Method for detecting index components of Zixue Powder and application thereof

Abstract

This invention relates to the field of analytical technology for traditional Chinese medicine preparations, and particularly to a method for detecting the indicator components of Zixue Powder and its application. The method provided by this invention is ultra-high performance liquid chromatography (UHPLC), wherein the chromatographic conditions include: mobile phase A being an aqueous solution containing 0.1%–0.2% v / v formic acid, and mobile phase B being methanol or acetonitrile, with linear gradient elution. This method can achieve good separation between linalool, isoflavone, angoloside C, cimicifugain, glycyrrhizin, cinnamic acid, isoglycyrrhizin, harpagoside, and glycyrrhizic acid, as well as between these components and other compounds, obtaining chromatographic peaks with high resolution and good peak shape. Combined with other detection methods or chromatographic conditions, further qualitative and quantitative analysis of the above-mentioned indicator components can be performed, providing a basis for the scientific evaluation and effective control of the quality of Zixue Powder.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis of traditional Chinese medicine preparations, and particularly to a method for detecting index components of Zixue Powder and its application. Background Art

[0002] Zixue Powder is composed of 16 herbs, namely gypsum, calcite, magnetite, talc, scrophulariaceae, rhizoma cimicifugae, licorice, lilac, costus root, lignum aquilariae resinatum, mirabilite, nitre, concentrated powder of buffalo horn, antelope horn powder, artificial musk, and cinnabar. It is a representative formula of the cold-resuscitation agent, with the effects of clearing heat and resuscitating, stopping convulsions and calming the mind. Clinically, it is used to treat infantile high fever and convulsions, septic shock, etc. Zixue Powder has a large number of herbs in its formula and complex components, but the chemical basic research is relatively weak. At present, the 2020 edition of the Chinese Pharmacopoeia only conducts qualitative identification on licorice, rhizoma cimicifugae, and muskone in the formula, and the relevant literature only reports the content analysis of muskone, which cannot comprehensively reflect the quality of Zixue Powder. Therefore, it is necessary to establish a more comprehensive quality detection method to improve its quality standard, so as to more effectively monitor the quality of Zixue Powder. Summary of the Invention

[0003] In view of the above technical problems, the present invention provides a method for detecting index components of Zixue Powder and its application. The method for detecting index components can well separate the index components through specific liquid chromatography conditions, and then qualitative and quantitative analysis can be carried out. The obtained data can more comprehensively reflect the quality of Zixue Powder.

[0004] To achieve the above invention purpose, the embodiments of the present invention adopt the following technical solutions:

[0005] In the first aspect, the present invention provides a method for detecting index components of Zixue Powder. The method uses ultra-high performance liquid chromatography to separate the index components. The chromatographic conditions of the ultra-high performance liquid chromatography include:

[0006] Chromatographic column: reversed-phase chromatographic column;

[0007] Mobile phase A is an aqueous solution of formic acid containing 0.1% - 0.2% v / v, and mobile phase B is methanol or acetonitrile. Linear gradient elution is carried out, and the gradient elution program is:

[0008]

[0009]

[0010] Flow rate: 0.5 mL / min;

[0011] Column temperature: 40 - 50 °C.

[0012] The above chromatographic conditions can achieve good separation between (+)-Agarospirol, Isoferulic acid, Angelopolyside C, Prim-O-glucosylcimifugin, Liquiritin, Cinnamic acid, Isoliquiritin, Harpagide, Glycyrrhizic acid and other compounds, obtaining chromatographic peaks with high resolution and good peak shapes. Combining with other detection methods or chromatographic conditions, qualitative analysis and quantitative detection of the above index components can be further carried out, providing a basis for scientifically evaluating and effectively controlling the quality of Zixue Powder.

[0013] Combined with the first aspect, the index components include (+)-Agarospirol, Isoferulic acid, Angelopolyside C, Prim-O-glucosylcimifugin, Liquiritin, Cinnamic acid, Isoliquiritin, Harpagide, Glycyrrhizic acid.

[0014] The formula of Zixue Powder consists of three categories of drugs: animal, mineral, and plant. Among them, the proportion of mineral drugs is relatively large and the components are relatively simple, which are easy to control. The proportion of animal drugs is relatively small. Therefore, in this invention, the components in the plant drugs with a large proportion and complex components are selected as the key components to measure the quality of Zixue Powder. There are 6 plant medicinal materials in the formula of Zixue Powder, among which: Scrophulariae Radix nourishes yin and cools blood; Cimicifugae Rhizoma clears heat and detoxifies; Aucklandiae Radix, Caryophylli Flos, and Aquilariae Lignum promote qi and open the orifices; Glycyrrhizae Radix补脾益气, and when used together with other medicinal flavors, it plays a medicinal effect. The above 9 index components are all quality control indicators or active components of the 6 medicinal materials. Among them, (+)-Agarospirol is the index component of the medicinal material Aquilariae Lignum, Angelopolyside C, Cinnamic acid, and Harpagide come from the medicinal material Scrophulariae Radix, Prim-O-glucosylcimifugin comes from the medicinal material Cimicifugae Rhizoma, Isoferulic acid comes from Scrophulariae Radix and Cimicifugae Rhizoma, Liquiritin, Isoliquiritin, and Glycyrrhizic acid come from the medicinal material Glycyrrhizae Radix.

