Construction method of fingerprint spectrum of angelica sinensis-astragalus membranaceus blood replenishing oral liquid, standard fingerprint spectrum of angelica sinensis-astragalus membranaceus blood replenishing oral liquid and application

Abstract

The invention discloses a construction method of a fingerprint spectrum of a Guiqi blood-enriching oral liquid as well as a standard fingerprint spectrum and application thereof, relates to the technical field of traditional Chinese medicine fingerprint spectrums, and solves the technical problem that no fingerprint spectrum item exists in the existing quality standard of the Guiqi blood-enriching oral liquid. The construction method comprises the following steps: preparation of a test solution, preparation of a negative sample solution, preparation of a reference solution, determination, similarity evaluation and establishment of the fingerprint spectrum. The quality control level of the angelica sinensis-astragalus membranaceus blood replenishing oral liquid can be improved, and the uniformity and stability of the clinical curative effect are guaranteed.

Description

Technical Field

[0001] This invention relates to the field of fingerprinting technology for traditional Chinese medicine, and specifically to a method for constructing a fingerprint spectrum of Guiqi Buxue Oral Liquid, its standard fingerprint spectrum, and its application. Background Technology

[0002] Guiqi Buxue Oral Liquid is a marketed OTC Class B traditional Chinese medicine. It is composed of twelve medicinal materials, including Astragalus membranaceus, Angelica sinensis, processed Polygonum multiflorum, Codonopsis pilosula, Lycium barbarum, Ligustrum lucidum, Poria cocos, Lentinus edodes, Tremella fuciformis, Salvia miltiorrhiza, Panax notoginseng, and Glycyrrhiza uralensis. This preparation has the effects of invigorating qi and nourishing blood, promoting blood circulation and removing blood stasis. Clinically, it is used to treat symptoms such as shortness of breath, fatigue, palpitations, insomnia, dizziness and vertigo caused by qi and blood deficiency and blood stasis. It reflects the characteristics of multi-component and multi-target synergistic effects of traditional Chinese medicine compound.

[0003] Currently, the quality control and standard implementation of Guiqi Buxue Oral Liquid are based on the Ministry of Health standard WS-5803(B-0803)-2014Z. This standard covers items such as prescription, preparation method, properties, identification, inspection, content determination, and indications, providing a guarantee for the basic quality control of the product. Specifically, the identification item uses thin-layer chromatography to qualitatively identify ingredients such as Danshen and Gancao; the content determination item uses high-performance liquid chromatography, with astragaloside A as a single indicator component for quantitative control. These methods, to a certain extent, reflect the authenticity of the product and some quality attributes.

[0004] However, traditional Chinese medicine compound preparations have complex chemical compositions, and their overall efficacy is the result of the combined effects of multiple components. Current quality standard models, especially those relying on the content determination of single or a few indicator components, are insufficient for comprehensively and holistically evaluating and controlling the intrinsic quality of products. Differences in chemical composition profiles (i.e., "fingerprints") between different batches can occur due to factors such as the source of medicinal materials and fluctuations in production process parameters, thus affecting the stability and consistency of efficacy. Furthermore, existing standards lack effective means for fine-grained monitoring and evaluation of the production process. For example, in key purification stages such as alcohol precipitation and water precipitation, traditional quality control methods are insufficient to scientifically assess the effects of process optimization.

[0005] Therefore, given the current quality standards for Guiqi Buxue Oral Liquid lack comprehensive control, there is an urgent need to research and establish its own, stable, and reliable fingerprint analysis method. Summary of the Invention

[0006] To address the above shortcomings, this invention provides a method for constructing a fingerprint spectrum of Guiqi Buxue Oral Liquid, along with its standard fingerprint spectrum and applications. The method includes evaporative light detection and ultraviolet light detection, solving the technical problem that the existing quality standards for Guiqi Buxue Oral Liquid lack a "fingerprint spectrum" item. This improves the quality control level of Guiqi Buxue Oral Liquid and ensures the uniformity and stability of clinical efficacy. The specific technical solution is as follows: In a first aspect, the present invention provides a method for constructing a fingerprint spectrum of Guiqi Buxue Oral Liquid, comprising the following steps: (1) Preparation of test solution: The test solution was prepared by method one and method two, respectively. (2) Preparation of negative sample solution: Negative sample solution was prepared using method one and method two respectively; (3) Preparation of reference solutions: Reference solutions were prepared using method one and method two, respectively; (4) Determination method: The test solution, negative sample solution and reference solution prepared by method one were injected into the ultra-high performance liquid chromatograph and determined by fingerprint chromatogram method one. The test solution, negative sample solution and reference solution prepared by method two were injected into the ultra-high performance liquid chromatograph and determined by fingerprint chromatogram method two. The chromatograms of the test sample, negative sample and reference solution prepared by method one and method two were recorded respectively. (5) Generating fingerprint spectrum: Using the "Similarity Evaluation System for Chromatographic Fingerprint Spectrum of Traditional Chinese Medicine" formulated by the National Pharmacopoeia Commission, the chromatograms obtained in step (4) are imported into the similarity evaluation system for chromatographic fingerprint spectrum of traditional Chinese medicine, and data processing is performed to generate the fingerprint spectrum of Guiqi Buxue Oral Liquid. (6) Identify common peaks: Based on the chromatograms of the negative sample solution and the reference solution, identify and determine the common peaks in the fingerprint spectrum of Guiqi Buxue Oral Liquid obtained in step (5).

[0007] Preferably, in step (2), the method for preparing the negative samples is as follows: 12 negative samples are prepared respectively, each lacking Astragalus membranaceus, Angelica sinensis, processed Polygonum multiflorum, Codonopsis pilosula, Lycium barbarum, Ligustrum lucidum, Poria cocos, Lentinus edodes, Tremella fuciformis, Salvia miltiorrhiza, Panax notoginseng, and Glycyrrhiza uralensis. 70 ml of each negative sample is accurately measured and placed in a separatory funnel. The samples are extracted three times with n-butanol, 30 ml each time. The n-butanol solutions are combined, evaporated to dryness, and the residue is dissolved in an appropriate amount of methanol and transferred to a 2 ml volumetric flask. Methanol is added to dilute to the mark, and the mixture is shaken well to obtain 12 negative samples lacking Astragalus membranaceus, Angelica sinensis, processed Polygonum multiflorum, Codonopsis pilosula, Lycium barbarum, Ligustrum lucidum, Poria cocos, Lentinus edodes, Tremella fuciformis, Salvia miltiorrhiza, Panax notoginseng, and Glycyrrhiza uralensis.

[0008] Preferably, in step (1), the method for preparing the test solution is as follows: accurately measure 40 ml of Guiqi Buxue Oral Liquid, place it in a separatory funnel, extract it three times with n-butanol, 30 ml each time, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in an appropriate amount of methanol and transfer it to a 5 ml volumetric flask, dilute it to the mark with methanol, shake well, and obtain the test solution.

[0009] Preferably, in step (2), the method for preparing the negative sample solution is as follows: accurately measure 40 ml of the negative sample, place it in a separatory funnel, extract it three times with n-butanol, 30 ml each time, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in an appropriate amount of methanol and transfer it to a 5 ml volumetric flask, dilute to the mark with methanol, shake well, and obtain the negative sample solution.

[0010] Preferably, in step (3), the method for preparing the reference solution specifically involves: accurately weighing ginsenoside Rb1, astragaloside A, glycyrrhizin, privetin, and stilbene glycoside reference standards and placing them in different volumetric flasks, dissolving them in methanol and diluting them to the mark, shaking well, and preparing reference solutions containing 0.956±0.005mg, 0.491±0.005mg, 1.054±0.005mg, 0.243±0.005mg, and 0.208±0.005mg of ginsenoside Rb1, astragaloside A, glycyrrhizin, privetin, and stilbene glycoside per ml, respectively.

