Quality control method of shuangyizhiqushi huoluo capsule and application thereof

Abstract

The application discloses a kind of double ant wetness and activate capsule's fingerprint spectrum construction method, the construction method includes the following steps: preparation of traditional Chinese medicine compound test solution;Preparation of reference solution;According to the results of the traditional Chinese medicine compound test solution and the reference solution by high performance liquid chromatography, the full period dual-wavelength fusion fingerprint spectrum is fitted using complete addition method, the average spectrum of fusion spectrum is calculated using average method, and the traditional Chinese medicine compound fingerprint spectrum is obtained.The application establishes dual-wavelength fusion fingerprint spectrum method, respectively collects chromatographic data under 210 nm and 270 nm wavelength, relies on traditional Chinese medicine chromatographic fingerprint spectrum super information characteristic digitization evaluation system 4.0 to carry out data fusion and systematically quantified fingerprint method (Systematically Quantified Fingerprint Method, SQFM) evaluation, and combines cluster analysis and principal component analysis and other chemical pattern recognition techniques to carry out quality consistency evaluation on multiple batches of samples, so as to construct more comprehensive quality control mode for the preparation.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a quality control method for Shuangyi Qushi Huoluo Capsules and its application. Background Technology

[0002] Shuangyi Qushi Tongluo Capsules are composed of multiple medicinal herbs with complex chemical components. Establishing a fingerprinting method that can comprehensively reflect its overall chemical characteristics is fundamental to achieving overall quality control of the preparation and promoting the improvement of quality standards. Currently, single-wavelength fingerprinting cannot simultaneously present various components with significantly different ultraviolet absorption characteristics in the compound, resulting in the loss of characteristic peak information and affecting the completeness of quality evaluation.

[0003] The quality control method and its application of the Shuangyi Qushi Huoluo Capsule claimed in this invention are not found in the prior art. Summary of the Invention

[0004] Based on this, the present invention provides a method for constructing the fingerprint spectrum of Shuangyi Qushi Huoluo Capsules, the method comprising the following steps: Preparation of the test solution of the traditional Chinese medicine compound: Take an appropriate amount of the intermediate of Shuangyi Qushi Huoluo Capsules, place it in a container, add solvent, seal tightly, weigh, extract for a period of time, cool and weigh again, use solvent to replenish the lost weight, shake well, centrifuge, take the supernatant, filter, take the filtrate to obtain the test solution of the traditional Chinese medicine compound; wherein, the traditional Chinese medicine compound includes black ants, black-winged termites, ginseng, astragalus, atractylodes macrocephala, angelica sinensis, salvia miltiorrhiza, processed aconite, chicken blood vine, morinda officinalis, epimedium, achyranthes bidentata, clematis chinensis, centipede, scorpion, safflower, wolfberry and coix seed; Preparation of reference solutions: Weigh appropriate amounts of protocatechuic acid, protocatechuic aldehyde, amygdalin, verrucoside glucoside, hyperoside, rosmarinic acid, salvianolic acid B, cuscutaidine A, cuscutaidine B, cuscutaidine C, icariin, and ginsenoside Rb1 reference standards, and add methanol to prepare reference solutions with concentrations of 10-50 μg / ml for each of the following: protocatechuic acid, protocatechuic aldehyde, amygdalin, verrucoside glucoside, hyperoside, rosmarinic acid, salvianolic acid B, cuscutaidine A, cuscutaidine B, cuscutaidine C, icariin, and ginsenoside Rb1. Based on the results of high performance liquid chromatography detection of the test solution and the reference solution of the traditional Chinese medicine compound, the full-time dual-wavelength fusion fingerprint spectrum was fitted by the complete addition method, and the average spectrum of the fusion spectrum was calculated by the average value method to obtain the fingerprint spectrum of the traditional Chinese medicine compound. The chromatographic conditions for this high-performance liquid chromatography (HPLC) detection are as follows: a column packed with octadecylsilane-bonded silica gel is used; mobile phase A is selected from one or more of acetonitrile, methanol, and tetrahydrofuran; mobile phase B is an aqueous acid solution, an aqueous alkaline solution, and / or an aqueous buffer solution; the gradient elution program is as follows: 0–20 min, 0%A→4%A; 20–45 min, 4%A→13%A; 45–60 min, 13%A→18%A; 60–80 min, 18%A→26%A; 80–95 min, 26%A→34%A; 95–120 min, 34%A→57%A; the flow rate is 0.5–1.2 ml / min; the column temperature is 25–35 °C; the detection wavelength is 200–400 nm; and the injection volume is 1–20 μl.

[0005] Furthermore, the intermediate of the Shuangyi Qushi Huoluo Capsule includes a traditional Chinese medicine extract, a first medicinal material powder, and a second medicinal material powder.

[0006] Furthermore, the first medicinal powder is ginseng fine powder.

[0007] Furthermore, the second medicinal powder is fine powder of Clematis chinensis.

[0008] Furthermore, the container is a stoppered conical flask.

[0009] Furthermore, the solvent is an alcohol, such as methanol.