[0015] Moreover, there are literature reports that Isoferulic acid can inhibit the production of inflammatory protein MIP-2 to play an anti-infective role, and Prim-O-glucosylcimifugin, Isoliquiritin, and Glycyrrhizic acid can inhibit the NF-κB signal, further reducing the expression of cytokines such as IL-1β, TNF-α, and HMGB1 to play an anti-inflammatory role. In the research process of this invention, the gene targets of the above 9 index components were collected through the SwissTargetPrediction platform, and a total of 291 component targets were obtained. Using the GeneCards database, 3806 inflammation-related targets were collected, and the intersection targets of the two were 229. Through PPI network analysis, the 9 components mainly play an anti-inflammatory role through targets such as STAT3, MAPK3, EGFR, JUN, and VEGFA. The above research results show that the above 9 components have an important impact on the medicinal effect of Zixuecao. Therefore, this invention selects (+)-Agarospirol, Isoferulic acid, Angelopolyside C, Prim-O-glucosylcimifugin, Liquiritin, Cinnamic acid, Isoliquiritin, Harpagide, and Glycyrrhizic acid as the index quantitative components of Zixue Powder.

[0016] Preferably, the chromatographic column is ZORBAX SB-C18 (4.6×100mm, 1.8μm). Using this chromatographic column can obtain ideal resolution and there are more chromatographic peaks.

[0017] Preferably, mobile phase B is methanol. In this detection method, using methanol as the organic phase enables better separation between the chromatographic peaks of each indicator component.

[0018] Preferably, the column temperature is 45°C.

[0019] In conjunction with the first aspect, the method for detecting the index components also includes characterizing the index components using mass spectrometry to achieve qualitative analysis of the substances separated by liquid chromatography.

[0020] Preferably, the method for detecting the index components is implemented using an ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometer.

[0021] Preferably, the mass spectrometry conditions for the mass spectrometry method include: using a positive / negative ion switching scanning mode, and a capillary voltage of 3.00 kV (ESI). + ) and -2.50kV (ESI) - Ion source temperature 120℃, cone voltage 40V, cone gas flow rate 50L / h, nitrogen degassing temperature 500℃, degassing volumetric flow rate 800L / h, low collision energy 6eV, high collision energy 10~45eV; mass range: m / z 50-1200amu.

[0022] In conjunction with the first aspect, the specific operation of the indicator component detection method includes:

[0023] S1. Extract the Zixue powder sample to be tested with a 50%–75% v / v methanol aqueous solution to prepare the test solution; dissolve the reference standard of the indicator component with a 50%–75% v / v methanol aqueous solution to prepare the reference standard solution.

[0024] S2. The test solution and the reference solution are detected using the ultra-high performance liquid chromatography method.

[0025] Preferably, the extraction method is ultrasonic extraction, with an ultrasonic temperature of 60°C and an ultrasonic time of 20–40 min. More preferably, the ultrasonic time is 30 min.

[0026] Preferably, the ratio of the Zixue powder sample to the methanol aqueous solution is 1:(10-50), g:mL.

[0027] Preferably, the concentration of the methanol aqueous solution is 65% to 75% v / v. More preferably, it is 75% v / v.

[0028] Secondly, the present invention also provides the application of the above-mentioned index component detection method in the detection of the content of index components in Zixuesan.

[0029] In conjunction with the second aspect, the chromatographic conditions also include multi-wavelength switching, and the wavelength switching procedure is as follows:

[0030]

[0031] Methodological validation showed that the method for detecting the content of Zixue powder index components established using the above-mentioned index component detection methods has a good linear relationship, low detection limit and quantitation limit, and RSD values ​​of precision, stability and repeatability are all less than 3.0%. The recovery rate is between 92.5% and 105.4%. It can accurately and efficiently detect the above-mentioned index components in Zixue powder, providing a research basis for improving the quality control standards of Zixue powder.

[0032] The beneficial effects of this invention are as follows: The method for detecting indicator components provided by this invention can detect nine indicator components, including linalool, isoflavonic acid, angoloside C, glycyrrhizin, cinnamic acid, harpagoside, isoglycyrrhizin, and glycyrrhizic acid. This invention utilizes this detection method to characterize the chemical components of Zixuesan under specific chromatographic and mass spectrometric conditions using ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS), identifying 75 non-volatile components, including 19 triterpenes and their glycosides, 17 flavonoids, 13 chromogens, 6 organic acids, 6 sesquiterpenes, 5 phenylpropanoids, 4 monoterpenes, and small amounts of diterpenes, sugars, and phenolic glycosides. This detection method provided by this invention can also be used for the content detection of Zixuesan indicator components. This invention utilizes the detection method to establish, for the first time, a quantitative analysis method for multiple components of Zixue San based on UPLC-PDA technology, using linalool, isoflavonic acid, angoloside C, cimicifugain, glycyrrhizin, cinnamic acid, harpagoside, isoglycyrrhizin, and glycyrrhizic acid as indicators. The systematic methodological verification results show that the method is feasible, meets the requirements of quantitative analysis, and the analysis results can provide a reference for improving the quality standard of Zixue San. Attached Figure Description

[0033] Figure 1 The image shows the total ion chromatogram in positive ion mode for the Zixuesan sample in Example 1.

[0034] Figure 2 The total ion chromatogram in negative ion mode of the Zixuesan sample in Example 1;

[0035] Figure 3 The total ion chromatogram in positive ion mode for the mixed reference standards in Example 1;

[0036] Figure 4 The total ion chromatogram in negative ion mode for the mixed control standard in Example 1;

[0037] Figure 5 The chromatograms were obtained under the chromatographic conditions of Examples 1, 3, and 4.

[0038] Figure 6 The chromatograms are obtained under the chromatographic conditions of Examples 1, 5, and 6;

[0039] Figure 7 The chromatograms are obtained using different chromatographic columns in Comparative Example 1 and Example 2. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0041] Zixue Powder has a wide variety of ingredients and complex components, but there is currently limited research data on its chemical composition and quality analysis, making it difficult to fully reflect its quality.