[0011] Preferably, the fingerprinting method one uses an evaporative light detector for detection, and the chromatographic conditions are: column: Elite-C 18 4.6 × 250 mm 5 μm; Mobile phase: acetonitrile-0.05% formic acid solution; Column temperature: 30℃; Flow rate: 1.0 ml / min; Drift tube temperature: 120℃; Carrier gas pressure: 3.6 bra; Elution gradient: Gradient elution according to the mobile phase ratios in Table 1; Table 1 Elution gradient table Preferably, the fingerprint spectrum obtained by the fingerprinting method one identified a total of 14 chromatographic peaks, characterizing six processed medicinal materials: Ligustrum lucidum, Astragalus membranaceus, Glycyrrhiza uralensis, processed Polygonum multiflorum, Lycium barbarum, and Panax notoginseng. Among them, peaks 1, 5, 6, and 9 (4 peaks in total) originated from Ligustrum lucidum processed medicinal materials; peaks 2, 12, and 14 originated from Astragalus membranaceus processed medicinal materials; peaks 3 and 13 originated from Glycyrrhiza uralensis processed medicinal materials; peak 4 originated from Polygonum multiflorum processed medicinal materials; peak 7 originated from Lycium barbarum processed medicinal materials; peaks 10 and 11 originated from Panax notoginseng processed medicinal materials; and peak 8 originated from Salvia miltiorrhiza processed medicinal materials. By comparing the chromatograms with those of the reference standard and negative sample, five characteristic peaks were identified: peak 3 was glycyrrhizin, peak 4 was stilbene glycoside, peak 6 was ligustrazine glycoside, peak 11 was ginsenoside Rb1, and peak 12 was astragaloside A.

[0012] Preferably, in step (1), the second method for preparing the test solution is as follows: accurately measure 30 ml of Guiqi Buxue Oral Liquid, accurately add 30 ml of methanol, weigh, sonicate for 30 min, take out, cool, weigh, replenish the lost weight with methanol, shake well, and obtain the test solution.

[0013] Preferably, in step (2), the second method for preparing the negative sample solution is as follows: accurately measure 30 ml of the negative sample, accurately add 30 ml of methanol, weigh, sonicate for 30 min, take out, cool, weigh, replenish the lost weight with methanol, shake well, and obtain the negative sample solution.

[0014] Preferably, in step (3), the second method for preparing the reference solution specifically involves: accurately weighing gallic acid, sodium tanshinone, protocatechuic aldehyde, ferulic acid, apigenin, codonopsis glycoside, ligustrazine lactone H, and salvianolic acid B reference standards respectively, placing them in different volumetric flasks, dissolving them in methanol and diluting them to the mark, shaking well, and preparing reference solutions containing 0.696±0.005 mg, 1.135±0.005 mg, 0.545±0.005 mg, 0.945±0.005 mg, 0.944±0.005 mg, 0.350±0.005 mg, 0.937±0.005 mg, and 0.450±0.005 mg of gallic acid, sodium tanshinone, protocatechuic aldehyde, ferulic acid, apigenin, codonopsis glycoside, ligustrazine lactone H, and salvianolic acid B per ml, respectively.

[0015] Preferably, the fingerprinting method two uses an ultraviolet detector for detection, and the chromatographic conditions are as follows: octadecylsilane-bonded silica gel as the packing material; column temperature of 35℃; flow rate of 1.0 ml / min; detection wavelength of 260 nm; acetonitrile as mobile phase A; 0.1% formic acid as mobile phase B; and gradient elution according to the mobile phase ratios in Table 2. Table 2 shows the proposed elution gradient table for fingerprint mapping method two. Preferably, the fingerprint spectrum obtained by the second fingerprinting method identified 15 common peaks, representing seven medicinal slices: Ligustrum lucidum, Astragalus membranaceus, Glycyrrhiza uralensis, processed Polygonum multiflorum, Salvia miltiorrhiza, Angelica sinensis, and Codonopsis pilosula. Among them, peak 8 originated from Ligustrum lucidum, peaks 5 and 14 from Astragalus membranaceus, peak 6 from Glycyrrhiza uralensis, peaks 1 and 7 from processed Polygonum multiflorum, peaks 2, 3, 10, 12, 13, and 15 from Salvia miltiorrhiza, peaks 4 and 11 from Angelica sinensis, and peak 9 from Codonopsis pilosula. By comparing with the chromatogram of the reference standard, eight characteristic peaks were identified, including peak 1 being gallic acid, peak 2 being sodium tanshinone, peak 3 being protocatechuic aldehyde, peak 4 being ferulic acid, peak 6 being apigenin glycyrrhizin, peak 9 being codonopsis glycoside, peak 11 being ligustrazine lactone H, and peak 12 being salvianolic acid B.

[0016] Preferably, the determination of the construction method involves precisely injecting 10 µl or 15 µl of each of the test solution, negative sample solution, and reference solution into an ultra-high performance liquid chromatograph for determination.

[0017] Preferably, the construction method further includes examining the extraction solvent, extraction times, and sampling amount of Guiqi Buxue Oral Liquid, and also includes testing the robustness, precision, repeatability, and stability of the test sample.

[0018] Preferably, the construction method further includes investigating the flow rate, column temperature, mobile phase system, formic acid concentration, and elution gradient of the chromatographic conditions.

[0019] Secondly, the present invention provides a standard fingerprint spectrum for Guiqi Buxue Oral Liquid, wherein the standard fingerprint spectrum is composed of the fingerprint spectrum obtained by the fingerprint spectrum construction method one and the fingerprint spectrum obtained by the fingerprint spectrum construction method two.

[0020] Thirdly, the present invention also provides the application of the fingerprint spectrum of Guiqi Buxue Oral Liquid described above in the quality control of Guiqi Buxue Oral Liquid, as a reference fingerprint spectrum in the quality monitoring of Guiqi Buxue Oral Liquid.

[0021] Thirdly, the present invention also provides a quality control method for Guiqi Buxue Oral Liquid, comprising: testing the chromatogram of the Guiqi Buxue Oral Liquid sample to be tested, then importing it into a Chinese medicine chromatographic fingerprint similarity evaluation system, using the aforementioned standard fingerprint as a reference for similarity evaluation, and determining whether its quality is qualified by the similarity.

[0022] The preparation method of the negative samples (lacking medicinal ingredients) used in this invention is as follows: the samples are prepared according to the preparation method under the national standard WS-5803(B-8303)-2014Z. According to the prescription and preparation method of Guiqi Buxue Oral Liquid, 12 negative samples (lacking medicinal ingredients) are prepared respectively, which are lacking astragalus, angelica, processed he shou wu, codonopsis, wolfberry, privet fruit, poria, shiitake mushroom, tremella, salvia miltiorrhiza, notoginseng, and licorice.

[0023] The present invention achieves at least the following beneficial effects: 1. This invention is beneficial to improving the quality control level of Guiqi Buxue Oral Liquid products. It can also be applied to the evaluation index of alcohol precipitation and water precipitation process optimization in the production process. It can also shorten the production cycle and reduce energy consumption and production costs through filing. It helps to promote the refinement, standardization and optimization of the production process of Guiqi Buxue Oral Liquid, and has important practical significance and application value.

[0024] 2. This invention establishes for the first time a method for constructing the fingerprint spectrum of Guiqi Buxue Oral Liquid, characterizing a total of 29 characteristic peaks, identifying 13 characteristic peaks, and characterizing 9 medicinal ingredients. The method has good sensitivity, repeatability, and stability, and can more effectively control the quality of Guiqi Buxue Oral Liquid, ensuring that the quality of the medicine is stable, uniform, effective, and controllable between batches, thus ensuring efficacy and medication safety. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This invention provides fingerprint chromatograms of Guiqi Buxue Oral Liquid and control fingerprint chromatograms. Figure 2 This is a chromatogram showing the selection of methanol concentration and detection wavelength in the pseudo-fingerprint spectral method of the present invention; Figure 3 This is a chromatogram showing the flow rate and column temperature selection for this invention; Figure 4 This is a chromatogram of the mobile phase system and formic acid concentration selection of the present invention; Figure 5 Chromatograms were used to examine the extraction solvent, number of extractions, extraction method, and extraction time of this invention. Figure 6 This is a chromatogram used to investigate the elution gradient and sample volume of this invention. Figure 7 The chromatogram of the test sample (Guiqi Buxue Oral Liquid) of this invention is shown below. Figure 8 This is a chromatogram for evaluating the durability of the present invention. Detailed Implementation

[0027] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Unless otherwise defined, all technical terms used below have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of the present invention. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in the present invention are commercially available or can be prepared by existing methods.

