Method for establishing fingerprint of Baoxin Avicel wood preparation and its application

Abstract

The present invention discloses a method for establishing a fingerprint of a Baoxin Avicel agent. The method comprises: performing high performance liquid chromatography analysis on a Baoxin Avicel agent sample to be tested, and establishing the fingerprint of the Baoxin Avicel agent based on the obtained liquid chromatography analysis result. The method can reflect most of the components contained in the Baoxin Avicel agent, or all of the index components, and can comprehensively and effectively reflect the quality information of the Baoxin Avicel agent, and the method has simple steps, convenient and fast operation, low detection cost, good reproducibility, large amount of information, good systematicity, characteristic and stability, and can be effectively used to detect the quality or authenticity of the Baoxin Avicel agent.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis. Specifically, the present invention relates to a method for establishing a fingerprint of a Baoxin Avicillin preparation and an application thereof. More specifically, the present invention relates to a method for establishing a fingerprint of a Baoxin Avicillin preparation, a fingerprint of a Baoxin Avicillin preparation, a method for determining the components of a Baoxin Avicillin preparation, a method for detecting the quality or authenticity of a Baoxin Avicillin preparation and a method for determining the production process of a Baoxin Avicillin preparation. Background Art

[0002] Baoxin Avicim oral liquid is an oral preparation that has the effects of strengthening the heart and liver, calming the nerves and tranquilizing the mind. It is mainly used clinically for palpitations, shortness of breath, dizziness, loss of appetite, mental weakness, etc. However, the current relevant quality control standards are relatively simple, with no content determination items. How to characterize the main chemical components of the test product from an overall macroscopic perspective and the effective quality control method still needs to be improved.

[0003] At the same time, how to effectively identify the quality and authenticity of Baoxin Avicii wood preparations is also an important issue that has yet to be resolved. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art.

[0005] To this end, in a first aspect of the present invention, the present invention proposes a method for establishing a fingerprint of a Baoxin Avicel preparation. According to an embodiment of the present invention, the method comprises: performing high performance liquid chromatography analysis on a Baoxin Avicel preparation sample to be tested, and establishing the fingerprint of the Baoxin Avicel preparation based on the obtained liquid chromatography analysis results. The fingerprint according to the embodiment of the present invention can comprehensively and effectively reflect the quality information of the preparation.

[0006] According to an embodiment of the present invention, the method for establishing the fingerprint of the Baoxin Avicel agent may further include at least one of the following additional technical features:

[0007] According to an embodiment of the present invention, the liquid chromatography analysis adopts the following conditions: the aqueous phase of the mobile phase is a phosphoric acid aqueous solution, and optionally, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.1%; the organic phase of the mobile phase is acetonitrile; and the elution method is gradient elution. By selecting a 0.1% phosphoric acid aqueous solution as the aqueous phase and acetonitrile as the organic phase, a fingerprint of the Baoxin Avicel agent with balanced peak area response, stable baseline, and small matrix interference can be obtained.

[0008] According to an embodiment of the present invention, the procedure of the gradient elution is: 0-35min, 0-21% acetonitrile, 100-79% of a 0.1% aqueous phosphoric acid solution by volume; 35-40min, 21% acetonitrile, 79% of a 0.1% aqueous phosphoric acid solution by volume; 40-60min, 21-55% acetonitrile, 79-45% of a 0.1% aqueous phosphoric acid solution by volume; 60-65min, 55-0% acetonitrile, 45-100% of a 0.1% aqueous phosphoric acid solution by volume; 65-75min, 0% acetonitrile, 100% of a 0.1% aqueous phosphoric acid solution by volume. According to a specific embodiment of the present invention, by adopting the elution procedure, the obtained rosmarinic acid has the largest separation degree, and the chromatographic peak purity is high, the chromatographic peaks are all analyzed, the number of chromatographic peaks is large, and the baseline fluctuation is small.

[0009] According to an embodiment of the present invention, the liquid chromatography analysis further adopts the following conditions: a chromatographic column with a column length of 150 mm, an inner diameter of 4.6 mm, and a particle size of 2.7 μm is used, preferably, the chromatographic column is a Waters CORTECS T3 chromatographic column. According to a specific embodiment of the present invention, when Waters CORTECS T3 is selected as the chromatographic column, the number of chromatographic peaks obtained is the largest, the peak heights of each peak are relatively balanced, the separation degree of rosmarinic acid is high, and the peak purity is relatively good.

[0010] According to an embodiment of the present invention, the detection wavelength is 230-251 nm, preferably, the detection wavelength is 235 nm. According to a specific embodiment of the present invention, when the detection wavelength is 235 nm, the baseline of the sample is flatter and the chromatographic peak is more balanced.

[0011] According to an embodiment of the present invention, the sample to be tested is obtained by diluting the Baoxin Avicel agent in 90% to 100% methanol by volume, preferably dissolving it in 98% to 100% methanol, so as to obtain the sample to be tested. According to a specific embodiment of the present invention, the most characteristic peaks can be obtained by dissolving it in 98% to 100% methanol.

[0012] According to an embodiment of the present invention, the Baoxin Avicel preparation is an oral preparation, and the volume ratio of the Baoxin Avicel preparation to 90% to 100% methanol is 1:(0.5-2), preferably 1:1.

[0013] According to an embodiment of the present invention, the injection volume of the sample to be tested is 10 μL. According to a specific embodiment of the present invention, when the injection volume is 10 μL, the response of each chromatographic peak is more balanced and the peak area response is higher, the baseline fluctuation is smaller, and the separation degree and peak purity of rosmarinic acid meet the requirements.

[0014] According to an embodiment of the present invention, the column temperature of the chromatographic column is 25-35° C. According to a specific embodiment of the present invention, when 30° C. is selected as the column temperature, the peak number and rosmarinic acid separation effect are better, the peak purity is higher, and the baseline fluctuation is smaller.

[0015] According to an embodiment of the present invention, the flow rate of the mobile phase is 0.8-1.2 mL / min. According to a specific embodiment of the present invention, when the flow rate of the mobile phase is 1.0 mL / min, the number of peaks obtained is large, the separation degree of rosmarinic acid is high, and the baseline fluctuation is small.