[0042] To address this problem, this invention provides a method for detecting the indicator components of Zixue Powder. This method uses ultra-high performance liquid chromatography (UHPLC) to separate the indicator components, achieving good separation among linalool, isoflavonic acid, angoloside C, cimicifugain, glycyrrhizin, cinnamic acid, isoglycyrrhizin, harpagoside, and glycyrrhizic acid, as well as among other compounds. Combined with other detection methods or chromatographic conditions, the qualitative and quantitative analysis of the above-mentioned indicator components can be further performed. The obtained detection results help to more comprehensively reflect the quality of Zixue Powder and provide methods and basis for improving the quality standards of Zixue Powder.

[0043] The technical solution of the present invention will be further described below through specific embodiments.

[0044] The analytical instruments used in the following examples:

[0045] ACQUITY I class ultra-high performance liquid chromatograph (Waters Technology, Inc., USA); Waters ACQUITY UPLC / Xevo G2-QTOF ultra-high performance liquid chromatograph / high resolution tandem mass spectrometer (Waters Technology, Inc., USA).

[0046] The materials and reagents used in the following examples:

[0047] Reference standards: Agaric tetraol (Aga, 111980-201904), isoflavonic acid (IA, 111698-201904), and harbazoside (111729-201707) were purchased from the National Institutes for Food and Drug Control, with a purity of ≥98%. Glycyrrhizin (Liq, ZM0314BD14), isoglycyrrhizin (Iso, Z02M3L1), Angoloside C (Ang, A03GB156840), Cimicifugain (Cim, Y11O9H72162), glycyrrhizic acid (Gly, Y02J11L113432), glycyrrhizin (Z13J11X108109), apigenin glycyrrhizin (YA0409HA14), isoglycyrrhizin (R07D8F50056), cinnamic acid (TCA, AA0806LB14), harpagoside (Har, Y21D10H106624), sucrose (H22J9R53193), styracin (F27J7S18516), and glycyrrhizin chalcone A were purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a purity ≥98%.

[0048] The samples of Scrophularia, Licorice, Cimicifuga, Clove, Costus Root, Agarwood, Concentrated Buffalo Horn Powder, Antelope Horn, Artificial Musk Slices, and Zixue Powder were all provided and identified by Tianjin Hongrentang Pharmaceutical Co., Ltd. They were all tested according to the requirements of each medicinal material item in the 2020 edition of the Chinese Pharmacopoeia, met the standards, and were pulverized and sieved for later use.

[0049] Zixue Powder (S1, S2, S3) is provided by Tianjin Hongrentang Pharmaceutical Co., Ltd.

[0050] The reagents used in the following examples:

[0051] Methanol (chromatographic grade, Sigma-Aldrich, USA), acetonitrile (chromatographic grade, Sigma-Aldrich, USA), formic acid (chromatographic grade, Shanghai Aladdin Biochemical Technology Co., Ltd.), water (Watsons drinking water).

[0052] Unless otherwise specified, all other instruments, reagents and medicines used in the following examples are commercially available.

[0053] Example 1

[0054] This invention provides a qualitative detection method for the indicator components of Zixue Powder.

[0055] 1. Liquid chromatography and mass spectrometry conditions

[0056] 1.1 Liquid Chromatography Conditions:

[0057] Waters Acquity I-Class ultra-high performance liquid chromatograph; column: Agilent ZORBAX SB-C18 column (4.6×100mm, 1.8μm, Agilent, USA); flow rate: 0.5mL / min; column temperature: 45℃; injection volume: 2μL; mobile phase: 0.1% v / v formic acid aqueous solution (A)-methanol (B), gradient elution program as shown in Table 1.

[0058] Table 1 Gradient elution program

[0059]

[0060] 1.2 Mass spectrometry conditions:

[0061] Xevo G2-XS QTOF mass spectrometer, MS E Both positive and negative scanning modes are supported, with a mass range of m / z 50-1200 amu. Specific mass spectrometry parameters are: capillary voltage 3.00 kV (ESI). + ) and -2.50kV (ESI) - The ion source temperature was 120℃, the cone voltage was 40V, the cone gas flow rate was 50L / h, the nitrogen degassing temperature was 500℃, the degassing volumetric flow rate was 800L / h, the low collision energy was 6eV, and the high collision energy was 10~45eV. Calibration solution: sodium formate solution.

[0062] 2. Solution preparation

[0063] 2.1 Preparation of the test solution

[0064] Accurately weigh 1.0g each of the following ingredients: Scrophularia ningpoensis, Glycyrrhiza uralensis, Cimicifuga foetida, Clove, Aucklandia lappa, Aquilaria sinensis, concentrated buffalo horn powder, antelope horn, artificial musk slices, and Zixue San sample powder. Place the powder in a 50mL Erlenmeyer flask, add 25mL of 75% v / v methanol aqueous solution, seal tightly, and weigh. Extract by ultrasonication at 60℃ for 30min, cool, and weigh again. Replenish the lost weight with 75% v / v methanol aqueous solution, shake well, centrifuge at 12700rpm for 10min, and collect the supernatant.

[0065] 2.2 Preparation of reference solution

[0066] Accurately weigh appropriate amounts of harpaquinone, glycyrrhizin, cimicifugain, isoglycyrrhizin, linalool, apigenin, angoloside C, glycyrrhizin, isofraguritic acid, isoglycyrrhizin, harpaquinone, glycyrrhizic acid, cinnamic acid, glycyrrhizin chalcone A, and gentianin, dissolve them in methanol, and prepare standards for harpaquinone 0.521 mg / mL, glycyrrhizin 1.000 mg / mL, cimicifugain 2.002 mg / mL, apigenin 1.162 mg / mL, linalool 2.002 mg / mL, and isoglycyrrhizin 1.110 mg / mL, respectively. A stock solution of reference standards containing L, Angoloside C 1.999 mg / mL, glycyrrhizin 0.503 mg / mL, isoflavonic acid 2.001 mg / mL, isoglycyrrhizin 0.501 mg / mL, harpagoside 1.004 mg / mL, glycyrrhizic acid 3.004 mg / mL, cinnamic acid 1.000 mg / mL, glycyrrhizin chalcone A 0.304 mg / mL, and gentianin 1.120 mg / mL was prepared; sucrose standard was dissolved in water to prepare a stock solution with a concentration of 0.974 mg / mL.