[0028] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0029] Guiqi Buxue Oral Liquid currently follows the Ministry of Health standard WS-5803(B-0803)-2014Z. The current standard includes items such as prescription, preparation method, properties, identification, inspection, content determination, and indications, but does not yet include a "fingerprint spectrum" item. Fingerprint spectrum technology, as a comprehensive and quantifiable product quality control method, can obtain the overall distribution information of multiple chemical components in a preparation through chromatography, spectroscopy, and other methods, forming a characteristic and reproducible "chemical fingerprint." This not only more comprehensively characterizes the product's quality features and distinguishes between genuine and counterfeit products, but also effectively monitors batch-to-batch consistency through quantitative methods such as similarity evaluation. More importantly, applying fingerprint spectrum to production process research can establish the correlation between key process parameters and the product's chemical fingerprint, providing scientific evaluation indicators for process optimization (such as precise control of alcohol precipitation and water precipitation processes). This allows for the achievement of goals such as improving process stability, shortening production cycles, and reducing energy consumption and costs through process registration.

[0030] The inventors are dedicated to researching fingerprint spectroscopy methods for Guiqi Buxue Oral Liquid in order to improve the quality control level of the product. They also hope that the fingerprint spectroscopy of Guiqi Buxue Oral Liquid can be used as an evaluation index for optimizing alcohol precipitation and water precipitation processes in the production process. Furthermore, they hope that the production cycle can be shortened and energy consumption and production costs can be reduced through registration.

[0031] Example 1: A standard fingerprint spectrum for Guiqi Buxue Oral Liquid, wherein the standard fingerprint spectrum is composed of a fingerprint spectrum obtained by the fingerprint spectrum construction method one and a fingerprint spectrum obtained by the fingerprint spectrum construction method two; wherein; The establishment of fingerprint mapping method one is specifically as follows: A method for constructing a fingerprint spectrum of Guiqi Buxue Oral Liquid includes the following steps: (1) Preparation of test solution: Accurately measure 30 ml of Guiqi Buxue Oral Liquid, place it in a separatory funnel, and extract it three times with n-butanol, 30 ml each time. Combine the n-butanol ester solutions, evaporate to dryness, dissolve the residue in an appropriate amount of methanol and transfer it to a 5 ml volumetric flask, dilute to the mark with methanol, shake well, and obtain the test solution. (2) Preparation of negative sample solution: First, prepare the negative sample according to the method in “2.2”, then accurately measure 30 ml of each negative sample, place it in a separatory funnel, and extract it three times with n-butanol, 30 ml each time. Combine the n-butanol ester solutions, evaporate to dryness, dissolve the residue in an appropriate amount of methanol and transfer it to a 5 ml volumetric flask, dilute with methanol to the mark, and shake well to obtain the solution. (3) Preparation of reference solutions: Accurately weigh the reference standards of ginsenoside Rb1, astragaloside A, glycyrrhizin, privetin, and stilbene glycoside and place them in different volumetric flasks. Dissolve and dilute them to the mark with methanol, shake well, and prepare reference solutions containing 0.956±0.005 mg, 0.491±0.005 mg, 1.054±0.005 mg, 0.243±0.005 mg, and 0.208±0.005 mg of ginsenoside Rb1, astragaloside A, glycyrrhizin, privetin, and stilbene glycoside per ml, respectively. (4) Determination method: Accurately pipette 15µl of each batch of test sample solution, negative sample solution and reference solution into the ultra-high performance liquid chromatograph for determination. Perform chromatographic analysis according to the chromatographic conditions and record the chromatograms of the test sample, negative sample and reference solution respectively. The chromatographic conditions for the determination were as follows: Column: Elite-C 18 4.6 × 250 mm 5 μm; Mobile phase: acetonitrile-0.05% formic acid solution; Column temperature: 30℃; Flow rate: 1.0 ml / min; Drift tube temperature: 120℃; Carrier gas pressure: 3.6 bra; Using acetonitrile as mobile phase A and 0.05% formic acid as mobile phase B, elution gradient: gradient elution according to the mobile phase ratios in Table 1; (5) Generating fingerprint spectrum: Using the "Similarity Evaluation System for Chromatographic Fingerprint Spectrum of Traditional Chinese Medicine" formulated by the National Pharmacopoeia Commission, the chromatogram obtained in step (4) is imported into the similarity evaluation system for chromatographic fingerprint spectrum of traditional Chinese medicine, and data processing is performed to generate the fingerprint spectrum of Guiqi Buxue Oral Liquid. (6) Identification of common peaks: Based on the chromatograms of the negative sample solution and the reference solution, the common peaks in the fingerprint spectrum of Guiqi Buxue Oral Liquid obtained in step (5) were identified and their attribution was determined; a total of 14 chromatographic peaks were identified, representing 6 kinds of medicinal slices: Ligustrum lucidum, Astragalus membranaceus, Glycyrrhiza uralensis, processed Polygonum multiflorum, Lycium barbarum, and Panax notoginseng; among them, peaks 1, 5, 6, and 9 were from Ligustrum lucidum, peaks 2, 12, and 14 were from Astragalus membranaceus, peaks 3 and 13 were from Glycyrrhiza uralensis, peak 4 was from Polygonum multiflorum, peak 7 was from Lycium barbarum, peaks 10 and 11 were from Panax notoginseng, and peak 8 was from Salvia miltiorrhiza; by comparing with the chromatograms of the reference and negative samples, 5 characteristic peaks were identified: peak 3 was glycyrrhizin, peak 4 was stilbene glycoside, peak 6 was ligustrazine, peak 11 was ginsenoside Rb1, and peak 12 was astragaloside A.

[0032] The establishment of fingerprint pattern method two is specifically as follows: A method for constructing a fingerprint spectrum of Guiqi Buxue Oral Liquid includes the following steps: (1) Preparation of test solution: Accurately measure 30 ml of Guiqi Buxue Oral Liquid, accurately add 30 ml of methanol, weigh, sonicate for 30 min, take out, cool, weigh, replenish the lost weight with methanol, shake well to obtain test solution; (2) Preparation of negative sample solution: First, prepare the negative sample according to the method in “2.2”, then accurately measure 30 ml of Guiqi Buxue Oral Liquid, accurately add 30 ml of methanol, weigh, sonicate for 30 min, take out, cool, weigh, make up the lost weight with methanol, shake well, and obtain the test sample solution. (3) Preparation of reference solutions: Accurately weigh gallic acid, sodium tanshinone, protocatechuic aldehyde, ferulic acid, apigenin, codonopsis glycoside, ligustrazine lactone H, and salvianolic acid B reference standards and place them in different volumetric flasks. Dissolve and dilute to the mark with methanol, shake well, and prepare reference solutions containing 0.696±0.005 mg, 1.135±0.005 mg, 0.545±0.005 mg, 0.945±0.005 mg, 0.944±0.005 mg, 0.350±0.005 mg, 0.937±0.005 mg, and 0.450±0.005 mg of gallic acid, sodium tanshinone, protocatechuic aldehyde, ferulic acid, apigenin, codonopsis glycoside, ligustrazine lactone H, and salvianolic acid B per ml, respectively. (4) Determination method: Accurately pipette 10µl of each batch of test sample solution, negative sample solution and reference solution into the ultra-high performance liquid chromatograph for determination. Perform chromatographic analysis according to the chromatographic conditions and record the chromatograms of the test sample, negative sample and reference solution respectively. The chromatographic conditions for the determination were as follows: octadecylsilane-bonded silica gel was used as the packing material; the column temperature was 35℃; the flow rate was 1.0 ml / min; the detection wavelength was 260 nm; acetonitrile was used as mobile phase A, and 0.1% formic acid was used as mobile phase B, with gradient elution performed according to the mobile phase ratios in Table 2. (5) Generating fingerprint spectrum: Using the "Similarity Evaluation System for Chromatographic Fingerprint Spectrum of Traditional Chinese Medicine" formulated by the National Pharmacopoeia Commission, the chromatogram obtained in step (4) is imported into the similarity evaluation system for chromatographic fingerprint spectrum of traditional Chinese medicine, and data processing is performed to generate the fingerprint spectrum of Guiqi Buxue Oral Liquid. (6) Identification of common peaks: Based on the chromatograms of the negative sample solution and the reference solution, the common peaks in the fingerprint chromatogram of Guiqi Buxue Oral Liquid obtained in step (5) were identified and their attribution was determined; a total of 15 common peaks were identified, representing 7 kinds of medicinal slices including Ligustrum lucidum, Astragalus membranaceus, Glycyrrhiza uralensis, processed Polygonum multiflorum, Salvia miltiorrhiza, Angelica sinensis, and Codonopsis pilosula; among them, peak 8 originated from Ligustrum lucidum, peaks 5 and 14 originated from Astragalus membranaceus, peak 6 originated from Glycyrrhiza uralensis, and peaks 1 and 7 originated from Astragalus membranaceus. The peaks were derived from processed Polygonum multiflorum slices; peaks 2, 3, 10, 12, 13, and 15 were derived from processed Salvia miltiorrhiza slices; peaks 4 and 11 were derived from processed Angelica sinensis slices; and peak 9 was derived from processed Codonopsis pilosula slices. By comparing the chromatograms with the reference standard, eight characteristic peaks were identified, including peak 1 being gallic acid, peak 2 being sodium tanshinone, peak 3 being protocatechuic aldehyde, peak 4 being ferulic acid, peak 6 being apigenin glycyrrhizin, peak 9 being codonopsis glycoside, peak 11 being ligustrazine lactone H, and peak 12 being salvianolic acid B.