[0010] Furthermore, the volume percentage concentration of methanol is 40% to 60%, for example, about 50%.

[0011] Furthermore, the mass / volume (g / ml) ratio of the intermediate of the Shuangyi Qushi Huoluo capsule to the solvent is 0.01 to 0.05, for example, about 0.02.

[0012] Furthermore, the extraction is either ultrasonic extraction or reflux extraction, for example, ultrasonic extraction.

[0013] Furthermore, the frequency of the ultrasound extraction is 20-60 kHz, for example, about 40 kHz.

[0014] Furthermore, the power of the ultrasound extraction is 100~300 W, for example, about 200 W.

[0015] Furthermore, the ultrasonic extraction time is 25-35 minutes, for example, about 30 minutes.

[0016] Furthermore, the centrifuge speed is 3000~5000 r / min, for example, about 4000 r / min.

[0017] Furthermore, the centrifugation time is 5 to 15 minutes, for example, about 10 minutes.

[0018] Furthermore, the centrifugation was performed at room temperature.

[0019] Furthermore, the filtration is a microporous membrane filtration.

[0020] Furthermore, the pore size of this microporous filter membrane is 0.22 μm.

[0021] Further, the preparation of the reference solution: Weigh appropriate amounts of protocatechuic acid, protocatechuic aldehyde, amygdalin, verrucoside glucoside, hyperoside, rosmarinic acid, salvianolic acid B, cuscuta A, cuscuta B, cuscuta C, icariin, and ginsenoside Rb1 reference standards, and place them separately in a first volumetric flask. Add methanol to dissolve and dilute to the mark, shake well, and use as stock solutions of the above reference standards. Accurately pipette the above stock solutions and place them in a second volumetric flask. Add methanol to dilute to the mark, shake well, and prepare a reference solution with a concentration of 10~50 μg / ml for each of the following: protocatechuic acid, protocatechuic aldehyde, amygdalin, verrucoside glucoside, hyperoside, rosmarinic acid, salvianolic acid B, cuscuta A, cuscuta B, cuscuta C, icariin, and ginsenoside Rb1.

[0022] Furthermore, the first volumetric flask is a 5 mL volumetric flask.

[0023] Furthermore, the second volumetric flask is a 10 mL volumetric flask.

[0024] Furthermore, the volume of the stock solution is 0.1 to 1 mL, for example, about 0.5 mL.

[0025] Furthermore, the concentration of protocatechuic acid in the reference solution was approximately 18.1 μg / ml.

[0026] Furthermore, the concentration of protocatechuic aldehyde in the reference solution was approximately 18.5 μg / ml.

[0027] Furthermore, the concentration of amygdalin in the reference solution was approximately 17.1 μg / ml.

[0028] Furthermore, the concentration of verbascoside in the reference solution was approximately 23.0 μg / ml.

[0029] Furthermore, the concentration of hyperoside in the reference solution was approximately 16.3 μg / ml.

[0030] Furthermore, the concentration of rosmarinic acid in the reference solution was approximately 18.2 μg / ml.

[0031] Furthermore, the concentration of salvianolic acid B in the reference solution was approximately 20.4 μg / ml.

[0032] Furthermore, the concentration of ascorbic acid A in the reference solution was approximately 19.1 μg / ml.

[0033] Furthermore, the concentration of astragaloside B in the reference solution was approximately 20.2 μg / ml.

[0034] Furthermore, the concentration of astragaloside C in the reference solution was approximately 32.3 μg / ml.

[0035] Furthermore, the concentration of icariin in the reference solution was approximately 19.4 μg / ml.

[0036] Furthermore, the concentration of ginsenoside Rb1 in the reference solution was approximately 22.1 μg / ml.

[0037] Furthermore, the flow rate is 0.6 to 1.0 ml / min, for example, about 0.8 ml / min.

[0038] Furthermore, the column temperature is 28~32℃, for example, about 30℃.

[0039] Furthermore, the single wavelengths of this detection wavelength are 210 nm and 270 nm, respectively.

[0040] Furthermore, the injection volume is 5~15 μl, for example, about 10 μl.

[0041] Furthermore, the chromatographic column is a Waters XSelect HSS T3 column.

[0042] Furthermore, the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

[0043] Furthermore, mobile phase A is acetonitrile.

[0044] Furthermore, the aqueous acid solution, aqueous alkaline solution, and / or aqueous buffer salt solution are selected from one or more weak acids and their salts, weak bases and their salts of different concentrations.

[0045] Furthermore, the aqueous acid solution, aqueous alkaline solution, and / or aqueous buffer solution are selected from different concentrations of formic acid, glacial acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid.

[0046] Furthermore, the acid aqueous solution is a 0.01% to 1% acid aqueous solution.

[0047] Furthermore, the acidic aqueous solution is a 0.01%~0.2% phosphoric acid aqueous solution.

[0048] Furthermore, the acidic aqueous solution is an aqueous solution of approximately 0.1% phosphoric acid.