[0067] Take an appropriate amount of each reference standard stock solution, measure it accurately, place it in a 10 mL volumetric flask, add 75% v / v methanol aqueous solution to dilute to the mark, shake well, and obtain a mixed reference standard solution. The mixed reference solution contained the following components: harpagoside 0.0521 mg / mL, glycyrrhizin 0.0500 mg / mL, cimicifugain 0.0501 mg / mL, isoglycyrrhizin 0.0555 mg / mL, linalool 0.0501 mg / mL, apigenin 0.0581 mg / mL, angoloside C 0.0500 mg / mL, glycyrrhizin 0.0503 mg / mL, isofragucic acid 0.0500 mg / mL, isoglycyrrhizin 0.0501 mg / mL, harpagoside 0.0502 mg / mL, glycyrrhizic acid 0.0511 mg / mL, cinnamic acid 0.0500 mg / mL, glycyrrhizin chalcone A 0.0304 mg / mL, sucrose 0.0487 mg / mL, and gentianin 0.0560 mg / mL.

[0068] 3. Data Processing

[0069] Information on the chemical components of various herbs used in Zixue Powder was collected and organized, and combined with the built-in traditional Chinese medicine database of UNIFI to establish a chemical component database for Zixue Powder. The mass spectrometry data were processed using UNIFI software, and compound data were further compared with reference standards and relevant literature. Energy intensity threshold settings: high energy 40, low energy 400; retention time error 0.1 min; mass number error ±2 mDa; positive ion mode, with [M+H] as the primary energy source. + [M+K] + [M+Na] + [M+NH4] +For adductor ions, the corrected mass number is 556.2766; [MH] is selected in negative ion mode. - [M-H+HCOOH] - For adduct ions, the corrected mass number is 554.2620.

[0070] 4. Experimental Results

[0071] UPLC-Q-TOF-MS was used to analyze the 75% v / v methanol extract of Zixue San. Combining the analysis with reference standards, compound retention times, secondary mass spectrometry fragments, and relevant literature information, a total of 75 compounds were identified in Zixue San using a self-built UNIFI database. These compounds included chromones, flavonoids, triterpenes and their saponins, monoterpenes and their glycosides, and organic acids. Further analysis of the identified compounds and their association with the medicinal materials in the formula revealed that among the 75 compounds, 27 were from Cimicifuga rhizome, 20 from licorice root, 12 from Scrophularia root, 13 from agarwood, and 4 from costus root. The total ion chromatograms of the Zixue San sample and mixed reference standards under different modes are shown below. Figures 1-4 As shown, specific compound information is provided in Table 2.

[0072]

[0073]

[0074]

[0075]

[0076] Example 2

[0077] This invention provides a method for determining the content of multiple components in Zixue powder.

[0078] 1. Experimental Methods

[0079] 1.1 Chromatographic conditions

[0080] Waters Acquity I-Class ultra-high performance liquid chromatograph; Column: Agilent ZORBAX SB-C18 (4.6×100mm, 1.8μm, Agilent, USA); Flow rate: 0.5mL / min; Column temperature: 45℃; Injection volume: 2μL; Mobile phase: 0.1% v / v formic acid aqueous solution (A)-methanol (B), gradient elution program as shown in Table 1 of Example 1; Multi-wavelength switching detection, wavelengths as shown in Table 3.

[0081] Table 3 Timing Wavelength Switching Table

[0082]

[0083] 1.2 Preparation of the test solution

[0084] Accurately weigh 1.0 g of Zixue powder and place it in a 50 mL Erlenmeyer flask. Add 25 mL of 75% v / v methanol aqueous solution, weigh, seal tightly, and sonicate at 60℃ for 30 min. Remove, cool, and weigh again. Replenish the lost weight with 75% v / v methanol aqueous solution, shake well, take an appropriate amount, centrifuge at 12700 rpm for 10 min, take 5 mL of supernatant and concentrate, redissolve with 1 mL of 75% v / v methanol aqueous solution, and filter through a 0.45 μm filter membrane to obtain the Zixue powder test solution.

[0085] 1.3 Preparation of reference solution

[0086] Accurately weigh appropriate amounts of reference standards for agaricone, isoflavone, angoloside C, cimicifugain, glycyrrhizin, cinnamic acid, isoglycyrrhizin, harpagoside, and glycyrrhizic acid, dissolve them in methanol, and prepare stock solutions containing agaricone 2.002 mg / mL, isoflavone 2.001 mg / mL, angoloside C 1.999 mg / mL, cimicifugain 2.002 mg / mL, glycyrrhizin 0.503 mg / mL, cinnamic acid 1.000 mg / mL, isoglycyrrhizin 0.501 mg / mL, harpagoside 1.004 mg / mL, and glycyrrhizic acid 3.004 mg / mL.

[0087] Accurately measure appropriate amounts of each reference stock solution and place them in a 10 mL volumetric flask. Add 75% v / v methanol aqueous solution to the mark and shake well to obtain a mixed reference solution. The mixed reference solution contains linalool 160.16 μg / mL, isoflavonic acid 460.23 μg / mL, angoloside C 175.91 μg / mL, cimicifugain 120.12 μg / mL, glycyrrhizin 35.21 μg / mL, cinnamic acid 70.00 μg / mL, isoglycyrrhizin 30.06 μg / mL, harpagoside 70.28 μg / mL, and glycyrrhizic acid 781.04 μg / mL.