[0033] Application of fingerprint spectrum in the quality control of Guiqi Buxue Oral Liquid, and its application as a reference fingerprint spectrum in the quality monitoring of Guiqi Buxue Oral Liquid.

[0034] Example 2 A quality control method for Guiqi Buxue Oral Liquid: In Example 1, a standard fingerprint spectrum of Guiqi Buxue Oral Liquid is constructed. The sample of Guiqi Buxue Oral Liquid to be tested is obtained by following the steps (1) to (4) of fingerprint spectrum method one or fingerprint spectrum method two in Example 1. Then, the chromatogram is imported into the Chinese medicine chromatographic fingerprint spectrum similarity evaluation system. The similarity is evaluated with reference to the standard fingerprint spectrum. The quality is judged by the similarity. If the sample can be similarly matched with the standard fingerprint spectrum, it is determined that the sample is qualified.

[0035] Test case In order to obtain a reliable fingerprint spectrum of Guiqi Buxue Oral Liquid, the inventors of this application conducted the following experiments: 1. Instruments and reagents The system used was a Waters 1260 high-performance liquid chromatograph, an ELSD 6100 detector, and octadecylsilane-bonded silica columns (Agilent ZORBAX C18 column (250×4.6mm 5µm), Waters Spherisorb ODS C18 column (250×4.6mm 5µm), and Elite Supersil ODS2 C18 column (250×4.6mm 5µm)); a KQ5200DB digital ultrasonic instrument; reference standards for gallic acid, sodium tanshinone, protocatechuic aldehyde, ferulic acid, apigenin, glycyrrhizin, codonopsis glycoside, ligustrazine lactone H, salvianolic acid B, ginsenoside Rb1, astragaloside A, glycyrrhizin, ligustrazine glycoside, and stilbene glycoside, all sourced from the China National Institutes for Food and Drug Control; methanol and acetonitrile (chromatographic grade); water (ultrapure water); formic acid and phosphoric acid (chromatographic grade); methanol, n-butanol, and ethyl acetate (analytical grade).

[0036] 2. Test methods 2.1 Preparation of Guiqi Blood-Nourishing Oral Liquid: The preparation method of Guiqi Blood-Nourishing Oral Liquid used in this invention is as follows: It is prepared according to the method under national standard WS-5803(B-8303)-2014Z, using 12 herbs: Astragalus membranaceus 120g, Angelica sinensis 80g, processed Polygonum multiflorum 60g, Codonopsis pilosula 80g, Lycium barbarum 60g, Ligustrum lucidum 60g, Poria cocos 60g, Lentinus edodes 60g, Tremella fuciformis 40g, Salvia miltiorrhiza 60g, Panax notoginseng 20g, and Glycyrrhiza uralensis 15g. Except for Lentinus edodes and Tremella fuciformis... The remaining ten ingredients, including Astragalus membranaceus, were decocted three times with water: 2.5 hours the first time, 1.5 hours the second time, and 1 hour the third time. The decoctions were filtered, the filtrates were combined, and concentrated to a relative density of 1.10–1.15 (60°C). The decoctions were cooled, and ethanol was added to make the alcohol content reach 50%. The decoctions were allowed to stand for 24 hours, then filtered. The ethanol was recovered from the filtrate under reduced pressure and concentrated to a relative density of 1.15–1.20 (60°C). Twice the amount of water was added, the mixture was stirred, and the decoctions were refrigerated for 48 hours. The filtrates were then filtered and reserved for later use. Shiitake mushrooms and white fungus were decocted three times with water: 2 hours the first time, 1 hour the second time, and 1 hour the third time. The decoctions were filtered, the filtrates were combined, and concentrated to a relative density of 1.05–1.10 (60°C). After cooling, ethanol was added to make the alcohol content 50%. The mixture was allowed to stand for 24 hours, then filtered. The ethanol in the filtrate was recovered under reduced pressure and concentrated to a relative density of 1.10–1.15 (60°C). Twice the amount of water was added, stirred well, and refrigerated for 48 hours. The mixture was then filtered. The two solutions were mixed, and 0.6g of protein sugar and 3g of sodium benzoate were added and stirred to dissolve. Water was then added to 1000ml, mixed well, filtered, bottled, and sterilized to obtain the Guiqi Buxue Oral Liquid.

[0037] 2.2 Preparation of negative samples (lacking medicinal ingredients): The prescription for Guiqi Buxue Oral Liquid is as follows: Astragalus membranaceus 120g, Angelica sinensis 80g, processed Polygonum multiflorum 60g, Codonopsis pilosula 80g, Lycium barbarum 60g, Ligustrum lucidum 60g, Poria cocos 60g, Lentinus edodes 60g, Tremella fuciformis 40g, Salvia miltiorrhiza 60g, Panax notoginseng 20g, and Glycyrrhiza uralensis 15g. According to the prescription, 12 negative samples were prepared, which were lacking Astragalus membranaceus, Angelica sinensis, processed Polygonum multiflorum, Codonopsis pilosula, Lycium barbarum, Ligustrum lucidum, Poria cocos, Lentinus edodes, Tremella fuciformis, Salvia miltiorrhiza, Panax notoginseng, and Glycyrrhiza uralensis.

[0038] 2.3 Establishment of fingerprint mapping method This experiment examined and compared the baseline characteristics, chromatographic peak separation effects, and number of chromatographic peaks of the two methods, and preliminarily determined two fingerprinting methods: the first method is the evaporative light detector method, and the second method is the ultraviolet detector method.

[0039] 2.3.1 Investigation of chromatographic conditions In the following experiments, the test solution, negative sample solution, and control solution corresponding to the simulated fingerprinting method one were prepared according to steps (1), (2), and (3) of the simulated fingerprinting method one, respectively. The test solution, negative sample solution, and control solution corresponding to the simulated fingerprinting method two were prepared according to steps (1), (2), and (3) of the simulated fingerprinting method two, respectively.

[0040] Each evaluation method used baseline conditions, chromatographic peak separation effect, and the number of chromatographic peaks as evaluation indicators.

[0041] (1) Flow rate selection: In the selection of flow rates, 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min were screened. The results showed that in the first method of proto-fingerprint chromatogram, a flow rate of 1.0 ml / min resulted in a stable baseline and good peak resolution, so 1.0 ml / min was selected. In the second method of proto-fingerprint chromatogram, a flow rate of 1.0 ml / min resulted in a large number of peaks and good peak resolution, so 1.0 ml / min was selected. The results are shown in [Figure number missing]. Figure 3 .

[0042] (2) Column temperature selection: In column temperature selection, pseudo-fingerprinting method one screened column temperatures of 25℃, 30℃, and 35℃, while pseudo-fingerprinting method two screened column temperatures of 30℃, 35℃, and 40℃. The results showed that for pseudo-fingerprinting method one, a column temperature of 30℃ resulted in a stable baseline and good peak resolution, making 30℃ the optimal column temperature. For pseudo-fingerprinting method two, a column temperature of 30℃ resulted in a higher number of peaks and better peak resolution, making 35℃ the optimal column temperature. The results are shown in [Figure number missing]. Figure 3 .

