material. The specific preparation method of the Juanbi Decoction reference material is as follows:

[0053] Take 3.75g of Notopterygium root, 3.75g of Angelica pubescens root, 3.75g of Gentiana macrophylla root, 7.50g of Piper kadsura stem, 2.63g of Ligusticum chuanxiong rhizome, 1.88g of Cinnamon bark, 3.00g of Vinegar-processed Boswellia carterii root, 3.00g of Aucklandia lappa root, 11.25g of Angelica sinensis root, 11.25g of Morus alba branch, and 1.88g of prepared Glycyrrhiza uralensis root. Place them in a clay pot, add water and decoct twice. For the first decoction, add 10 times the amount of water, soak for 30 minutes, bring to a boil over high heat, and simmer over low heat for 30 minutes. Filter and reserve the filtrate. For the second decoction, add 8 times the amount of water, bring to a boil over high heat, and simmer over low heat for 20 minutes. Filter and combine the filtrates. Concentrate under reduced pressure to a concentration with a ratio of approximately 0.6:1 (g:mL) of the amount of medicinal slices added to the volume of the concentrated decoction. Freeze-dry to obtain a dry paste, which is the material basis of Juanbi Decoction (i.e., dry paste powder).

[0054] This embodiment describes a method for quality testing of a traditional Chinese medicine formula called Juanbi Decoction, which specifically includes the following steps:

[0055] S1, Solution Preparation

[0056] S11. Preparation of the test solution

[0057] Take about 1g of the substance standard of Juanbi Decoction, accurately add 10mL of 80vol% methanol aqueous solution, sonicate (power 800W, frequency 40kHz) for 30 minutes, shake well, filter, and take the filtrate to obtain the test solution.

[0058] Meanwhile, in the preparation of the test solution, the extract of Juanbi Decoction can also be used to prepare the test solution. The preparation method is as follows: Take 3.75g of Qianghuo, 3.75g of Duhuo, 3.75g of Qinjiu, 7.50g of Haifengteng, 2.63g of Chuanxiong, 1.88g of Rougui, 3.00g of vinegar-processed Ruxiang, 3.00g of Muxiang, 11.25g of Danggui, 11.25g of Sangzhi, and 1.88g of Zhigancao. Place them in a clay pot, add water and decoct twice. For the first decoction, add 10 times the amount of water, soak for 30 minutes, bring to a boil over high heat, simmer over low heat for 30 minutes, filter, and reserve the filtrate. For the second decoction, add 8 times the amount of water, bring to a boil over high heat, simmer over low heat for 20 minutes, filter, combine the filtrates, and obtain the extract. Accurately measure 5 mL of the extract and place it in a stoppered conical flask. Accurately add 20 mL of methanol, sonicate (800 W power, 40 kHz frequency) for 30 minutes, shake well, filter, and collect the filtrate as the test solution.

[0059] Alternatively, the test solution can be prepared using concentrated Juanbi Decoction. The preparation method is as follows: Take 3.75g of Qianghuo, 3.75g of Duhuo, 3.75g of Qinjiu, 7.50g of Haifengteng, 2.63g of Chuanxiong, 1.88g of Rougui, 3.00g of vinegar-processed Ruxiang, 3.00g of Muxiang, 11.25g of Danggui, 11.25g of Sangzhi, and 1.88g of Zhigancao. Place them in a clay pot, add water and decoct twice. For the first decoction, add 10 times the amount of water, soak for 30 minutes, bring to a boil over high heat, simmer over low heat for 30 minutes, filter, and reserve the filtrate. For the second decoction, add 8 times the amount of water, bring to a boil over high heat, simmer over low heat for 20 minutes, filter, combine the filtrates, and concentrate under reduced pressure to a concentration of approximately 0.6:1 (g:mL). Accurately measure 2 mL of the concentrated solution and place it in a stoppered conical flask. Accurately add 8 mL of methanol, sonicate (800 W power, 40 kHz frequency) for 30 minutes, shake well, filter, and collect the filtrate to obtain the test solution.