[0088] 2. Data Processing

[0089] The mixed reference solution was serially diluted to prepare a series of mixed reference solutions of different concentrations. The peak area of ​​each compound was determined using the chromatographic conditions described in section 1.1. A standard curve was plotted with the compound reference concentration as the abscissa (x, μg / mL) and the peak area as the ordinate, yielding a linear regression equation. The peak area of ​​each compound in the test solution was then determined using the chromatographic conditions described in section 1.1. Substituting this value into the linear regression equation, the content of each compound in the test solution was calculated, and subsequently, the content of each compound in the Zixue San powder was calculated.

[0090] Example 3

[0091] This invention provides a method for determining the content of multiple components in Zixue powder.

[0092] 1. Experimental Methods

[0093] 1.1 Chromatographic conditions

[0094] Waters Acquity I-Class ultra-high performance liquid chromatograph; column: Agilent ZORBAX SB-C18 (4.6×100mm, 1.8μm, Agilent, USA); flow rate: 0.5mL / min; column temperature: 45℃; injection volume: 2μL; mobile phase: 0.1% v / v formic acid aqueous solution (A)-acetonitrile (B), gradient elution program as shown in Table 1 of Example 1. Multi-wavelength switching detection, wavelengths as shown in Table 3 of Example 2.

[0095] 1.2 Preparation of the test solution: Same as in Example 2.

[0096] 1.3 Preparation of the reference solution: Same as in Example 2.

[0097] 2. Data processing: Same as in Example 2.

[0098] Example 4

[0099] This invention provides a method for determining the content of multiple components in Zixue powder.

[0100] 1. Experimental Methods

[0101] 1.1 Chromatographic conditions

[0102] Waters Acquity I-Class ultra-high performance liquid chromatograph; column: Agilent ZORBAX SB-C18 column (4.6×100mm, 1.8μm, Agilent, USA); flow rate: 0.5mL / min; column temperature: 45℃; injection volume: 2μL; mobile phase: 0.2% v / v formic acid aqueous solution (A)-methanol (B), gradient elution program as shown in Table 1 of Example 1.

[0103] 1.2 Preparation of the test solution: Same as in Example 2.

[0104] 1.3 Preparation of the reference solution: Same as in Example 2.

[0105] 2. Data processing: Same as in Example 2.

[0106] Example 5

[0107] This invention provides a method for determining the content of multiple components in Zixue powder.

[0108] 1. Experimental Methods

[0109] 1.1 Chromatographic conditions

[0110] Waters Acquity I-Class ultra-high performance liquid chromatograph; column: Agilent ZORBAX SB-C18 column (4.6×100mm, 1.8μm, Agilent, USA); flow rate: 0.5mL / min; column temperature: 40℃; injection volume: 2μL; mobile phase: 0.1% v / v formic acid aqueous solution (A)-methanol (B), gradient elution program as shown in Table 1 of Example 1.

[0111] 1.2 Preparation of the test solution: Same as in Example 2.

[0112] 1.3 Preparation of the reference solution: Same as in Example 2.

[0113] 2. Data processing: Same as in Example 2.

[0114] Example 6

[0115] This invention provides a method for determining the content of multiple components in Zixue powder.

[0116] 1. Experimental Methods

[0117] 1.1 Chromatographic conditions

[0118] Waters Acquity I-Class ultra-high performance liquid chromatograph; column: Agilent ZORBAX SB-C18 column (4.6×100mm, 1.8μm, Agilent, USA); flow rate: 0.5mL / min; column temperature: 50℃; injection volume: 2μL; mobile phase: 0.1% v / v formic acid aqueous solution (A)-methanol (B), gradient elution program as shown in Table 1 of Example 1.

[0119] 1.2 Preparation of the test solution: Same as in Example 2.

[0120] 1.3 Preparation of the reference solution: Same as in Example 2.

[0121] 2. Data processing: Same as in Example 2.

[0122] Example 7

[0123] This invention provides a method for determining the content of multiple components in Zixue powder.

[0124] 1. Experimental Methods

[0125] 1.1 Chromatographic conditions: Same as in Example 2.

[0126] 1.2 Preparation of the test solution:

[0127] Accurately weigh 2.5g of Zixue powder and place it in a 50mL Erlenmeyer flask. Add 25mL of 75% v / v methanol aqueous solution, seal tightly, and weigh. Extract by ultrasonication at 60℃ for 30min, cool, and weigh again. Replenish the lost weight with 75% v / v methanol aqueous solution, shake well, and centrifuge at 12700rpm for 10min. Take the supernatant to obtain the product.

[0128] 1.3 Preparation of the reference solution: Same as in Example 2.

[0129] 2. Data processing: Same as in Example 2.

[0130] Example 8

[0131] This invention provides a method for determining the content of multiple components in Zixue powder.

[0132] 1. Experimental Methods

[0133] 1.1 Chromatographic conditions: Same as in Example 2.

[0134] 1.2 Preparation of the test solution:

[0135] Accurately weigh 0.5g of Zixue powder and place it in a 50mL Erlenmeyer flask. Add 25mL of 75% v / v methanol aqueous solution, seal tightly, and weigh. Extract by ultrasonication at 60℃ for 30min, cool, and weigh again. Replenish the lost weight with 75% v / v methanol aqueous solution, shake well, and centrifuge at 12700rpm for 10min. Take the supernatant to obtain the product.

[0136] 1.3 Preparation of the reference solution: Same as in Example 2.

[0137] 2. Data processing: Same as in Example 2.