[0043] (3) Selection of mobile phase system: In the selection of mobile phase systems, the simulated fingerprinting method one screened mobile phase systems such as acetonitrile-water, acetonitrile-0.05% formic acid, and methanol-0.05% formic acid. The simulated fingerprinting method two screened mobile phase systems such as acetonitrile-water, acetonitrile-0.1% formic acid, and methanol-0.1% formic acid. The results showed that in simulated fingerprinting method one, the mobile phase system of acetonitrile-0.05% formic acid resulted in a stable baseline, good peak resolution, and a large number of peaks; therefore, acetonitrile-0.05% formic acid was selected as the mobile phase system. In simulated fingerprinting method two, the mobile phase system of acetonitrile-0.1% formic acid showed good peak resolution and a large number of peaks; therefore, acetonitrile-0.05% formic acid was selected as the mobile phase system. The results are shown in [Figure number missing]. Figure 4 .

[0044] (4) Selection of formic acid concentration: In selecting the formic acid concentration, formic acid concentrations such as acetonitrile-0.05%, acetonitrile-0.1%, and acetonitrile-0.15% were screened. The results showed that for fingerprinting method one, the baseline, peak resolution, and number of peaks were basically consistent across different formic acid concentrations; however, high formic acid concentrations could damage the chromatographic column. Therefore, to protect the column, a formic acid concentration of 0.05% was selected. For fingerprinting method two, a formic acid concentration of 1.0% resulted in good peak resolution and a large number of peaks. Again, high formic acid concentrations could damage the column; therefore, a formic acid concentration of 0.05% was selected. The results are shown in [Figure number missing]. Figure 4 .

[0045] (5) Elution gradient selection: In the selection of elution gradients, the simulated fingerprinting method one screened three elution gradients from Table 1 (elution gradient 3 of simulated fingerprinting method one), Table 3 (elution gradient 1 of simulated fingerprinting method one), and Table 4 (elution gradient 2 of simulated fingerprinting method one). The simulated fingerprinting method two screened three elution gradients from Table 2 (elution gradient 3 of simulated fingerprinting method two), Table 5 (elution gradient 1 of simulated fingerprinting method two), and Table 6 (elution gradient 2 of simulated fingerprinting method two). The results showed that for simulated fingerprinting method one, the elution gradient in Table 1 had good peak resolution and a large number of peaks, therefore, the elution gradient in Table 1 was selected. For simulated fingerprinting method two, the elution gradient in Table 2 had a stable baseline, good peak resolution, and a large number of peaks, therefore, the elution gradient in Table 2 was selected. The results are shown in […]. Figure 6 .

[0046] Table 3. Method for simulated fingerprinting – Chromatographic conditions: Elution gradient 1 Table 4. Fingerprinting Method 1: Chromatographic Conditions and Elution Gradient 2 Table 5. Chromatographic conditions for the simulated fingerprinting method 2: Elution gradient 1 Table 6. Chromatographic conditions for simulated fingerprinting method 2: Elution gradient 2 (6) Selection of detection wavelength: In the elution gradient selection, wavelengths of 220, 260, 280, and 320 nm were screened, with baseline characteristics, peak separation efficiency, and the number of peaks used as evaluation indicators. The results showed that a detection wavelength of 260 nm provided good peak separation and a large number of peaks; therefore, 260 nm was selected as the detection wavelength. The results are shown in [Figure number missing]. Figure 2 .

[0047] Based on a comprehensive review of the methods and the results of this study, and considering various factors, the chromatographic conditions selected for the proposed fingerprinting method are as follows: Column: Elite-C18 4.6×250mm 5μm; Mobile phase: Acetonitrile-0.05% formic acid solution; Column temperature: 30℃; Flow rate: 1.0 ml / min; Elution gradient: see Table 1; Drift tube temperature: 120℃; Carrier gas pressure: 3.6 bra.

[0048] The chromatographic conditions for the simulated fingerprint method 2 are as follows: Column: Elite-C18 4.6×250mm 5μm; Mobile phase: Acetonitrile-0.1% formic acid solution; Column temperature: 35℃; Flow rate: 1.0 ml / min; Detection wavelength: 260nm; Elution gradient: see Table 2.

[0049] 2.3.2 Test Sample Preparation Investigation 2.3.2.1 Investigation of Sample Preparation for Simulated Fingerprint Method 1 The following evaluation criteria are baseline conditions, chromatographic peak separation effect, number of chromatographic peaks and area size.

[0050] (1) Investigation of extraction solvent: Accurately measure 30ml of Guiqi Buxue Oral Liquid, making two portions, and place them in separatory funnels. Extract three times each with ethyl acetate and n-butanol, 30ml each time. Combine the ethyl acetate and n-butanol extracts, evaporate to dryness, dissolve the residue in an appropriate amount of methanol, transfer to a 5ml volumetric flask, dilute to the mark with methanol, shake well, filter, and inject the filtrate according to the method of simulated fingerprint chromatogram. The results show that the chromatographic peak resolution and the number of chromatographic peaks are better with n-butanol as the extraction solvent. Therefore, n-butanol is selected as the extraction solvent. The results are shown in […]. Figure 5 .

[0051] (2) Examination of the number of extractions: Accurately measure 30ml of Guiqi Buxue Oral Liquid, dividing it into four portions. Place each portion in a separatory funnel and extract with n-butanol, 30ml each time, for extractions 1, 2, 3, and 4. Combine the n-butanol extracts, evaporate to dryness, dissolve the residue in an appropriate amount of methanol, transfer to a 5ml volumetric flask, dilute to the mark with methanol, shake well, filter, and inject the filtrate according to the proposed fingerprinting method. The results showed that the baseline, peak separation, and number of peaks were basically consistent for extractions 1, 2, 3, and 4. The peak area increased with the number of extractions, but the peaks were basically consistent for extractions 3 and 4. Therefore, extraction was performed 3 times. The results are shown in the figure. Figure 5 .

[0052] (3) Sampling quantity consideration: Accurately measure 30, 50, 70, and 100 ml of Guiqi Buxue Oral Liquid, a total of four portions, and place them in separatory funnels. Extract each portion three times with n-butanol, 50 ml each time. Combine the n-butanol extracts, evaporate to dryness, dissolve the residue in an appropriate amount of methanol, transfer to a 5 ml volumetric flask, dilute to the mark with methanol, shake well, filter, and inject the filtrate according to the method of simulated fingerprint chromatogram. The results show that the baseline, chromatographic peak separation, and number of peaks are basically consistent for sample volumes of 30, 50, 70, and 100 ml. The peak area increases with increasing sample volume, but the peaks are basically the same for 70 ml and 100 ml. Therefore, the sample volume of 70 ml was selected. The results are shown in the figure. Figure 6 .

[0053] 2.3.2.2 Investigation on the preparation of test samples for pseudo-fingerprint spectroscopy method two Each evaluation item uses baseline conditions, chromatographic peak separation effect, and the number of chromatographic peaks as evaluation indicators.

[0054] (1) Investigation of extraction solvent: Four 30ml portions of Guiqi Buxue Oral Liquid were precisely measured. 15ml, 30ml, 70ml, and 120ml of methanol were added to each portion, resulting in methanol concentrations of 30%, 50%, 70%, and 80%, respectively. The portions were weighed, sonicated for 30 minutes, removed, cooled, and weighed again. The lost weight was replenished with methanol, the mixture was shaken well, and filtered. The filtrate was then injected using the second method of the simulated fingerprint chromatogram. The results showed that a 50% methanol concentration resulted in good peak resolution and a large number of peaks; therefore, 50% methanol was chosen as the extraction solvent. The results are shown in the figure. Figure 2 .

[0055] (2) Examination of extraction methods: Three portions of Guiqi Buxue Oral Liquid (30ml each) were precisely measured and 30ml of methanol were added. Each portion was weighed and extracted separately using three different methods: shaking extraction for 30min, ultrasonic extraction for 30min, and reflux extraction for 30min. After extraction, the portions were removed, cooled, and weighed again. The lost weight was replenished with methanol, and the mixture was shaken well and filtered. The filtrate was then injected using the second method of the simulated fingerprint chromatogram. The results showed that the baseline characteristics, peak resolution, and number of peaks were basically consistent across the three extraction methods. However, ultrasonic extraction was chosen due to its convenience and speed. The results are shown in the figure below. Figure 5 .