[0016] According to a specific embodiment of the present invention, a method for establishing a fingerprint of a Baoxin Avicin wood agent comprises the following steps: diluting the Baoxin Avicin wood agent in methanol to obtain a sample to be tested, and obtaining a fingerprint of the Baoxin Avicin wood agent by high performance liquid chromatography. Wherein, the volume fraction of the methanol is 90% to 100%, preferably 98%-100% methanol for dilution. The volume ratio of the Baoxin Avicin wood agent to the methanol is 1:0.5 to 2, preferably 1:1. The injection volume of the sample to be tested is 15-20 μL, preferably 10 μL. The detection wavelength is 230-251 nm, and the detection wavelength is preferably 235 nm. A chromatographic column with a column length of 150 mm, an inner diameter of 4.6 mm, and a particle size of 2.7 μm is used, and preferably the chromatographic column model is a Waters CORTECS T3 chromatographic column. The column temperature of the chromatographic column is set to 25 to 35 ° C, preferably 30 ° C. The flow rate of the mobile phase is set to 0.8-1.2 mL / min, preferably 1.0 mL / min. The elution method is gradient elution. The procedure of the gradient elution is:

[0017] Time (min) Acetonitrile (%) 0.1% by volume phosphoric acid aqueous solution (%) 0～35 0→21 100→79 35～40 21 79 40～60 21→55 79→45 60～65 55→0 45→100 65～75 0 100

[0018] The inventors found that the fingerprint of the Baoxin Avicennia preparation can be obtained quickly and effectively under the conditions, and the separation degree of each component in the Baoxin Avicennia preparation is high. The obtained fingerprint can comprehensively reflect the quality information of the Baoxin Avicennia preparation. The method has simple steps, convenient and fast operation, low detection cost, good reproducibility and specificity, high precision, and good systematicity and characteristic.

[0019] In the second aspect of the present invention, the present invention provides a fingerprint spectrum of Baoxin Avicennia wood preparation. According to an embodiment of the present invention, the fingerprint spectrum is measured according to the method described in the first aspect.

[0020] In the third aspect of the present invention, the present invention proposes a fingerprint spectrum of Baoxin Avicel wood preparation. According to an embodiment of the present invention, the spectrum has characteristic peaks at at least one of the following positions:

[0021] 8.514±5%min, 8.840±5%min, 10.320±5%min, 10.633±5%min, 10.937±5%min, 13.827±5%min, 16.817±5%min, 17.215±5%min, 18.032±5 %min, 18.243±5%min, 19.420±5%min, 21.769±5%min, 23.429±5%min, 24.272±5%min, 25.888±5%min, 26.481±5%min, 28.256±5%min, 2 8.718±5%min, 30.160±5%min, 31.544±5%min, 31.840±5%min, 32.214±5%min, 32.380±5%min, 34.260±5%min, 35.014±5%min, 36.039± 5%min, 36.534±5%min, 37.921±5%min, 38.313±5%min, 38.654±5%min, 46.815±5%min, 47.493±5%min, 47.995±5%min, 55.999±5%min,

[0022] Among them, the peaks at the positions are represented as follows: 19.420±5%min represents caffeic acid, 23.429±5%min represents crocin, 25.888±5%min represents agarwood tetraol, 31.544±5%min represents isoquercetin, 36.039±5%min represents hesperidin, 37.921±5%min represents rosmarinic acid, 46.815±5%min represents cynarin, and 55.999±5%min represents linalool.

[0023] According to an embodiment of the present invention, the fingerprint has at least 34 characteristic peaks, numbered 1 to 34 from left to right, and taking the chromatographic peak of rosmarinic acid numbered 28 as a reference, 1: relative retention time: 0.225±5%, relative peak area: 0.453±5%, 2: relative retention time: 0.233±5%, relative peak area: 0.098±5%, 3: relative retention time: 0.272±5%, relative peak area: 0.461±5%, 4: relative retention time: 0.281±5%, relative peak area: 0.136±5%, 5: relative retention time: 0.289±5%, relative peak area: 0.293±5%, 6: relative retention time: 0.365±5%, relative peak area: 0 .154±5%, 7: Relative retention time: 0.444±5%, relative peak area: 0.116±5%, 8: Relative retention time: 0.454±5%, relative peak area: 0.092±5%, 9: Relative retention time: 0.476±5%, relative peak area: 0.478±5%, 10: Relative retention time: 0.481±5%, relative peak area: 0.054±5%, 11: Relative retention time: 0.512±5%, relative peak area: 0.133±5%, 12: Relative retention time: 0.574±5%, relative peak area: 0.310±5%, 13: Relative retention time: 0.618±5%, relative peak area: 0.145±5%, 14: Relative retention time: 0.618±5%, relative peak area: 0.145±5%, Relative retention time: 0.640 ± 5%, relative peak area: 0.088 ± 5%, 15: Relative retention time: 0.683 ± 5%, relative peak area: 0.174 ± 5%, 16: Relative retention time: 0.698 ± 5%, relative peak area: 0.090 ± 5%, 17: Relative retention time: 0.745 ± 5%, relative peak area: 0.930 ± 5%, 18: Relative retention time: 0.757 ± 5%, relative peak area: 0.108 ± 5%, 19: Relative retention time: 0.796 ± 5%, relative peak area: 0.242 ± 5%, 20: Relative retention time: 0.832 ± 5%, relative peak area: 0.311 ± 5%, 21: Relative retention time: 0.840 ± 5%, relative peak area: 0.179±5%, 22: relative retention time: 0.850±5%, relative peak area: 0.085±5%, 23: relative retention time: 0.854±5%, relative peak area: 0.472±5%, 24: relative retention time: 0.904±5%, relative peak area: 0.096±5%, 25: relative retention time: 0.923±5%, relative peak area: 0.095±5%, 26: relative retention time: 0.950±5%, relative peak area: 0.280±5%, 27: relative retention time: 0.963±5%, relative peak area: 0.225±5%, 28: relative retention time: 1.000±5%, relative peak area: 1.000±5%, 29: relative retention time: 1.010±5%, relative peak area: 0.112±5%, 30: relative retention time: 1.019±5%, relative peak area: 0.204±5%, 31: relative retention time: 1.235±5%, relative peak area: 0.108±5%, 32: relative retention time: 1.252±5%, relative peak area: 0.058±5%, 33: relative retention time: 1.266±5%, relative peak area: 0.119±5%, 34: relative retention time: 1.477±5%, relative peak area: 0.063±5%. Therefore, the fingerprint can be used as a standard spectrum, including most of the components contained in the effective part of Baoxin Avicel preparation, or all of the index components, which can comprehensively and effectively reflect the quality information of qualified Baoxin Avicel preparation. .

[0024] In a fourth aspect of the present invention, the present invention proposes a method for determining the composition of the Baoxin Avicel agent. According to an embodiment of the present invention, the method comprises: performing high performance liquid chromatography analysis on a sample of the Baoxin Avicel agent to be tested, and determining the composition of the Baoxin Avicel agent based on the obtained liquid chromatography analysis results, wherein the high performance liquid chromatography analysis conditions are as defined above. The method according to an embodiment of the present invention can obtain the effective ingredient information of the Baoxin Avicel agent and determine its composition and content.

[0025] According to an embodiment of the present invention, the content of the agent component of the Baoxin Avicennia wood is determined based on the obtained liquid chromatography analysis results.