[0138] Example 9

[0139] This invention provides a method for determining the content of multiple components in Zixue powder.

[0140] 1. Experimental Methods

[0141] 1.1 Chromatographic conditions: Same as in Example 2.

[0142] 1.2 Preparation of the test solution:

[0143] Accurately weigh 1.0g of Zixue powder and place it in a 50mL Erlenmeyer flask. Add 25mL of 75% v / v methanol aqueous solution, seal tightly, and weigh. Extract by ultrasonication at 60℃ for 20min, cool, and weigh again. Replenish the lost weight with 75% v / v methanol aqueous solution, shake well, and centrifuge at 12700rpm for 10min. Take the supernatant to obtain the product.

[0144] 1.3 Preparation of the reference solution: Same as in Example 2.

[0145] 2. Data processing: Same as in Example 2.

[0146] Example 10

[0147] This invention provides a method for determining the content of multiple components in Zixue powder.

[0148] 1. Experimental Methods

[0149] 1.1 Chromatographic conditions: Same as in Example 2.

[0150] 1.2 Preparation of the test solution:

[0151] Accurately weigh 1.0g of Zixue powder and place it in a 50mL Erlenmeyer flask. Add 25mL of 75% v / v methanol aqueous solution, seal tightly, and weigh. Extract by ultrasonication at 60℃ for 40min, cool, and weigh again. Replenish the lost weight with 75% v / v methanol aqueous solution, shake well, and centrifuge at 12700rpm for 10min. Take the supernatant to obtain the product.

[0152] 1.3 Preparation of the reference solution: Same as in Example 2.

[0153] 2. Data processing: Same as in Example 2.

[0154] Example 11

[0155] This invention provides a method for determining the content of multiple components in Zixue powder.

[0156] 1. Experimental Methods

[0157] 1.1 Chromatographic conditions: Same as in Example 2.

[0158] 1.2 Preparation of the test solution:

[0159] Accurately weigh 1.0g of Zixue powder and place it in a 50mL Erlenmeyer flask. Add 25mL of 65% v / v methanol aqueous solution, seal tightly, and weigh. Extract by ultrasonication at 60℃ for 30min, cool, and weigh again. Replenish the lost weight with 65% v / v methanol aqueous solution, shake well, and centrifuge at 12700rpm for 10min. Take the supernatant to obtain the product.

[0160] 1.3 Preparation of the reference solution: Same as in Example 2.

[0161] 2. Data processing: Same as in Example 2.

[0162] Example 12

[0163] This invention provides a method for determining the content of multiple components in Zixue powder.

[0164] 1. Experimental Methods

[0165] 1.1 Chromatographic conditions: Same as in Example 2.

[0166] 1.2 Preparation of the test solution:

[0167] Accurately weigh 1.0g of Zixue powder and place it in a 50mL Erlenmeyer flask. Add 25mL of 50% v / v methanol aqueous solution, seal tightly, and weigh. Extract by ultrasonication at 60℃ for 30min, cool, and weigh again. Replenish the lost weight with 50% v / v methanol aqueous solution, shake well, and centrifuge at 12700rpm for 10min. Take the supernatant to obtain the product.

[0168] 1.3 Preparation of the reference solution: Same as in Example 2.

[0169] 2. Data processing: Same as in Example 2.

[0170] Test Example 1

[0171] The test solution was analyzed under the chromatographic conditions of Examples 1, 3-6, and the resulting chromatograms are shown below. Figure 5 , Figure 6 As shown:

[0172] The chromatographic conditions of Examples 1, 3, and 4 were all able to separate the nine index components, but the separation degree of each compound in the chromatograms of Examples 1 and 4 was better than that in Example 3.

[0173] Examples 5 and 6 also achieved the separation of 9 index components, but compared with Examples 5 and 6, the separation degree of each compound in the chromatogram of Example 1 is better.

[0174] Test Example 2

[0175] The test solutions of Examples 2, 7, and 8 were analyzed under the chromatographic conditions of Example 2, and the peak area (A / g) per unit mass was calculated. The results are shown in Table 4.

[0176] Table 4. Peak areas of nine index components in the test solutions of Examples 2, 7, and 8.

[0177]

[0178] All embodiments yielded high peak areas, with Example 2 exhibiting the highest peak area, indicating that the sample solution preparation method of Example 2 was more efficient in extracting each component.

[0179] Test Example 3

[0180] The test solutions of Examples 2, 9, and 10 were analyzed under the chromatographic conditions of Example 2, and the peak area (A / g) per unit mass was calculated. The results are shown in Table 5.

[0181] Table 5. Peak areas of nine index components in the test solutions of Examples 2, 9, and 10.

[0182]

[0183] All embodiments yielded high peak areas, with Example 2 exhibiting the highest peak area, indicating that the sample solution preparation method of Example 2 was more efficient in extracting each component.

[0184] Test Example 4

[0185] The test solutions of Examples 2, 11, and 12 were analyzed under the chromatographic conditions of Example 2, and the peak area (A / g) per unit mass was calculated. The results are shown in Table 6.

[0186] Table 6 shows the peak areas of the nine index components in the test solutions of Examples 2, 11, and 12.

[0187]

[0188] All embodiments yielded high peak areas, with Example 2 exhibiting the highest peak area, indicating that the sample solution preparation method of Example 2 was more efficient in extracting each component.

[0189] Test Example 5

[0190] This test case provides a methodological review of the multi-component content determination method of Example 2.

[0191] 1. Studies on linearity, limit of quantitation, and limit of detection.