[0060] S12. Preparation of single-herb control solution

[0061] Take appropriate amounts of each herb powder (passed through a No. 3 sieve) (including approximately 1g of Notopterygium root, 1g of Angelica pubescens root, 1g of Gentiana macrophylla root, 3g of Angelica sinensis root, 0.7g of Ligusticum chuanxiong rhizome, 2g of Piper kadsura stem, 0.8g of Boswellia carterii root, 0.5g of Cinnamomum cassia root, 0.8g of Aucklandia lappa root, 3g of Morus alba branch, and 0.5g of Glycyrrhiza uralensis root), place them separately in round-bottom flasks, add 50mL of water, decoct for 30 minutes, cool, centrifuge (5000 rpm) for 5 minutes, collect the supernatant, evaporate to dryness, dissolve the residue in 50mL of 80 vol% methanol aqueous solution, transfer to a 100mL stoppered conical flask, seal tightly, sonicate (800W power, 40kHz frequency) for 30 minutes, remove, cool, shake well, filter, and collect the filtrate to obtain the single herb reference solution.

[0062] S13. Preparation of reference solution

[0063] Take an appropriate amount of ferulic acid reference standard, accurately weigh it, place it in a brown volumetric flask, add 75 vol% methanol aqueous solution to prepare a solution containing 75 μg of reference standard per 1 mL, which is the reference standard solution.

[0064] S2, Quality Inspection

[0065] The test solution was subjected to liquid chromatography-mass spectrometry (LC-MS) analysis to obtain the UPLC-UV chromatogram and UPLC-MS total ion chromatogram (positive and negative ion mode TIC chromatograms) of Juanbi Decoction. This is the chromatogram of Juanbi Decoction. See details below. Figure 1 . Figure 1 The substances in chromatographic peaks 1 to 41 marked in the figure were all well separated and detected.

[0066] The chromatographic conditions for liquid chromatography-mass spectrometry (LC-MS) analysis are as follows:

[0067] The chromatographic column was packed with octadecylsilane-bonded silica gel (Waters Acquity). BEH C18 chromatographic column, with a length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.7 μm;

[0068] The flow rate was 0.4 mL / min;

[0069] The column temperature is 40℃;

[0070] The detection wavelength is 310nm;

[0071] The injection volume was 3 μL;

[0072] Acetonitrile was used as mobile phase A, and 0.05 wt% formic acid aqueous solution was used as mobile phase B;

[0073] The elution method is gradient elution, and the elution conditions are:

[0074] 0–12 min, 1% → 11% mobile phase A, 99% → 89% mobile phase B;

[0075] 12–14 min, 11% → 13% mobile phase A, 89% → 87% mobile phase B;

[0076] 14–15 min, 13% → 14% mobile phase A, 87% → 86% mobile phase B;

[0077] 15–18.5 min, 14% → 16% mobile phase A, 86% → 84% mobile phase B;

[0078] 18.5–21.5 min, 16% mobile phase A, 84% mobile phase B;

[0079] 21.5–25 min, 16% → 19% mobile phase A, 84% → 81% mobile phase B;

[0080] 25–26 min, 19% → 26% mobile phase A, 81% → 74% mobile phase B;

[0081] 26–32 min, 26% mobile phase A, 74% mobile phase B;

[0082] 32–33.5 min, 26% → 44% mobile phase A, 74% → 56% mobile phase B;

[0083] 33.5–45 min, 44% → 46% mobile phase A, 56% → 54% mobile phase B;

[0084] 45–46 min, 46% → 66% mobile phase A, 54% → 34% mobile phase B;

[0085] 46–55 min, 66% mobile phase A, 34% mobile phase B.

[0086] The mass spectrometry conditions for liquid chromatography-mass spectrometry (LC-MS) are as follows:

[0087] Mass spectrometry was performed in both positive and negative ion modes, with a drying gas temperature of 350℃, a drying gas flow rate of 10 L / min, a nebulizer gas pressure of 35 psi, a sheath gas temperature of 350℃, a sheath gas flow rate of 12 L / min, and capillary voltages of 3500 V (positive mode) and 3500 V (negative mode). Primary mass spectrometry was performed in MS mode with a mass scan range of 100–1200 m / z. Secondary mass spectrometry was performed in Auto MSMS mode, with the collision voltage adjusted appropriately for different compounds. The obtained LC-MS data were acquired using Agilent MassHunter (B.08.00) software. Data processing was performed using Agilent Qualitative Navigator (B.08.00) and Qualitative Workflows (B.08.00) software.

[0088] Simultaneously, liquid chromatography-mass spectrometry (LC-MS) analysis was performed on each single-herb reference solution and each reference standard solution, and the results were compared and analyzed with those of the test solution to clarify the herb origin and characterize the main chromatographic peaks of 41 peak substances. Among them, the peaks of 10 herbs, namely Notopterygium incisum, Gentiana macrophylla, Angelica pubescens, Ligusticum chuanxiong, Angelica sinensis, Vinegar-processed Boswellia carterii, Morus alba twigs, Glycyrrhiza uralensis (processed), Cinnamomum cassia, Aucklandia lappa, and Piper kadsura, could be identified in the LC-MS fingerprint chromatograms of the material reference.