[0026] In the fifth aspect of the present invention, the present invention proposes a method for detecting the quality or authenticity of Baoxin Avicii wood agent. According to an embodiment of the present invention, the method comprises: comparing the obtained Baoxin Avicii wood agent fingerprint with the Baoxin Avicii wood agent control fingerprint. It should be noted that the "Baoxin Avicii wood agent control fingerprint" described in the present application refers to the fingerprint of a Baoxin Avicii wood agent product of known qualified quality, which can be obtained by the method for constructing a fingerprint described in the present application, or can be obtained by purchase. Based on the comparison results, the quality or authenticity of the Baoxin Avicii wood agent to be tested is determined.

[0027] According to an embodiment of the present invention, the above method for detecting the quality or authenticity of the Baoxin Avicii wood agent may further include at least one of the following additional technical features:

[0028] According to an embodiment of the present invention, the method for determining the control fingerprint of the Baoxin Avicennia agent is the same as the method for determining the fingerprint of the Baoxin Avicennia agent to be tested. According to an embodiment of the present invention, the method for determining the control fingerprint of the Baoxin Avicennia agent is the same as the method for determining the fingerprint of the Baoxin Avicennia agent to be tested, so that the interference of factors such as environment and instruments can be eliminated, the judgment result has high credibility, good reproducibility and high stability.

[0029] According to an embodiment of the present invention, when the similarity between the fingerprint of the tested Baoxin Avicel agent and the control fingerprint of the Baoxin Avicel agent is not less than 0.9, it indicates that the quality of the tested Baoxin Avicel agent is qualified.

[0030] According to an embodiment of the present invention, when the similarity between the fingerprint of the Baoxin Avicennia wood preparation to be tested and the control fingerprint of the Baoxin Avicennia wood preparation is not less than 0.9, it indicates that the Baoxin Avicennia wood preparation to be tested is authentic.

[0031] According to an embodiment of the present invention, the similarity judgment standard is to import the fingerprint of the sample to be tested into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012.130723 version) software, set the reference spectrum, perform multi-point correction and Mark peak matching, and the software automatically calculates the similarity result.

[0032] In the fourth aspect of the present invention, the present invention proposes a method for determining the production process of Baoxin Avicel agent. According to an embodiment of the present invention, the method comprises: (1) preparing Baoxin Avicel agent by using a candidate process; (2) obtaining the fingerprint of the prepared Baoxin Avicel agent by using the method described above; (3) comparing the fingerprint of the Baoxin Avicel agent to be tested with the reference fingerprint of the Baoxin Avicel agent; and (4) determining the final production process of Baoxin Avicel agent based on the comparison result. Through the above process screening, a production process for producing high-quality Baoxin Avicel agent can be obtained.

[0033] According to an embodiment of the present invention, the above method for determining the production process of Baoxin Avicel wood agent may further include at least one of the following additional technical features:

[0034] According to a specific embodiment of the invention, a candidate process is used to prepare a Baoxin Avicillin agent, and then the Baoxin Avicillin agent is diluted in 98%-100% methanol at a ratio of 1:1 to obtain a sample to be tested, and a fingerprint of the Baoxin Avicillin agent is obtained by high performance liquid chromatography. The injection volume of the sample to be tested is 10 μL. The detection wavelength is 235 nm. A Waters CORTECS T3 chromatographic column with a column length of 150 mm, an inner diameter of 4.6 mm, and a particle size of 2.7 μm is used. The column temperature of the chromatographic column is set to 25-35°C. The flow rate of the mobile phase is set to 0.8-1.2 mL / min. The elution method is gradient elution. The procedure for the gradient elution is:

[0035]

[0036]

[0037] The fingerprint of the prepared Baoxin Avicel preparation is obtained under the conditions of the high performance liquid chromatography, and then the fingerprint of the Baoxin Avicel preparation to be tested is compared with the control fingerprint of the Baoxin Avicel preparation, and finally the production process of the final Baoxin Avicel preparation is determined based on the comparison result. Through the above process screening, the production process of producing high-quality Baoxin Avicel preparation can be obtained.

[0038] According to an embodiment of the present invention, the method for determining the control fingerprint of the Baoxin Avicennia agent is the same as the method for determining the fingerprint of the Baoxin Avicennia agent to be tested, which can eliminate the interference of factors such as environment and instruments, has high credibility, good reproducibility and high stability.

[0039] According to an embodiment of the present invention, if the similarity between the fingerprint of the Baoxin Avicii agent to be tested and the control fingerprint of the Baoxin Avicii agent is not less than 0.9, the candidate process is selected as the final production process of the Baoxin Avicii agent, and if the similarity between the fingerprint of the Baoxin Avicii agent to be tested and the control fingerprint of the Baoxin Avicii agent is less than 0.9, the candidate process should be optimized. According to an embodiment of the present invention, the above similarity comparison method can ensure the high-quality production process of the Baoxin Avicii agent.

[0040] According to an embodiment of the present invention, the method for optimizing the process includes adjusting at least one of the following processing parameters: formulation and storage conditions. This can optimize the production process of Baoxin Avicel formulation more concisely and effectively.

[0041] Those skilled in the art will appreciate that the method and effect of determining the position peaks in the above-mentioned spectrum are also applicable to the method of establishing the characteristic spectrum of the Baoxin Avicel preparation and the method of determining the composition of the Baoxin Avicel preparation.

[0042] The above and / or additional aspects and advantages of the present invention will become apparent from the description of the embodiments in conjunction with the following drawings or will be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The chromatogram of the test solution in the mobile phase aqueous phase investigation according to Example 1 of the present invention is shown;

[0044] Figure 2 The chromatogram of the test solution in the mobile phase organic phase investigation according to Example 2 of the present invention is shown;

[0045] Figure 3 The chromatogram of the test solution in the chromatographic column investigation according to Example 3 of the present invention is shown;

[0046] Figure 4 The chromatogram of the test solution in the gradient elution procedure investigation according to Example 4 of the present invention is shown;

[0047] Figure 5 The chromatogram of the test solution in the column temperature investigation according to Example 5 of the present invention is shown;

[0048] Figure 6 The chromatogram of the test solution in the flow rate investigation according to Example 6 of the present invention is shown;

[0049] Figure 7 The chromatogram of the test solution in the detection wavelength investigation according to Example 7 of the present invention is shown;

[0050] Figure 8 The chromatogram of the test solution in the sample injection amount investigation according to Example 8 of the present invention is shown;

[0051] Fig. 9 The chromatogram of the test solution in the dilution multiple investigation according to Example 9 of the present invention is shown;

[0052] Fig.10 The fingerprint of Baoxin Aiweiximu oral liquid established according to Example 10 of the present invention is shown;

[0053] Fig.11 The fingerprints of different batches of Baoxin Avicennia oral liquid according to Example 11 of the present invention are shown. DETAILED DESCRIPTION

[0054] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in this field or the product specifications are used. The instruments or instruments used without specifying the manufacturer are all conventional products that can be obtained commercially.