[0056] (3) Examination of extraction time: Three 30ml portions of Guiqi Buxue Oral Liquid were precisely measured, and 30ml of methanol was precisely added. The portions were weighed and extracted using ultrasound for 15, 30, and 60 minutes respectively. After extraction, the portions were removed, cooled, and weighed again. The lost weight was replenished with methanol, and the mixture was shaken well and filtered. The filtrate was then injected using the second method of the simulated fingerprint chromatogram. The baseline characteristics, peak resolution, and number of peaks were basically consistent across the three extraction times. However, to save time, ultrasound extraction for 15 minutes was selected. The results are shown in the figure. Figure 5 .

[0057] (4) Sampling quantity consideration: Accurately measure 20, 30, 40, 50, and 60 ml of Guiqi Buxue Oral Liquid, and accurately add 20, 30, 40, 50, and 60 ml of methanol respectively. Weigh the solutions, sonicate for 15 minutes, remove, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and inject the filtrate according to the second method of the simulated fingerprint chromatogram. The results showed that the baseline characteristics, peak resolution, and number of peaks were basically consistent across the five sample volumes. However, to save on usage, a sample volume of 30 ml was selected. The results are shown in the figure. Figure 6 .

[0058] 2.3.3 Characteristic Peak Identification 2.3.3.1 Characteristic Peak Identification in Simulated Fingerprint Spectrum Method (1) Preparation of the test solution: Accurately measure 70ml of Guiqi Buxue Oral Liquid, place it in a separatory funnel, and extract it three times with n-butanol, 50ml each time. Combine the n-butanol ester solutions, evaporate to dryness, dissolve the residue in an appropriate amount of methanol, transfer it to a 5ml volumetric flask, dilute to the mark with methanol, and shake well.

[0059] (2) Preparation of negative control solutions: Accurately measure 70 ml of the negative sample under “2.2”, and prepare 12 negative control solutions respectively according to the test sample preparation method of the proposed fingerprint spectrum method 1, which are lacking Astragalus membranaceus, Angelica sinensis, processed Polygonum multiflorum, Angelica sinensis, Codonopsis pilosula, Lycium barbarum, Ligustrum lucidum, Poria cocos, Lentinus edodes, Tremella fuciformis, Salvia miltiorrhiza, Panax notoginseng, and Glycyrrhiza uralensis.

[0060] (3) Preparation of reference solution: Preparation of ginsenoside Rb1 reference solution: Accurately weigh 9 mg of ginsenoside Rb1 reference standard (actual sample weight 9.56 mg) and place it in a 10 ml volumetric flask. Dissolve and dilute to the mark with methanol, and shake well. Preparation of astragaloside A reference solution: Accurately weigh 9 mg of astragaloside A reference standard (actual sample weight 9.83 mg) and place it in a 20 ml volumetric flask. Dissolve and dilute to the mark with methanol, and shake well. Preparation of glycyrrhizin reference solution: Accurately weigh 10 mg of glycyrrhizin reference standard (actual sample weight 1... 0.54 mg) was placed in a 10 ml volumetric flask, dissolved in methanol and diluted to the mark, and shaken well to obtain the solution; For the preparation of privetin reference solution: 12 mg of privetin reference standard (actual sample weight was 12.14 mg) was accurately weighed and placed in a 50 ml volumetric flask, dissolved in methanol and diluted to the mark, and shaken well to obtain the solution; For the preparation of stilbene glycoside reference solution: 10 mg of stilbene glycoside reference standard (actual sample weight was 10.42 mg) was accurately weighed and placed in a 50 ml volumetric flask, dissolved in methanol and diluted to the mark, and shaken well to obtain the solution.

[0061] Take the above-mentioned test solution, negative control solution, and reference solution, and inject them according to the chromatographic method of Method 1 for simulated fingerprinting, and record the chromatograms. This method identified 14 chromatographic peaks, characterizing six medicinal slices: Ligustrum lucidum, Astragalus membranaceus, Glycyrrhiza uralensis, processed Polygonum multiflorum, Lycium barbarum, and Panax notoginseng. Among them, peaks 1, 5, 6, and 9 (4 peaks) originated from Ligustrum lucidum slices; peaks 2, 12, and 14 originated from Astragalus membranaceus slices; peaks 3 and 13 originated from Glycyrrhiza uralensis slices; peak 4 originated from Polygonum multiflorum slices; peak 7 originated from Lycium barbarum slices; peaks 10 and 11 originated from Panax notoginseng slices; and peak 8 originated from Salvia miltiorrhiza slices. By comparing with the chromatograms of the reference solution, five characteristic peaks were identified: peak 3 is glycyrrhizin, peak 4 is stilbene glycoside, peak 6 is ligustrazine glycoside, peak 11 is ginsenoside Rb1, and peak 12 is astragaloside A. The negative control solution and reference solution did not have corresponding chromatographic peaks. The chromatogram of Guiqi Buxue Oral Liquid is shown below. Figure 7 .

[0062] 2.3.3.2 Characteristic Peak Identification in Pseudo-Fingerprint Method Two (1) Preparation of test solution: Accurately measure 30ml of Guiqi Buxue Oral Liquid, accurately add 30ml of methanol, weigh, sonicate for 15min, take out, cool, weigh, replenish the lost weight with methanol, shake well, and the test solution is obtained.

[0063] (2) Preparation of negative control solution: Accurately measure 70 ml of the negative sample under item "2.2", and prepare 12 negative control solutions according to the test sample preparation method of method two of the proposed fingerprint spectrum, which are lacking Astragalus membranaceus, Angelica sinensis, processed Polygonum multiflorum, Angelica sinensis, Codonopsis pilosula, Lycium barbarum, Ligustrum lucidum, Poria cocos, Lentinus edodes, Tremella fuciformis, Salvia miltiorrhiza, Panax notoginseng, and Glycyrrhiza uralensis.

[0064] (3) Preparation of reference solution: Preparation of gallic acid reference solution: Accurately weigh 13 mg of gallic acid reference standard (actual sample weight is 13.92 mg) and place it in a 20 ml volumetric flask. Dissolve and dilute to the mark with methanol, and shake well. Preparation of tanshinone sodium reference solution: Accurately weigh 11 mg of tanshinone sodium reference standard (actual sample weight is 11.35 mg) and place it in a 10 ml volumetric flask. Dissolve and dilute to the mark with methanol, and shake well. Preparation of protocatechuic aldehyde reference solution: Accurately weigh 10 mg of protocatechuic aldehyde reference standard (actual sample weight is 10.91 mg) and place it in a 10 ml volumetric flask. Dissolve and dilute to the mark with methanol, and shake well. Preparation of ferulic acid reference solution: Accurately weigh 9 mg of ferulic acid reference standard (actual sample weight is 9.45 mg) and place it in a 10 ml volumetric flask. Dissolve and dilute to the mark with methanol, and shake well. Preparation of apigenin reference solution: Accurately weigh 9 mg of apigenin reference standard (actual sample weight 9.44 mg) and place it in a 10 ml volumetric flask, dissolve and dilute to the mark with methanol, and shake well. Preparation of codonopsis glycoside reference solution: Accurately weigh 8 mg of codonopsis glycoside reference standard (actual sample weight 8.75 mg) and place it in a 25 ml volumetric flask, dissolve and dilute to the mark with methanol, and shake well. Preparation of ligustrazine H reference solution: Accurately weigh 9 mg of ligustrazine H reference standard (actual sample weight 9.37 mg) and place it in a 10 ml volumetric flask, dissolve and dilute to the mark with methanol, and shake well. Preparation of salvianolic acid B reference solution: Accurately weigh 9 mg of apigenin reference standard (actual sample weight 9.00 mg) and place it in a 20 ml volumetric flask, dissolve and dilute to the mark with methanol, and shake well.

[0065] Take the above-mentioned test solution, negative control solution, and reference solution, and inject them according to the chromatographic method of the simulated fingerprint chromatogram method two, and record the chromatograms. Fifteen chromatographic peaks were identified, characterizing seven medicinal slices: Ligustrum lucidum, Astragalus membranaceus, Glycyrrhiza uralensis, processed Polygonum multiflorum, Salvia miltiorrhiza, Angelica sinensis, and Codonopsis pilosula. Among them, peak 8 originated from Ligustrum lucidum, peaks 5 and 14 from Astragalus membranaceus, peak 6 from Glycyrrhiza uralensis, peaks 1 and 7 from processed Polygonum multiflorum, peaks 2, 3, 10, 12, 13, and 15 from Salvia miltiorrhiza, peaks 4 and 11 from Angelica sinensis, and peak 9 from Codonopsis pilosula. By comparing with the chromatograms of the reference standards, eight characteristic peaks were identified, including peak 1 (gallic acid), peak 2 (sodium tanshinone), peak 3 (protocatechuic aldehyde), peak 4 (ferulic acid), peak 6 (apigenin glycyrrhizin), peak 9 (codonopsis glycoside), peak 11 (ligustrazine lactone H), and peak 12 (tanshinone B). Negative control solution and control solution showed no corresponding chromatographic peaks. The chromatogram of Guiqi Buxue Oral Liquid is shown below. Figure 7 .