[0192] The mixed reference solution from Example 2 was serially diluted to prepare a series of mixed reference solutions of different concentrations. The peak areas of each compound were determined using the chromatographic conditions described in Example 2. A standard curve was plotted with the compound reference concentration as the abscissa (x, μg / mL) and the peak area as the ordinate, yielding a linear regression equation. The limit of detection (LOD) was set at a signal-to-noise ratio (S / N) of 3, and the limit of quantitation (LOQ) at S / N = 10. The results are shown in Table 7. The results indicate that the nine index components in Zixue San exhibited good linearity within their respective concentration ranges, with correlation coefficients r > 0.999.

[0193] Table 7. Linear regression equations, correlation coefficients, linear ranges, limits of detection, and limits of quantitation for the nine compounds in Zixue Powder.

[0194]

[0195]

[0196] 2. Precision test

[0197] (1) Intraday precision test

[0198] The Zixue San test solution was prepared according to the method described in section 1.2 of Example 2. The peak areas of each compound were determined using the chromatographic conditions of Example 2. The same test solution was injected repeatedly 6 times, and the RSD values ​​were calculated to examine the intra-day precision. The results showed that the intra-day precision RSD values ​​of the nine index components in Zixue San were 0.1%-1.1%, indicating that the method has good intra-day precision. The results are shown in Table 8.

[0199] Table 8. Results of intraday precision determination of index component content in *Zi Xue* (a type of herb).

[0200]

[0201] (2) Daytime precision test

[0202] The Zixue San test solution was prepared according to the method described in section 1.2 of Example 2. The peak areas of each compound were determined using the chromatographic conditions of Example 2. The same test solution was injected six times over three days, and the RSD values ​​were calculated to assess the inter-day precision. The results showed that the inter-day precision RSD values ​​of the nine index components in Zixue San ranged from 0.6% to 2.8%, indicating that the method has good inter-day precision. The results are shown in Table 9.

[0203] Table 9. Results of Daytime Precision Measurement of Index Component Content in *Zi Xue*

[0204]

[0205] 3. Stability test

[0206] The Zixuesan test solution was prepared according to the method described in section 1.2 of Example 2. The solution was injected at 0, 2, 4, 6, 8, 10, and 12 hours using the chromatographic conditions of Example 2. The peak areas of each compound were measured, and the RSD values ​​were calculated to investigate the stability of the index components in the Zixuesan test solution. The results showed that the RSD values ​​of the nine index components in Zixuesan ranged from 0.37% to 1.68%, indicating good stability of the sample within 12 hours. The results are shown in Table 10.

[0207] Table 10 Results of stability test for determination of index components in *Zixue* (a type of medicinal herb).

[0208]

[0209]

[0210] 4. Repeatability test

[0211] Six test solutions were prepared in parallel according to the method described in section 1.2 of Example 2. The peak areas of each compound were determined using the chromatographic conditions of Example 2, and the compound contents and RSD values ​​were calculated. The results are shown in Table 11. The average contents of linalool, isoflavonic acid, angoloside C, cimicifugain, glycyrrhizin, cinnamic acid, isoglycyrrhizin, harpagoside, and glycyrrhizic acid were 0.1018, 0.2946, 0.1095, 0.0730, 0.0208, 0.0441, 0.0170, 0.0469, and 0.4889 mg / g, respectively. The RSD values ​​of each compound ranged from 0.6% to 2.5%, indicating that the method has good repeatability.

[0212] Table 11. Repeatability test results of the content of indicator components in *Zi Xue* (n=6)

[0213]

[0214] 5. Recovery test

[0215] Accurately weigh 20.02, 20.01, 19.99, 20.02, 5.03, 10.00, 5.01, 10.04, and 30.04 mg of agaric tetraol, isoflavonic acid, angoloside C, cimicifugain, glycyrrhizin, cinnamic acid, isoglycyrrhizin, harpagoside, and glycyrrhizic acid reference standards, respectively. Dissolve in methanol and dilute to the mark to prepare solutions with concentrations of 2.002, 2.001, 1.999, 2.002, 0.503, 1.000, and 0 mg, respectively. The following reference solutions were prepared at concentrations of 0.501, 1.004, and 3.004 mg / mL: linalool, isoflavonic acid, angoloside C, cimicifugain, glycyrrhizin, cinnamic acid, isoglycyrrhizin, harpagoside, and glycyrrhizic acid. Appropriate amounts of each reference solution were placed in 10 mL volumetric flasks and diluted to the mark with methanol to obtain mixed reference solutions with concentrations of 160.16, 460.23, 175.91, 120.12, 35.21, 70.00, 30.06, 70.28, and 781.04 μg / mL.

[0216] Accurately weigh 0.50 g of Zixue San powder and add 1.0 mL of each mixed reference solution with an equivalent mass to that of each component in the sample. Prepare six test solutions in parallel according to the method in section "1.2" of Example 2, and analyze them under the chromatographic conditions of Example 2. Calculate the content, recovery rate, and RSD value of each compound. The results are shown in Table 12. The results show that the recoveries of the nine compounds in Zixue San ranged from 92.5% to 105.4%, and the RSD values ​​were all ≤3.0%, indicating that the recovery rates of each compound were good and the method was feasible.

[0217] Table 12 Results of the recovery test of index components in Zixue Powder (n=6)

[0218]

[0219]

[0220]

[0221] In summary, in the methodological studies, linalool, isoflavonic acid, angoloside C, cimicifugain, glycyrrhizin, cinnamic acid, isoglycyrrhizin, harpagoside, and glycyrrhizic acid showed good linearity within their respective concentration ranges; the intra-day precision RSD values ​​were all <1.1%; the inter-day precision RSD values ​​were all <2.8%; the stability RSD values ​​were all <1.7%; the repeatability RSD values ​​were all <2.5%; and the recoveries ranged from 92.5% to 105.4%, with RSD values ​​≤3.0%. Therefore, the method for determining the content of multiple components of Zixue San provided in Example 2 is reasonable and feasible.