[0055] The test sample Baoxin Aiweiximu oral liquid used in the following examples includes 10 medicinal ingredients such as silkworm cocoons, fragrant blue orchid, lavender, ox tongue grass, and saffron.

[0056] Example 1: Investigation of mobile phase water

[0057] Preparation of test solution: Accurately pipette 5 mL of Baoxin Avicim oral solution into a 10 mL volumetric flask, add methanol to dilute to the scale, shake well, filter using a 0.22 μm microporous filter membrane, collect the filtrate to obtain the test solution.

[0058] Preparation of reference solution: Accurately weigh 2 mg of rosmarinic acid reference substance, place it in a 100 mL volumetric flask, add 50% methanol to the scale, oscillate and mix to obtain the rosmarinic acid reference solution.

[0059] Test method: Use acetonitrile-phosphoric acid aqueous solution, acetonitrile-acetic acid aqueous solution, phosphoric acid-water and phosphoric acid-ammonia aqueous solution as mobile phases, and test the test solution and reference solution according to the following chromatographic conditions:

[0060] Chromatographic column 1: YMC-Triart C18 (length 150 mm, inner diameter 4.6 mm, particle size 5 μm), flow rate 1.0 mL / min, column temperature 30 °C, detection wavelength 251 nm, injection volume 10 μL per test;

[0061] Table 1: Gradient elution program 1

[0062] Time (min) Organic phase (%) Water phase (%) 0～50 5～30 95～70 50～52 30～90 70～10 52～57 90 10 57～60 90～5 10～95 60～70 5 95

[0063] Results: Figure 1 As shown, when water or 0.05% ammonia water by volume is used as the aqueous mobile phase, the number of peaks in the test sample chromatogram is small and the response values ​​of each peak are uneven; when 0.1% phosphoric acid by volume and 0.2% acetic acid by volume are used as the aqueous mobile phase, the number of peaks in the test sample chromatogram is relatively complete, but when 0.1% phosphoric acid by volume is used as the aqueous mobile phase, the separation degree of rosmarinic acid is greater, which meets the requirements and the peak purity is relatively good, which is conducive to the separation of the target compound.

[0064] Example 2: Investigation of the organic phase of the mobile phase

[0065] Preparation of test solution: Accurately pipette 5 mL of Baoxin Avicim oral solution into a 10 mL volumetric flask, add methanol to dilute to the scale, shake well, filter using a 0.22 μm microporous filter membrane, collect the filtrate to obtain the test solution.

[0066] Preparation of reference solution: Accurately weigh 2 mg of rosmarinic acid reference substance, place it in a 100 mL volumetric flask, add 50% methanol to the scale, oscillate and mix to obtain the rosmarinic acid reference solution.

[0067] Test method: Use acetonitrile-0.1% phosphoric acid aqueous solution and methanol-0.1% phosphoric acid aqueous solution as mobile phases, and test the test solution and reference solution according to the following chromatographic conditions:

[0068] Chromatographic column 1: YMC-Triart C18 (length 150 mm, inner diameter 4.6 mm, particle size 5 μm); flow rate: 1.0 mL / min; column temperature: 30°C; detection wavelength: 251 nm; injection volume for each detection: 10 μL.

[0069] Table 2: Gradient elution program 2

[0070] Time (min) Organic phase (%) Water phase (%) 0～50 5～30 95～70 50～52 30～90 70～10 52～57 90 10 57～60 90～5 10～95 60～70 5 95

[0071] Results: Figure 2 As shown, when acetonitrile is used as the organic mobile phase, the number of chromatographic peaks of the test sample is relatively complete, the separation degree of rosmarinic acid is higher and the peak purity is better, which is conducive to the separation of the target compound; when methanol is used as the organic phase, the result is not ideal.

[0072] Example 3: Investigation of chromatographic columns

[0073] Preparation of test solution: Accurately pipette 5 mL of Baoxin Avicim oral solution into a 10 mL volumetric flask, add methanol to dilute to the scale, shake well, filter using a 0.22 μm microporous filter membrane, collect the filtrate to obtain the test solution.

[0074] Preparation of reference solution: Accurately weigh 2 mg of rosmarinic acid reference substance, place it in a 100 mL volumetric flask, add 50% methanol to the scale, oscillate and mix to obtain the rosmarinic acid reference solution.

[0075] Test method: Use chromatographic column 1: YMC-Triart C18 (length 150mm, inner diameter 4.6mm, particle size 5μm), chromatographic column 2: XSelect HSS T3 (length 150mm, inner diameter 4.6mm, particle size 5μm), chromatographic column 3: WatersCORTECS T3 (length 150mm, inner diameter 4.6mm, particle size 2.7μm) and chromatographic column 4: ACE Excel 5C18-AR (length 150mm, inner diameter 4.6mm, particle size 5μm) as chromatographic columns, and test the test solution and reference solution according to the following chromatographic conditions:

[0076] Organic phase: acetonitrile; aqueous phase: 0.1% phosphoric acid by volume; flow rate: 1.0 mL / min; column temperature: 30°C; detection wavelength: 251 nm; injection volume per test: 10 μL;

[0077] Table 3: Gradient elution program 3

[0078] Time (min) Organic phase (%) Water phase (%) 0～50 5～30 95～70 50～52 30～90 70～10 52～57 90 10 57～60 90～5 10～95 60～70 5 95

[0079] Results: Figure 3 As shown in the figure, the Waters CORTECS T3 column has the largest number of chromatographic peaks, the peak heights of each peak are relatively balanced, the separation of rosmarinic acid is high and the peak purity is relatively good; the YMC-Triart C18 column has the least number of chromatographic peaks, the peak heights of each peak are relatively balanced, the separation of rosmarinic acid is high and the peak purity is relatively good; the XSelect HSS T3 column has a medium number of chromatographic peaks, the peak heights of each peak are relatively balanced, but rosmarinic acid is not separated, and the differences in purity angle and purity threshold are large; the ACE Excel 5C18-AR column has a medium number of chromatographic peaks, the peak heights of each peak are relatively balanced, the separation of rosmarinic acid is high, but the differences in purity angle and purity threshold are large.

[0080] Example 4: Investigation of gradient elution procedure

[0081] Preparation of test solution: Accurately pipette 5 mL of Baoxin Avicim oral solution into a 10 mL volumetric flask, add methanol to dilute to the scale, shake well, filter using a 0.22 μm microporous filter membrane, collect the filtrate to obtain the test solution.

[0082] Preparation of reference solution: Accurately weigh 2 mg of rosmarinic acid reference substance, place it in a 100 mL volumetric flask, add 50% methanol to the scale, oscillate and mix to obtain the rosmarinic acid reference solution.