[0089] First-order mass spectrometry analysis was performed using high-resolution TOF-MS for precise mass number determination, and the results were analyzed... Figure 1 Compounds in peaks 1-41 were analyzed, and a total of 41 compounds were identified. The results are shown in Table 1 and Table 2. Figure 2 As shown, these compounds, in positive mode, can generate a molecular ion peak [M+H]. + , and [M+Na] + [M+K] + In isoadductor mode, a molecular ion peak [MH] can be generated; however, in negative mode, a molecular ion peak can be generated. - [M+Cl] - [M+HCOO] - Isoadated ions. Based on this information about adducted ions, the precise molecular weight and molecular formula of the compound can be accurately deduced (mass measurement error ≤ 5 ppm, indicating correct matching results), thus aiding in subsequent structural identification. Among these, in... Figure 2 In the primary mass spectrometry information of the 41 compound peaks, most compounds responded in both positive and negative ion modes, while some compounds could only be detected in one mode.

[0090] The test solution was further analyzed by Auto MS / MS (negative mode predominant) under the same chromatographic conditions, i.e., secondary mass spectrometry analysis, to confirm the structures of some compounds in Table 1. The results are shown in Table 1 and... Figure 3 As shown, the results of the first-stage mass spectrometry identification in Table 1 were further confirmed to some extent by the second-stage mass spectrometry analysis.

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100] UPLC-QTOF-MS analysis of the reference solution of Juanbi Decoction revealed 41 components. By comparing the retention times with literature data and reference standards, and analyzing the accurate molecular weights provided by UPLC-QTOF-MS, 38 of these components were identified. The medicinal ingredients from which the compounds originated were also attributed.

[0101] a) Coumarins

[0102] Eleven coumarin compounds were identified, among which the characteristic components of Notopterygium incisum were imperatorin isomer, imperatorin, noteratorin alcohol isomer, and noteratorin alcohol; the characteristic components of Angelica pubescens were isoimperatorin, angelicol G / D / B, angelicol A, angelicol C / F / E / H, angelicin, osthol, and dihydroaperitol angelicate.

[0103] Furthermore, the accurate chemical structures of four compounds—notopterygium alcohol, osthol, dihydroaperitol angelic acid ester, and purslane—were confirmed by comparison with standard samples.

[0104] b. Iridoid glycosides

[0105] Five iridoid glycosides were identified, namely loganic acid, gentiopicrin, gentiopicrin, 6'-O-β-D-glucose gentiopicrin, and gentiopicrin as the main characteristic components. Among them, gentiopicrin and loganic acid were confirmed to have accurate chemical structures by comparison with standards.

[0106] c. Phenylpropanoids

[0107] Five phenylpropanoid compounds were identified. Chlorogenic acid is a common component of Angelica sinensis, Angelica pubescens, Notopterygium incisum, and Aucklandia lappa. Neochlorogenic acid is a common component of Angelica pubescens and Aucklandia lappa. Isochlorogenic acid A, Isochlorogenic acid B, and Isochlorogenic acid C are specific components of Aucklandia lappa.

[0108] d. Flavonoids

[0109] Five flavonoids were identified, with morin A being a specific component of mulberry twig, and glycyrrhizin, apigenin / isoapigenin glycyrrhizin, isoglycyrrhizin, and gentiopicrin being specific components of licorice. The accurate chemical structure of glycyrrhizin was confirmed by comparison with standards.

[0110] f. Organic acids

[0111] Three organic acid compounds were identified: vanillic acid, a specific component of Angelica sinensis, and glycyrrhizic acid, a specific component of Glycyrrhiza uralensis. Ferulic acid is a common component of Angelica sinensis, Angelica pubescens, and Ligusticum chuanxiong. The accurate chemical structure of ferulic acid was confirmed by comparison with standards.

[0112] g, amino acids

[0113] One amino acid compound was identified, and tryptophan is a unique component of Angelica sinensis.

[0114] h, glycosides

[0115] One saponin-like compound was identified, and adenosine is a specific component of Angelica sinensis.