[0066] 2.3.4 Methodological Examination (1) Robustness test: Test solutions from the same batch were used, and the robustness of Agilent ZORBAX C18, Waters Spherisorb ODS C18, and Elite Supersil ODS2 C18 columns were tested according to the chromatographic method of Method 1 for the simulated fingerprinting. The results showed that all three columns of Method 1 for the simulated fingerprinting could separate 14 chromatographic peaks with ideal resolution, indicating good robustness of the method. The results are shown in [Figure number missing]. Figure 8 .

[0067] (2) Precision assessment: For simulated fingerprinting method one, 70 ml of the same batch of Guiqi Buxue Oral Liquid was precisely measured; for simulated fingerprinting method two, 30 ml of the same batch of Guiqi Buxue Oral Liquid was precisely measured. The test samples were prepared according to simulated fingerprinting method one and simulated fingerprinting method two, respectively. The samples were then injected six times consecutively under the chromatographic conditions of simulated fingerprinting method one and simulated fingerprinting method two. Peak 4 was used as the reference peak (S) for simulated fingerprinting method one, and peak 3 was used as the reference peak (S) for simulated fingerprinting method two. The RSDs of the relative peak area and relative retention time of each characteristic peak were all less than 5.0%, indicating good instrument precision. The results are shown in Table 7-10.

[0068] Table 7. Relative Peak Area for Precision Evaluation of Simulated Fingerprint Pattern Method 1 Table 8. Relative Retention Schedule for Precision Evaluation of Method 1 of Simulated Fingerprint Mapping Table 9. Relative Peak Area of ​​Simulated Fingerprint Spectrum Method Two Table 10 Relative Retention Schedule for Simulated Fingerprint Mapping Method 2 (3) Repeatability test: Accurately measure 70 ml of the same batch of Guiqi Buxue Oral Liquid, and take 6 portions according to the simulated fingerprint method 1; take 6 portions according to the simulated fingerprint method 2. Prepare test samples according to simulated fingerprint method 1 and simulated fingerprint method 2 respectively. Prepare 6 portions of each method in parallel. Inject and determine the samples sequentially according to the chromatographic conditions of simulated fingerprint method 1 and simulated fingerprint method 2. For simulated fingerprint method 1, peak 4 is used as the reference peak (S), and for simulated fingerprint method 2, peak 3 is used as the reference peak (S). The RSD of the relative peak area and relative retention time of each characteristic peak is less than 5.0%, indicating that the method has good repeatability. The results of simulated fingerprint method 1 are shown in Tables 11-12, and the results of simulated fingerprint method 2 are shown in Tables 13-14.

[0069] Table 11 Relative Peak Area Table for Simulated Fingerprint Spectrum Method 1 Table 12 Relative Retention Schedule for Simulated Fingerprint Mapping Method 1 Table 13 Relative Peak Area Table for Repeatability Testing of Simulated Fingerprint Method Two Table 14. Timetable for Relative Retention of Repeatability Testing of Simulated Fingerprint Mapping Method 2 (4) Stability test: Accurately measure 70 ml of the same batch of Guiqi Buxue Oral Liquid and prepare the test solution according to Method 1 of the simulated fingerprinting method. At 0 h, 3 h, 6 h, 12 h, 18 h, and 24 h after preparation, the solution is analyzed under the chromatographic conditions of Method 1, with peak 4 as the reference peak (S). Then, take 30 ml of the solution according to Method 2 and prepare the test solution accordingly. At 0 h, 2, 4, 8, 12, and 24 h after preparation, the solution is analyzed under the chromatographic conditions of Method 2, with peak 3 as the reference peak (S). The RSDs of the relative peak area and relative retention time of each characteristic peak are all less than 5.0%, indicating that the test solution is stable within 24 h. The results of Method 1 are shown in Tables 15-16, and the results of Method 2 are shown in Tables 17-18.

[0070] Table 15 Relative peak areas for stability assessment of pseudo-fingerprint spectral method one Table 16 Relative Retention Time Table for Stability Assessment of Simulated Fingerprint Pattern Method 1 Table 17 Relative peak areas for stability assessment of pseudo-fingerprint spectroscopy method two Table 18 Relative Retention Time Table for Stability Assessment of Method 2 of Pseudo-Fingerprint Spectra 2.3.5 Fingerprint mapping and similarity evaluation Take 15 batches of Guiqi Buxue Oral Liquid. For Method 1 of the simulated fingerprint chromatogram, accurately measure 70 ml and prepare the test solution according to Method 1. Inject and determine the chromatogram under the chromatographic conditions of Method 1, record the chromatogram, and calculate the relative retention time and relative peak area of ​​each characteristic peak. The results are shown in Tables 19-20. For Method 2 of the simulated fingerprint chromatogram, accurately measure 30 ml and prepare the test solution according to Method 2. Inject and determine the chromatogram under the chromatographic conditions of Method 2, record the chromatogram, and calculate the relative retention time and relative peak area of ​​each characteristic peak. The results are shown in Tables 21-22.

[0071] The fingerprint chromatograms of 15 batches of Guiqi Buxue Oral Liquid were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version) for data analysis. Using the chromatogram of sample S1 as the reference chromatogram, the mean method was used, with a time window width of 0.1 min, and multi-point correction was performed to generate the HPLC fingerprint chromatograms of 15 batches of Guiqi Buxue Oral Liquid and their corresponding characteristic chromatograms (R). See [link to data]. Figure 1 .

[0072] The simulated fingerprinting method identified 14 common peaks. Similarity was calculated using the control feature spectrum as a reference, and the results are shown in Table 23. The results showed that (1) the relative retention time RSD of each feature peak was between 0.15% and 0.93%, indicating that the elution time of each feature peak was stable; the relative peak area RSD of each feature peak was between 30.05% and 51.12%, indicating that different batches of Guiqi Buxue oral liquid samples had similar chemical composition as a whole, but the mass fraction of some components was different, which may be related to the batch differences of the raw materials; (2) the similarity of 15 Guiqi Buxue oral liquid samples was all >0.920, indicating that the established fingerprint spectrum of Guiqi Buxue oral liquid was stable and could reflect its fingerprint characteristics.

[0073] Table 23 Similarity table of 15 batches of Guiqi Buxue Oral Liquid in Method 1 of simulated fingerprinting. After completing the content testing of multiple batches of samples, it was found that the peak areas of astragaloside A and ginsenoside Rb1 in some batches of Guiqi Buxue Oral Liquid were low. At the same time, due to the different detector sensitivities, the target peak area values ​​were small. In order to better ensure the accuracy of the fingerprint spectrum, the fingerprint spectrum construction method of this invention was finally obtained.

[0074] The second method for prototyping fingerprint spectrum identified 15 common peaks. Similarity was calculated using the control feature spectrum as a reference, and the results are shown in Table 24. The results showed that (1) the relative retention time RSD of each feature peak was between 0.08% and 0.30%, indicating that the elution time of each feature peak was stable; the relative peak area RSD of each feature peak was between 17.81% and 45.45%, indicating that different batches of Guiqi Buxue oral liquid samples had similar chemical composition as a whole, but the mass fraction of some components was different, which may be related to the batch differences of the raw materials; (2) the similarity of 15 batches of Guiqi Buxue oral liquid was all >0.930, indicating that the established fingerprint spectrum of Guiqi Buxue oral liquid was stable and could reflect its fingerprint characteristics.

[0075] Table 24 Similarity table of 15 batches of Guiqi Buxue Oral Liquid in Method 2 of simulated fingerprinting. After completing the content detection of multiple batches of samples, it was found that the peak area of ​​some batches of Guiqi Buxue Oral Liquid was low. At the same time, due to the different detector sensitivities, the target peak area value was small. In order to better ensure the accuracy of the fingerprint spectrum, the fingerprint spectrum construction method of the present invention was finally obtained.