[0083] Test method: Use the gradient elution procedures in Tables 3 to 10 and test the test solution and reference solution according to the following chromatographic conditions:

[0084] Chromatographic column 3: Waters CORTECS T3 (length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm), flow rate: 1.0 mL / min, column temperature: 30°C; detection wavelength: 251 nm; injection volume: 10 μL; organic phase: acetonitrile, aqueous phase: 0.1% phosphoric acid by volume;

[0085] Table 4: Gradient elution program 4

[0086] Time (min) Organic phase (%) Water phase (%) 0～60 0～24 100～76 60～65 24～35 76～65 65～70 35～0 65～100 70～80 0 100

[0087] Table 5: Gradient elution program 5

[0088] Time (min) Organic phase (%) Water phase (%) 0～25 0～25 100～70 25～35 25～50 75～5 35～40 50～0 5～100 40～50 0 100

[0089] Table 6: Gradient elution program 6

[0090]

[0091] Table 7: Gradient elution program 7

[0092] Time (min) Organic phase (%) Water phase (%) 0～30 0～17 100～83 30～45 17 83 45～54 17～32 83～68 54～56 32～0 68～100 56～65 0 100

[0093] Table 8: Gradient elution program 8

[0094]

[0095]

[0096] Table 9: Gradient elution program 9

[0097] Time (min) Organic phase (%) Water phase (%) 0～35 0～25 100～75 35～38 25 75 38～45 25～60 75～40 45～50 60～0 40～100 50～60 0 100

[0098] Table 10: Gradient elution program 10

[0099] Time (min) Organic phase (%) Water phase (%) 0～35 0～21 100～79 35～40 21 79 40～60 21～55 79～45 60～65 55～0 45～100 65～75 0 100

[0100] Results: Figure 4 The gradient elution programs shown in Tables 3-10 correspond to Figure 4Conditions 1-8 in the elution procedure), under gradient elution program 3, the baseline fluctuated greatly and the peak purity did not meet the requirements; under gradient elution programs 4 and 7, although the separation of rosmarinic acid was good, the rosmarinic acid peak was impure and did not meet the requirements because the purity angle was much greater than the purity threshold; under gradient elution programs 5 and 6, although the number of peaks was large, the separation of rosmarinic acid was small; under gradient elution program 8, the separation of rosmarinic acid met the requirements, the highest organic phase was 32%, and there were still peaks after the organic phase was increased, so the organic phase needed to be further increased; under gradient elution program 9, the separation of rosmarinic acid met the requirements, but the peak purity did not meet the requirements, and the baseline fluctuated greatly; under gradient elution program 10, the separation of rosmarinic acid was the largest, and the chromatographic peak purity was high, all the chromatographic peaks were analyzed, the number of chromatographic peaks was large, and the baseline fluctuation was small.

[0101] Example 5: Investigation of column temperature

[0102] Preparation of test solution: Accurately pipette 5 mL of Baoxin Avicim oral solution into a 10 mL volumetric flask, add methanol to dilute to the scale, shake well, filter using a 0.22 μm microporous filter membrane, collect the filtrate to obtain the test solution.

[0103] Preparation of reference solution: Accurately weigh 2 mg of rosmarinic acid reference substance, place it in a 100 mL volumetric flask, add 50% methanol to the scale, oscillate and mix to obtain the rosmarinic acid reference solution.

[0104] Test method: Use 25℃, 30℃, and 35℃ as column temperatures, and test the test solution and reference solution according to the following chromatographic conditions:

[0105] Chromatographic column 3: Waters CORTECS T3 (length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm), flow rate: 1.0 mL / min, detection wavelength: 251 nm, injection volume: 10 μL, organic phase: acetonitrile, aqueous phase: 0.1% phosphoric acid by volume, elution conditions: gradient elution program 10 (as shown in Table 10).

[0106] Results: Figure 5 As shown, when the column temperature is 25℃, the separation degree of rosmarinic acid meets the requirements, but the peak is impure; when the column temperature is 30℃ and 35℃, the number of peaks, the separation effect of rosmarinic acid, and the peak purity are equivalent, but the baseline fluctuation at 30℃ is smaller.

[0107] Example 6: Investigation of flow rate

[0108] Preparation of test solution: Accurately pipette 5 mL of Baoxin Avicim oral solution into a 10 mL volumetric flask, add methanol to dilute to the scale, shake well, filter using a 0.22 μm microporous filter membrane, collect the filtrate to obtain the test solution.

[0109] Preparation of reference solution: Accurately weigh 2 mg of rosmarinic acid reference substance, place it in a 100 mL volumetric flask, add 50% methanol to the scale, oscillate and mix to obtain the rosmarinic acid reference solution.

[0110] Test method: The test solution and the reference solution were tested at flow rates of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min respectively according to the following chromatographic conditions:

[0111] Chromatographic column 3: Waters CORTECS T3 (length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm), column temperature: 30°C, detection wavelength: 251 nm, injection volume: 10 μL, organic phase: acetonitrile, aqueous phase: 0.1% phosphoric acid by volume, elution conditions: gradient elution program 10 (as shown in Table 10).

[0112] Results: Figure 6 As shown, with the increase of flow rate, the number of peaks decreased to a certain extent. When the flow rate was 1.0 mL / min, the number of peaks was large, the separation degree of rosmarinic acid was high, and the baseline fluctuation was small.

[0113] Example 7: Investigation of detection wavelength

[0114] Preparation of test solution: Accurately pipette 5 mL of Baoxin Avicim oral solution into a 10 mL volumetric flask, add methanol to dilute to the scale, shake well, filter using a 0.22 μm microporous filter membrane, collect the filtrate to obtain the test solution.

[0115] Preparation of reference solution: Accurately weigh 2 mg of rosmarinic acid reference substance, place it in a 100 mL volumetric flask, add 50% methanol to the scale, oscillate and mix to obtain the rosmarinic acid reference solution.

[0116] Test method: Use 220nm, 230nm, 235nm, 240nm, 251nm and 330nm as detection wavelengths respectively, and test the test solution and the reference solution according to the following chromatographic conditions:

[0117] Chromatographic column 3: Waters CORTECS T3 (length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm), column temperature: 30°C, flow rate: 1.0 mL / min, injection volume: 10 μL, organic phase: acetonitrile, aqueous phase: 0.1% phosphoric acid by volume, elution conditions: gradient elution program 10 (as shown in Table 10).

[0118] Results: Figure 7As shown in the figure, under the same integration parameters (including peak width, threshold, minimum peak height, minimum peak area, prohibited integration time, etc.), when the detection wavelength is 220nm, although the number of chromatographic peaks is the largest, the chromatographic peak response is slightly unbalanced, the chromatogram baseline fluctuates greatly, the baseline noise is large, and the wavelength is close to the solvent cutoff wavelength, which is not suitable as the optimal detection wavelength; when the detection wavelength is 230nm and 235nm, the number of peaks is not much different, and the difference in the number of peaks is mainly before 5min, but when the detection wavelength is 235nm, the baseline of the test sample is flatter and the chromatographic peaks are more balanced; when the detection wavelength is 240nm and 251nm, the baseline fluctuation is small and the chromatographic peak response is balanced, but the number of peaks is less than that of 235nm; when the detection wavelength is 330nm, the number of chromatographic peaks is small and the response value is uneven.