[0116] The quality testing method for Juanbi Decoction in this embodiment can be used for quality evaluation or control throughout the entire process of research, development, production, and clinical application of Juanbi Decoction.

[0117] Examples 2-7: Quality Testing Methods for Juanbi Decoction

[0118] Examples 2-7 are quality testing methods for Juanbi Decoction. Their steps are basically the same as those in Example 1, with the only difference being the process parameters. For details, please refer to Tables 2 and 3:

[0119] Table 2. Summary of process parameters in Examples 2-3

[0120]

[0121]

[0122] Table 3. Summary of process parameters in Examples 4-7

[0123]

[0124] The process steps and parameters for other parts of Examples 2 to 7 are the same as those in Example 1, and the test results obtained are also similar to those in Example 1, so they will not be repeated here.

[0125] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for quality testing of a traditional Chinese medicine formula called Juanbi Decoction, characterized in that, The quality inspection method includes the following steps: S1. When Juanbi Decoction is in solid form, take the solid Juanbi Decoction and add it to a methanol aqueous solution with a concentration of 70~85 vol%, sonicate, filter, and prepare the test solution; the solid Juanbi Decoction is Juanbi Decoction dry extract powder, and the weight-volume ratio of Juanbi Decoction to methanol aqueous solution is 0.9~1.1 g: 10 mL; When Juanbi Decoction is in liquid form, take Juanbi Decoction extract or Juanbi Decoction concentrate, add methanol, sonicate, filter, and obtain the test solution; the volume ratio of Juanbi Decoction extract / Juanbi Decoction concentrate to methanol is 1:3.8~4.

2. S2. The quality of Juanbi Decoction was determined by high performance liquid chromatography-tandem mass spectrometry using the test solution. The mobile phase A for the high-performance liquid chromatography detection is acetonitrile, and the mobile phase B is an aqueous solution of formic acid with a concentration of 0.04~0.06wt%. The elution method for the high-performance liquid chromatography detection is gradient elution; The elution conditions for the gradient elution are as follows: 0~12min, 1%→11% mobile phase A, 99%→89% mobile phase B; 12~14 min, 11%→13% mobile phase A, 89%→87% mobile phase B; 14~15 min, 13%→14% mobile phase A, 87%→86% mobile phase B; 15~18.5min, 14%→16% mobile phase A, 86%→84% mobile phase B; 18.5~21.5 min, 16% mobile phase A, 84% mobile phase B; 21.5~25 min, 16%→19% mobile phase A, 84%→81% mobile phase B; 25~26 min, 19%→26% mobile phase A, 81%→74% mobile phase B; 26~32 min, 26% mobile phase A, 74% mobile phase B; 32~33.5 min, 26%→44% mobile phase A, 74%→56% mobile phase B; 33.5~45 min, 44%→46% mobile phase A, 56%→54% mobile phase B; 45~46 min, 46%→66% mobile phase A, 54%→34% mobile phase B; 46~55 min, 66% mobile phase A, 34% mobile phase B; The detection wavelength for the high-performance liquid chromatography (HPLC) is 290~330nm; the chromatographic column is a Waters Acquity UPLC®BEH C18 column with a length of 100mm, an inner diameter of 2.1mm, and a particle size of 1.7μm. The mass spectrometry detection conditions are as follows: drying gas temperature 345~355℃, drying gas flow rate 8~12L / min, nebulizing gas pressure 34~36psi, sheath gas temperature 345~355℃, sheath gas flow rate 11~13L / min, capillary voltage in positive ion mode 3400~3600V, and capillary voltage in negative ion mode 3400~3600V. During the mass spectrometry detection process, the first-stage mass spectrometry uses MS mode with a mass scan range of 100~1200m / z, and the second-stage mass spectrometry uses Auto MSMS mode. The quality testing method detected a total of 41 peak substances, including imperatorin isomer, imperatorin, notopterygol isomer, notopterygol, isopyramekibrin, angelicol G / D / B, angelicol A, angelicol C / F / E / H, osthol, dihydroaperitol angelic acid ester, loganic acid, swertiamarin, gentiopicrin, swertiamarin, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, morin A, and vanillic acid.

2. The method for quality testing of the Juanbi Decoction according to claim 1, characterized in that, The high-performance liquid chromatography (HPLC) detection was performed at a flow rate of 0.38–0.45 mL / min and a column temperature of 38–45 °C.

3. The method for quality testing of the Juanbi Decoction according to claim 1, characterized in that, The mass spectrometry detection was performed in both positive ion mode and negative ion mode.

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