[0076] This invention investigated 15 batches of Guiqi Buxue Oral Liquid. The elution times of each characteristic peak were stable. While the 15 batches of Guiqi Buxue Oral Liquid samples generally exhibited similar chemical compositions, some differences in the mass fraction of certain components were observed, which may be related to batch variations in the original medicinal materials. The similarity of all 15 batches of Guiqi Buxue Oral Liquid was >0.930, indicating that the established fingerprint chromatogram of Guiqi Buxue Oral Liquid is of stable quality and can reflect its fingerprint characteristics. This invention establishes an HPLC fingerprint chromatogram method for Guiqi Buxue Oral Liquid, which has good repeatability and stability. The 29 labeled characteristic components can comprehensively reflect the material basis of Guiqi Buxue Oral Liquid, providing a more favorable technical means for improving quality standards and serving as a detection method for subsequent process optimization.

[0077] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for constructing a fingerprint spectrum of Guiqi Buxue Oral Liquid, characterized in that, Includes the following steps: (1) Preparation of test solution: The test solution was prepared by method one and method two, respectively. (2) Preparation of negative sample solution: Negative sample solution was prepared using method one and method two respectively; (3) Preparation of reference solutions: Reference solutions were prepared using method one and method two, respectively; (4) Determination method: The test solution, negative sample solution and reference solution prepared by method one were injected into the ultra-high performance liquid chromatograph and determined by fingerprint chromatogram method one. The test solution, negative sample solution and reference solution prepared by method two were injected into the ultra-high performance liquid chromatograph and determined by fingerprint chromatogram method two. The chromatograms of the test sample, negative sample and reference solution prepared by method one and method two were recorded respectively. (5) Generate fingerprint spectrum: Import the chromatogram obtained in step (4) into the Chinese medicine chromatographic fingerprint spectrum similarity evaluation system for data processing to generate the fingerprint spectrum of Guiqi Buxue oral liquid; (6) Identify common peaks: Based on the chromatograms of the negative sample solution and the reference solution, identify and determine the common peaks in the fingerprint spectrum of Guiqi Buxue Oral Liquid obtained in step (5).

2. The construction method according to claim 1, characterized in that, In step (1), the method for preparing the test sample solution is as follows: take Guiqi Buxue Oral Liquid, extract it with n-butanol by shaking 2-3 times, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in methanol, shake well, and obtain the test sample solution; in step (2), the method for preparing the negative sample solution is as follows: take the negative sample, extract it with n-butanol by shaking 2-3 times, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in methanol, shake well, and obtain the negative sample solution; in step (3), the method for preparing the reference solution is as follows: weigh out the reference standards of ginsenoside Rb1, astragaloside A, glycyrrhizin, ligustrazine, and stilbene glycoside, dissolve them in methanol, shake well, and prepare the reference solution.

3. The construction method according to claim 1, characterized in that, The fingerprinting method described above uses an evaporative light detector for detection, and the chromatographic conditions are as follows: column: Elite-C 18 4.6 × 250 mm 5 μm; Mobile phase: acetonitrile (A) - 0.05% formic acid solution (B); Column temperature: 30℃; Flow rate: 1.0 ml / min; Drift tube temperature: 120℃; Carrier gas pressure: 3.6 bra; Elution gradient: 0~10min, 15~18%A, 85~82%B; 10~15min, 18~19%A, 82~81%B; 15~25min, 19~20%A, 81~80%B; 25~30min, 20~23%A, 80~77%B; 30~54min, 23~25%A, 77~75%B; 54~59min, 25~30%A, 75~70%B; 59~71min, 30~32%A, 70~68%B; 71~80min, 32~34%A, 68~66%B; 80~90min, 34~35%A, 66~65%B; 90~93min, 35~37%A, 65~63%B; 93~96min, 37~42%A, 63~58%B; 96~96.1min, 42~15%A, 58~85%B; 96.1~102min, 15%A, 85%B.

4. The construction method according to claim 3, characterized in that, The fingerprint spectrum obtained by the fingerprinting method 1 identified 14 chromatographic peaks, representing six processed medicinal materials: Ligustrum lucidum, Astragalus membranaceus, Glycyrrhiza uralensis, processed Polygonum multiflorum, Lycium barbarum, and Panax notoginseng. Among them, peaks 1, 5, 6, and 9 (4 peaks) originated from Ligustrum lucidum, peaks 2, 12, and 14 from Astragalus membranaceus, peaks 3 and 13 from Glycyrrhiza uralensis, peak 4 from Polygonum multiflorum, peak 7 from Lycium barbarum, peaks 10 and 11 from Panax notoginseng, and peak 8 from Salvia miltiorrhiza. By comparing the chromatograms with those of the reference standard and negative sample, five characteristic peaks were identified: peak 3 was glycyrrhizin, peak 4 was stilbene glycoside, peak 6 was ligustrazine, peak 11 was ginsenoside Rb1, and peak 12 was astragaloside A.

5. The construction method according to claim 1, characterized in that, In step (1), the second method for preparing the test sample solution is as follows: take Guiqi Buxue Oral Liquid, add methanol, sonicate, weigh, replenish the lost weight with methanol, shake well, and obtain the test sample solution; in step (2), the second method for preparing the negative sample solution is as follows: take the negative sample, add methanol, sonicate, weigh, replenish the lost weight with methanol, shake well, and obtain the negative sample solution; in step (3), the second method for preparing the reference solution is as follows: take gallic acid, sodium tanshinone, protocatechuic aldehyde, ferulic acid, apigenin, codonopsis glycoside, ligustrazine lactone H, and salvianolic acid B reference standards respectively, dissolve them in methanol, shake well, and prepare the reference solution.

6. The construction method according to claim 1, characterized in that, The fingerprinting method two uses a UV detector for detection. The chromatographic conditions are as follows: octadecylsilane-bonded silica gel as the packing material; column temperature 35℃; flow rate 1.0 ml / min; detection wavelength 260 nm; acetonitrile as mobile phase A; 0.1% formic acid as mobile phase B; gradient elution as follows: 0–5 min, 3% A, 97% B; 5~10min, 3~5%A, 97~95%B; 10~16min, 5~10%A, 95~90%B; 16~35min, 10~15%A, 90~85%B; 35~50min, 15~20%A, 85~80%B; 50~60min, 20~30%A, 80~70%B; 60~70min, 30~40%A, 70~60%B.

7. The construction method according to claim 6, characterized in that, The fingerprint spectrum obtained by the second fingerprinting method identified 15 common peaks, representing seven processed medicinal herbs: Ligustrum lucidum, Astragalus membranaceus, Glycyrrhiza uralensis, processed Polygonum multiflorum, Salvia miltiorrhiza, Angelica sinensis, and Codonopsis pilosula. Among them, peak 8 originated from Ligustrum lucidum, peaks 5 and 14 from Astragalus membranaceus, peak 6 from Glycyrrhiza uralensis, peaks 1 and 7 from processed Polygonum multiflorum, peaks 2, 3, 10, 12, 13, and 15 from Salvia miltiorrhiza, peaks 4 and 11 from Angelica sinensis, and peak 9 from Codonopsis pilosula. By comparing with the chromatogram of the reference standard, eight characteristic peaks were identified, including peak 1 being gallic acid, peak 2 being sodium tanshinone, peak 3 being protocatechuic aldehyde, peak 4 being ferulic acid, peak 6 being apigenin glycyrrhizin, peak 9 being codonopsis glycoside, peak 11 being ligustilide H, and peak 12 being salvianolic acid B.

8. A standard fingerprint spectrum of Guiqi Buxue Oral Liquid, characterized in that, The standard fingerprint spectrum is composed of the fingerprint spectrum obtained by the construction method of claim 4 and the fingerprint spectrum obtained by the construction method of claim 7.

9. The application of the fingerprint spectrum of Guiqi Buxue Oral Liquid as described in claim 8 in the quality control of Guiqi Buxue Oral Liquid, characterized in that, Application of fingerprint spectroscopy as a reference in the quality control of Guiqi Buxue Oral Liquid.

10. A quality control method for Guiqi Buxue Oral Liquid, characterized in that, include: The chromatogram of the sample of Guiqi Buxue Oral Liquid to be tested was then imported into the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System. The similarity evaluation was carried out with reference to the standard fingerprint spectrum described in claim 8, and the quality was judged by the similarity.

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