[0119] Example 8: Investigation of injection volume

[0120] Preparation of test solution: Accurately pipette 5 mL of Baoxin Avicim oral solution into a 10 mL volumetric flask, add methanol to dilute to the scale, shake well, filter using a 0.22 μm microporous filter membrane, collect the filtrate to obtain the test solution.

[0121] Preparation of reference solution: Accurately weigh 2 mg of rosmarinic acid reference substance, place it in a 100 mL volumetric flask, add 50% methanol to the scale, oscillate and mix to obtain the rosmarinic acid reference solution.

[0122] Test method: Use 10μL, 15μL and 20μL as the injection volume, and test the test solution and reference solution according to the following chromatographic conditions:

[0123] Chromatographic column 3: Waters CORTECS T3 (length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm), column temperature: 30°C, flow rate: 1.0 mL / min, detection wavelength: 235 nm, organic phase: acetonitrile, aqueous phase: 0.1% phosphoric acid by volume, elution conditions: gradient elution program 10 (as shown in Table 10).

[0124] Results: Figure 8 As shown, compared with the injection volumes of 15 μl and 20 μl, when the injection volume is 10 μl, the responses of the chromatographic peaks are more balanced and the peak areas have a higher response, the baseline fluctuation is smaller, and the separation and peak purity of rosmarinic acid meet the requirements.

[0125] Example 9: Investigation of preparation of test solution

[0126] 1. Investigation of dilution solvent types

[0127] Accurately pipette 5 mL of Baoxin Avicim oral solution into a 10 mL volumetric flask, dilute to the mark with water, ethanol and methanol respectively, shake well to obtain the test solution.

[0128] The test solutions prepared above were respectively injected into a high performance liquid chromatograph and tested according to the following chromatographic conditions:

[0129] Chromatographic column 3: Waters CORTECS T3 (length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm), column temperature: 30°C, detection wavelength: 235 nm, flow rate: 1.0 mL / min, injection volume: 10 μL, organic phase: acetonitrile, aqueous phase: 0.1% phosphoric acid by volume, elution program: gradient elution program 10 (as shown in Table 10).

[0130] Table 11: Results of investigation of different types of dilution solvents - relative peak areas

[0131]

[0132]

[0133] Results: As shown in Table 11, the RSDs of the relative peak areas of the characteristic peaks of the test solutions using different dilution solvents were all within 5.0%, but when methanol was used as the dilution solvent, the number of peaks was the largest.

[0134] 2. Investigation of dilution solvent concentration

[0135] Accurately pipette 5 mL of Baoxin Avicim oral solution into a 10 mL volumetric flask, dilute to the scale with 100% methanol, 25% methanol, 50% methanol and 75% methanol by volume, shake well to obtain the test solution.

[0136] The test solutions prepared above were respectively injected into a high performance liquid chromatograph and tested according to the following chromatographic conditions:

[0137] Chromatographic column 3: Waters CORTECS T3 (length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm), column temperature: 30°C, detection wavelength: 235 nm, flow rate: 1.0 mL / min, injection volume: 10 μL, organic phase: acetonitrile, aqueous phase: 0.1% phosphoric acid by volume, elution program: gradient elution program 10 (as shown in Table 10).

[0138] Table 12: Results of investigation of different concentrations of dilution solvents - relative peak area

[0139]

[0140]

[0141] Among them, N / A means not applicable.

[0142] Results: As shown in Table 12, the RSD values ​​of the relative retention time of each characteristic peak of the test solution diluted with different concentrations of methanol were all within 5.0%, and the RSD values ​​of the relative peak area were all within 15.0%. When 100% methanol was used as the dilution solvent, the number of peaks was the largest and the relative peak area of ​​the chromatographic peak was higher overall.

[0143] 3. Investigation of dilution multiples

[0144] Accurately pipette Baoxin Avicim oral solution, dilute it 1.25 times, 2 times, 3.3 times and 5 times respectively with methanol as the dilution solvent, shake well to obtain the test solution.

[0145] The test solutions prepared above were respectively injected into a high performance liquid chromatograph and tested according to the following chromatographic conditions:

[0146] Chromatographic column 3: Waters CORTECS T3 (length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm), column temperature: 30°C, detection wavelength: 235 nm, flow rate: 1.0 mL / min, injection volume: 10 μL, organic phase: acetonitrile, aqueous phase: 0.1% phosphoric acid by volume, elution program: gradient elution program 10 (as shown in Table 10).

[0147] Results: Fig. 9 As shown in the figure, the separation and peak purity of rosmarinic acid at different dilution factors meet the requirements, and the dilution factor is inversely correlated with the number of peaks; when the dilution factor is 1.25, the number of peaks is the largest, but the baseline fluctuates greatly; when the dilution factor is 3 and 5 times, the number of peaks is small; when the dilution factor is 2 times, the number of peaks is large, and the baseline fluctuates less.

[0148] Example 10: Establishment of fingerprint of Baoxin Avicennia oral liquid

[0149] (1) Preparation of reference solution: Take rosmarinic acid reference substance and add methanol to prepare reference solution, wherein the concentration of rosmarinic acid in the reference solution is 60 μg / mL;

[0150] (2) Preparation of test solution: Accurately pipette 5 mL of Baoxin Avicim oral solution into a 10 mL volumetric flask, dilute to scale with methanol, shake well, filter, and collect the filtrate to obtain the solution;

[0151] (3) Chromatographic conditions: Column 3: Waters CORTECS T3 (length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm), column temperature: 30 °C, detection wavelength: 235 nm, flow rate: 1.0 mL / min, organic phase: acetonitrile, aqueous phase: 0.1% volume fraction of phosphoric acid, elution program: gradient elution program 10;

[0152] (4) Determination: Accurately pipette 10 μL of the reference solution and the test solution into a high performance liquid chromatograph. The fingerprint of the obtained Baoxin Avicel preparation contains Fig.10 The 34 characteristic peaks shown are identified as follows: 11 is caffeic acid, 13 is crocin, 15 is agalate tetraol, 20 is isoquercetin, 26 is hesperidin, 28 is rosmarinic acid, 31 is cynarin, and 34 is linalool.