[0222] Example 11

[0223] This embodiment provides the results of multi-component content determination for different batches of Zixue Powder.

[0224] Different batches of Zixuesan samples were taken, and Zixuesan test solutions were prepared according to the method in section "1.2" of Example 2. The peak areas of each compound in the test solution were determined using the chromatographic conditions of Example 2, and the content of each compound was calculated. The results are shown in Table 13. The content of the index components of Zixuesan in different batches is significantly different, which may be related to the different quality of the raw materials in different batches.

[0225] Table 13. Results of the determination of the content of key components of Zixue Powder using different processes (mg / g)

[0226]

[0227] Comparative Example 1

[0228] This comparative example provides a method for determining the content of multiple components in Zixue Powder.

[0229] 1. Experimental Methods

[0230] 1.1 Chromatographic conditions

[0231] Waters Acquity I-Class ultra-high performance liquid chromatograph; chromatographic column is Waters ACQUITY. C18 (2.1×100mm, 1.7μm); flow rate: 0.5mL / min; column temperature: 45℃; injection volume: 2μL; mobile phase: 0.1% v / v formic acid aqueous solution (A)-methanol (B), gradient elution program as shown in Table 1 of Example 1.

[0232] 1.2 Preparation of the test solution: Same as in Example 2.

[0233] 1.3 Preparation of the reference solution: Same as in Example 2.

[0234] 2. Data processing: Same as in Example 2.

[0235] The results are as follows Figure 7 As shown, the Zixue San sample exhibited better resolution on the Agilent ZORBAX SB C18 column than on the Waters C18 column, and also showed more chromatographic peaks.

[0236] Comparative Example 2

[0237] This comparative example provides a method for determining the content of multiple components in Zixue Powder.

[0238] 1. Experimental Methods

[0239] 1.1 Chromatographic conditions: Same as in Example 2.

[0240] 1.2 Preparation of the test solution:

[0241] Accurately weigh 1.0 g of Zixue powder and place it in a 50 mL Erlenmeyer flask. Add 25 mL of 25% v / v methanol aqueous solution, 50% v / v methanol aqueous solution, 75% v / v methanol aqueous solution, and methanol, respectively. Weigh the flask, sonicate at room temperature for 30 min, remove, cool, and weigh again. Replenish the weight loss with 25% v / v methanol aqueous solution, 50% v / v methanol aqueous solution, 75% v / v methanol aqueous solution, and methanol, respectively. Shake well, take an appropriate amount, centrifuge at 12700 rpm for 10 min, and collect the supernatant to obtain the test solution extracted with different solvents. Determine the peak area of ​​each component in the test solution according to the chromatographic conditions of Example 2, and calculate the peak area per unit mass (A / g). The results are shown in Table 14.

[0242] Table 14 Peak areas of nine index components in the test sample solution under different extraction solvents

[0243]

[0244]

[0245] As shown in Table 14, the peak areas of the index components in each test solution were lower than those in Examples 2 and 11. When 75% v / v methanol aqueous solution or methanol was used as the extraction solvent, the peak area of ​​Iso was higher than that of Iso in the test solution of Example 12, but the peak areas of Ang and Gly were significantly lower than those in Example 12.

[0246] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting the index components of Zixue San (a traditional Chinese medicine), characterized in that, The indicator components include linalool, isoflavonic acid, angoloside C, cimicifugain, glycyrrhizin, cinnamic acid, isoglycyrrhizin, harpagoside, and glycyrrhizic acid. The specific operation of the indicator component detection method includes: S1. Extract the Zixue powder sample to be tested with 50%~75% v / v methanol aqueous solution by ultrasonication at a temperature of 60℃ for 20~40 min to prepare the test solution; dissolve the reference standard of the indicator component in 50%~75% v / v methanol aqueous solution to prepare the reference standard solution. S2. The test solution and the reference solution were detected by ultra-high performance liquid chromatography. The chromatographic conditions for the ultra-high performance liquid chromatography method include: Chromatographic column: ZORBAX SB-C18, 4.6 × 100 mm, 1.8 μm; Mobile phase A is an aqueous solution containing 0.1%~0.2% v / v formic acid, and mobile phase B is methanol or acetonitrile. Linear gradient elution is performed, and the gradient elution program is as follows: Flow rate: 0.5 mL / min; Column temperature: 40~50 ℃; The method for detecting the index components also includes characterizing the index components using mass spectrometry.

2. The method for detecting the index components of Zixue Powder according to claim 1, characterized in that, The mobile phase B is methanol; and / or The column temperature is 45°C.

3. The method for detecting the index components of Zixue Powder according to claim 1, characterized in that, The method for detecting the index components was implemented using an ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometer.

4. The method for detecting the index components of Zixue Powder according to claim 3, characterized in that, The mass spectrometry conditions for the method include: a positive / negative ion switching scanning mode, capillary voltage of 3.00 kV and –2.50 kV, ion source temperature of 120 °C, cone voltage of 40 V, cone gas flow rate of 50 L / h, nitrogen degassing temperature of 500 °C, degassing volumetric flow rate of 800 L / h, low collision energy of 6 eV, and high collision energy of 10–45 eV; mass range: m / z 50–1200 amu.

5. The method for detecting the index components of Zixue Powder according to claim 1, characterized in that, The ratio of the Zixue powder sample to the methanol aqueous solution is 1:(10~50), g:mL; and / or The concentration of the methanol aqueous solution is 65%~75% v / v.

6. The application of the index component detection method according to any one of claims 1 to 5 in the detection of index component content in Zixuesan, characterized in that, The chromatographic conditions include a UV detector instead of a mass spectrometer detector, and also include multi-wavelength switching, with the wavelength switching procedure as follows: 。

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