[0153] Example 11: Method Validation

[0154] 1. Exclusivity

[0155] (1) Preparation of reference solution: Take rosmarinic acid reference substance and add methanol to prepare reference solution, wherein the concentration of rosmarinic acid in the reference solution is 60 μg / mL;

[0156] (2) Preparation of blank solution: Accurately pipette 5 mL of methanol blank solvent into a 10 mL volumetric flask, dilute to scale with methanol, shake well, filter, and collect the filtrate;

[0157] (3) Preparation of auxiliary material solution: Accurately pipette 5 mL of auxiliary material solution (accurately weigh an appropriate amount of refined honey, add an appropriate amount of solvent, shake well, filter, and collect the filtrate to obtain the solution) into a 10 mL volumetric flask, dilute to the mark with methanol, shake well, filter, and collect the filtrate to obtain the solution;

[0158] (4) Preparation of test solution: Accurately pipette 5 mL of Baoxin Avicim oral solution into a 10 mL volumetric flask, dilute to scale with methanol, shake well, filter, and collect the filtrate to obtain the solution;

[0159] (5) Preparation of the solution of the three-flavor negative preparation: accurately pipette 5 mL of the solution of the three-flavor negative preparation of Herba Glehniae, Lavender and Herba Cinerariae into a 10 mL volumetric flask, dilute to the mark with methanol, shake well, filter, and collect the filtrate to obtain the solution;

[0160] (6) Chromatographic conditions: Column 3: Waters CORTECS T3 (length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm), column temperature: 30°C, detection wavelength: 235 nm, flow rate: 1.0 mL / min, gradient elution program 10 (as shown in Table 10), organic phase: acetonitrile, aqueous phase: 0.1% by volume phosphoric acid;

[0161] (7) Determination: Accurately pipette 10 μL of reference solution, blank solution, excipient solution and test solution into a high performance liquid chromatograph and record the chromatograms and peak areas.

[0162] Results: As shown in Table 13, the blank solution, excipient solution and negative preparation did not interfere with the determination of rosmarinic acid in the test sample, and the separation degree of rosmarinic acid in the test sample solution was greater than 1.5, and the specificity of rosmarinic acid was good.

[0163] Table 13: Specificity test of Baoxin Avicim oral solution

[0164]

[0165] Among them, N / A means not detected or not applicable

[0166] 2. Precision

[0167] The test solution of Baoxin Avicim oral solution was prepared according to the method in Example 10, and the sample was injected 6 times continuously according to its chromatographic conditions to examine the precision of the instrument. The relative retention time and peak area of ​​34 common peaks contained in the sample were calculated with reference to the retention time and peak area of ​​28:.

[0168] Results: The RSD of the relative retention time of each common peak was less than 1%, and the RSD of the relative peak area was less than 5%, which met the technical requirements of the fingerprint spectrum and showed that the precision of the instrument was good.

[0169] 3. Repeatability

[0170] According to the method in Example 10, a reference solution and 6 samples of Baoxin Avicim oral liquid were prepared, and the chromatographic conditions were followed to measure the chromatograms and peak areas. The relative retention times and peak areas of the 34 common peaks in the samples were calculated with the retention time and peak area of ​​28 as reference.

[0171] Table 14: Repeatability test of Baoxin Avicim oral solution - relative retention time

[0172]

[0173]

[0174] Table 15: Repeatability test of Baoxin Avicim oral solution - relative peak area

[0175]

[0176]

[0177] Results: The fingerprints of the six samples were consistent, with 34 corresponding characteristic peaks. Taking the 28th chromatographic peak as the reference peak, the average relative retention times of the remaining chromatographic peaks were: 0.225, 0.233, 0.272, 0.281, 0.289, 0.365, 0.444, 0.454, 0.476, 0.481, 0.512, 0.574, 0.61 8, 0.640, 0.683, 0.698, 0.745, 0.757, 0.796, 0.832, 0.840, 0.850, 0.854, 0.904, 0.923, 0.950, 0.963, 1.000, 1.010, 1.019, 1.235, 1.252, 1.266 and 1.477; average relative peak The areas are: 0.453, 0.098, 0.461, 0.136, 0.293, 0.154, 0.116, 0.092, 0.478, 0.054, 0.133, 0.310, 0.145, 0.088, 0.174, 0.090, 0.930, 0.108, 0.242, 0.311, 0.179, 0 .085, 0.472, 0.096, 0.095, 0.280, 0.225, 1.000, 0.112, 0.204, 0.108, 0.058, 0.119, and 0.063. The RSDs of the relative retention times of the characteristic peaks in the fingerprints of the six test samples were all within 5.0%, and the RSD values ​​of the relative peak areas of the characteristic peaks were all within 15.0%.

[0178] 4. Fingerprint similarity evaluation

[0179] 19 batches of Baoxin Aiweiximu oral liquid (numbered 1-19) were tested under the conditions of Example 9, and the chromatograms recorded were imported into the Chinese medicine chromatographic fingerprint similarity evaluation system 2004A version, with 20210118 (S1) as the reference spectrum, the time window was 0.1min, and the obtained spectrum was shown in Fig.11 ,The similarity results are shown in Table 16.

[0180] Table 16: Similarity results:

[0181]

[0182]

[0183] Results: The similarity of 19 batches of Baoxin Avicim oral liquid was between 0.972 and 0.992, with good similarity.

[0184] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0185] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for establishing a fingerprint of a Baoxin Avicipi formulation, characterized in that: include: Performing HPLC analysis on the samples of the Baoxin Avicel agent to be tested; and The fingerprint of the Baoxin Avicel agent is established based on the obtained liquid chromatography analysis results; The sample to be tested is obtained by the following method: Diluting the Baoxin Avicel agent in 90% to 100% methanol by volume to obtain the sample to be tested; The Baoxin Avicipi preparation is an oral preparation; The liquid chromatography analysis adopts the following conditions: The aqueous phase of the mobile phase was 0.1% phosphoric acid aqueous solution; The organic phase of the mobile phase was acetonitrile; The elution mode is gradient elution; The procedure of the gradient elution is: 0-35min, 0-21% acetonitrile, 100-79% 0.1% phosphoric acid aqueous solution by volume; 35-40min, 21% acetonitrile, 79% 0.1% phosphoric acid aqueous solution by volume; 40-60min, 21-55% acetonitrile, 79-45% 0.1% phosphoric acid aqueous solution by volume; 60-65min, 55-0% acetonitrile, 45-100% 0.1% phosphoric acid aqueous solution by volume; 65-75min, 0% acetonitrile, 100% 0.1% phosphoric acid aqueous solution by volume; The detection wavelength is 230-251nm; The chromatographic column used in the liquid chromatography analysis is a Waters CORTECS T3 chromatographic column.

2. The method according to claim 1, characterized in that The liquid chromatography analysis uses a chromatographic column with a column length of 150 mm, an inner diameter of 4.6 mm, and a particle size of 2.7 μm.

3. The method according to claim 1, characterized in that The detection wavelength is 235 nm.

4. The method according to claim 1, characterized in that: The sample to be tested is obtained by the following method: The Baoxin Avicel agent is dissolved in 98%-100% methanol by volume to obtain the sample to be tested.

5. The method according to claim 1, characterized in that The volume ratio of the Baoxin Avicel agent to methanol with a volume fraction of 90% to 100% is 1: (0.5 to 2).

6. The method according to claim 1, characterized in that The volume ratio of the Baoxin Avicel agent to 90% to 100% methanol is 1:

1.

7. The method according to claim 1, characterized in that The injection volume of the sample to be tested is 10 μL.

8. The method according to claim 1, characterized in that The column temperature of the chromatographic column used in the liquid chromatography analysis is 25-35°C.

9. The method according to claim 1, characterized in that: The flow rate of the mobile phase is 0.8-1.2 mL / min.

10. The method according to claim 2, characterized in that The spectrum has characteristic peaks at at least one of the following positions: 8.514±5%min, 8.840±5%min, 10.320±5%min, 10.633±5%min, 10.937±5%min, 13.827±5%min, 16.817±5%min, 17.215±5%min, 18.032±5%min, 18.243±5%min, 19.420±5%min, 21.769±5%min, 23.429±5%min, 24.272±5%min, 25.888±5%min, 26.481±5%min, 28.256±5%min, 28.718±5%min, 30.160±5%min, 31.544±5%min, 31.840±5%min, 32.214±5%min, 32.380±5%min, 34.260±5%min, 35.014±5%min, 36.039±5%min, 36.534±5%min, 37.921±5%min, 38.313±5%min, 38.654±5%min, 46.815±5%min, 47.493±5%min, 47.995±5%min, 55.999±5%min, Among them, the peaks at the positions are represented as follows: 19.420±5%min represents caffeic acid, 23.429±5%min represents crocin, 25.888±5%min represents alaiotetrol, 31.544±5%min represents isoquercetin, 36.039±5%min represents hesperidin, 37.921±5%min represents rosmarinic acid, 46.815±5%min represents cynaroside, and 55.999±5%min represents linalool.

11. The method according to claim 10, characterized in that Taking the retention time of rosmarinic acid as reference, the relative retention time of the characteristic peak is: 0.225±5%，0.233±5%，0.272±5%，0.281±5%，0.289±5%，0.365±5%，0.444±5%，0.454±5%，0.476±5%，0.481±5%，0.512±5%，0.574±5%，0.618±5%，0.640±5%，0.683±5%，0.698±5%，0.745±5%，0.757±5%，0.796±5%，0.832±5%，0.840±5%，0.850±5%，0.854±5%，0.904±5%，0.923±5%，0.950±5%，0.963±5%，1.000±5%，1.010±5%，1.019±5%，1.235±5%，1.252±5%，1.266±5%，1.477±5%。 12. The method according to claim 10, characterized in that Taking the peak area of ​​rosmarinic acid as a reference, the relative peak areas of the characteristic peaks are 0.453±5%, 0.098±5%, 0.461±5%, 0.136±5%, 0.293±5%, 0.154±5%, 0.116±5%, 0.092±5%, 0.478±5%, 0.054±5%, 0.133±5%, 0.310±5%, 0.145±5%, 0.088±5%, 0.174±5%, 0.090 ±5%, 0.930±5%, 0.108±5%, 0.242±5%, 0.311±5%, 0.179±5%, 0.085±5%, 0.472±5%, 0.096±5%, 0.095±5%, 0.280±5%, 0.225±5%, 1.000±5%, 0.112±5%, 0.204±5%, 0.108±5%, 0.058±5%, 0.119±5%, 0.063±5%.

13. A method for determining the composition of Baoxin Avicii wood preparation, characterized in that: include: The samples of Baoxin Avicel agent to be tested were analyzed by high performance liquid chromatography; as well as Based on the obtained liquid chromatography analysis results, the composition of the Baoxin Avicel preparation is determined, Wherein, the high performance liquid chromatography analysis is as defined in any one of claims 1 to 12.

14. The method according to claim 13, characterized in that Further including: Based on the obtained liquid chromatography analysis results, the content of the Baoxin Avicennia wood agent component is determined.

15. A method for detecting the quality or authenticity of Baoxin Avicii wood preparation, characterized in that: include: Obtaining the fingerprint of the Baoxin Avicennia wood preparation to be tested by the method described in any one of claims 1 to 12; Compare the fingerprint of the tested Baoxin Avicennia agent with the control fingerprint of the Baoxin Avicennia agent; Based on the comparison results, the quality or authenticity of the Baoxin Avicii wood preparation to be tested is determined.

16. The method according to claim 15, characterized in that The determination method of the control fingerprint of the Baoxin Avicennia wood preparation is the same as the determination method of the fingerprint of the Baoxin Avicennia wood preparation to be tested.

17. The method according to claim 15, characterized in that The similarity between the fingerprint of the tested Baoxin Avicel preparation and the control fingerprint of the Baoxin Avicel preparation is not less than 0.9, which is an indication that the quality of the tested Baoxin Avicel preparation is qualified.

18. The method according to claim 15, characterized in that The similarity between the fingerprint spectrum of the Baoxin Avicin wood preparation to be tested and the control fingerprint spectrum of the Baoxin Avicin wood preparation is not less than 0.9, which is an indication that the Baoxin Avicin wood preparation to be tested is authentic.

19. A method for determining the production process of Baoxin Avicel, characterized in that: include: (1) Using the candidate process to prepare the Baoxin Avicel agent; (2) obtaining a fingerprint of the prepared Baoxin Avicii wood preparation using the method described in any one of claims 1 to 12; (3) comparing the fingerprint of the tested Baoxin Avicel agent with the reference fingerprint of the Baoxin Avicel agent; as well as (4) Based on the comparison results, determine the final production process of Baoxin Avicel preparation.

20. The method according to claim 19, characterized in that The determination method of the control fingerprint of the Baoxin Avicennia wood preparation is the same as the determination method of the fingerprint of the Baoxin Avicennia wood preparation to be tested.

21. The method according to claim 19, characterized in that In step (4), when the similarity between the fingerprint of the tested Baoxin Avicel preparation and the control fingerprint of the Baoxin Avicel preparation is not less than 0.9, the candidate process is selected as the final production process of the Baoxin Avicel preparation.

22. The method according to claim 19, characterized in that When in step (4), the similarity between the fingerprint of the tested Baoxin Avicel agent and the reference fingerprint of the Baoxin Avicel agent is less than 0.9, the candidate process is optimized, and steps (1) to (4) are repeated until the similarity between the fingerprint of the Baoxin Avicel agent and the reference fingerprint of the Baoxin Avicel agent is not less than 0.

9.

23. The method according to claim 22, characterized in that The optimization process includes adjusting the parameters of at least one of the following processes: Preparation prescription and storage conditions.

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