[0049] Furthermore, the buffer salt solution is a phosphate solution and / or a phosphate solution.

[0050] Furthermore, the pH value of this buffer salt solution is no greater than 7.0.

[0051] Furthermore, when the single wavelengths of the detection wavelength are 210 nm and 270 nm, the fingerprint spectrum includes peaks 1-38, with peak 22 serving as a reference peak. The average relative retention times of peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38 are approximately 0.070, 0.075, and 0.08, respectively. 5. Approximately 0.103, approximately 0.126, approximately 0.183, approximately 0.209, approximately 0.225, approximately 0.242, approximately 0.257, approximately 0.269, approximately 0.301, approximately 0.327, approximately 0.365, approximately 0.384, approximately 0.561, approximately 0.622, approximately 0.643, approximately 0.814, approximately 0.860, approximately 0.978, approximately 1.026, approximately 1.041, approximately 1.151, approximately 1.180, approximately 1.261, approximately 1.368, approximately 1.428, approximately 1.649, approximately 1.675, approximately 1.708, approximately 1.792, approximately 1.807, approximately 1.828, approximately 1.876, approximately 1.886, and approximately 1.907.

[0052] Furthermore, when the single wavelengths of this detection wavelength are 210 nm and 270 nm respectively... At nm, the fingerprint spectrum includes peaks 1-38, with peak 22 serving as a reference peak. The average relative peak areas of peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38 are approximately 5.454, 19.242, and 15.56, respectively. 9. Approximately 1.820, approximately 4.310, approximately 1.265, approximately 6.609, approximately 3.713, approximately 3.074, approximately 2.877, approximately 1.764, approximately 2.257, approximately 1.554, approximately 1.064, approximately 1.187, approximately 1.250, approximately 3.034, approximately 1.121, approximately 1.223, approximately 0.565, approximately 1.069, approximately 1.642, approximately 0.812, approximately 0.961, approximately 0.403, approximately 0.641, approximately 1.123, approximately 0.483, approximately 1.436, approximately 0.615, approximately 0.449, approximately 3.748, approximately 0.370, approximately 0.686, approximately 0.851, approximately 0.703, and approximately 0.495.

[0053] Furthermore, when the single wavelengths of the detection wavelength are 210 nm and 270 nm, peak 16 is protocatechuic acid, peak 17 is protocatechuic aldehyde, peak 19 is amygdalin, peak 24 is verbascoside, peak 25 is hyperoside, peak 28 is rosmarinic acid, peak 30 is salvianolic acid B, peak 33 is asparagine A, peak 34 is asparagine B, peak 35 is asparagine C, peak 36 is icariin, and peak 38 is ginsenoside Rb1.

[0054] According to another aspect of the present invention, a quality control method for Shuangyi Qushi Huoluo Capsules is provided, the quality control method comprising the following steps: (1) Establish the standard fingerprint spectrum of the reference sample of Shuangyi Qushi Huoluo Capsules according to the above fingerprint spectrum construction method; (2) Take the test solution of the traditional Chinese medicine compound and perform detection according to the chromatographic conditions in the above fingerprint chromatogram construction method to obtain the fingerprint chromatogram of the traditional Chinese medicine compound sample; and (3) Compare the fingerprint spectrum of the Chinese herbal compound sample to be tested obtained in step (2) with the standard fingerprint spectrum of the reference sample of the Shuangyi Qushi Huoluo capsule obtained in step (1), and evaluate it in combination with the system fingerprint quantitative method and / or chemical pattern recognition analysis. If it meets the requirements, it is a qualified product; if it does not meet the requirements, it is an unqualified product.

[0055] Furthermore, this chemical pattern recognition analysis includes cluster analysis and principal component analysis.

[0056] Furthermore, the evaluation index for the fingerprint quantification method in this system includes macro-qualitative similarity S. m Macro-quantitative similarity P m The relative deviation α between % and the sample fingerprint signal homogenization coefficient.

[0057] Furthermore, the compliance requirement includes: when using the system fingerprint quantitative method for evaluation, the evaluation indicators of the system fingerprint quantitative method must simultaneously meet the following three conditions: (1) Macro-qualitative similarity S m Not less than 0.70; (2) Macro-quantitative similarity P m % ranges from 70% to 130%; and (3) The relative deviation α of the sample fingerprint signal homogenization coefficient is not higher than 0.30.

[0058] According to another aspect of the present invention, the above-described construction method or the above-described quality control method is provided for use in the quality testing and / or quality evaluation and / or quality control of Shuangyi Qushi Huoluo Capsules.

[0059] The beneficial effects of this invention are: This invention establishes a dual-wavelength fusion fingerprinting method, which collects chromatographic data at wavelengths of 210 nm and 270 nm respectively. The data is fused and evaluated using the Systematically Quantified Fingerprint Method (SQFM) based on the digital evaluation system for super-information features of chromatographic fingerprinting of traditional Chinese medicine 4.0. Furthermore, chemical pattern recognition techniques such as cluster analysis and principal component analysis are combined to evaluate the quality consistency of multiple batches of samples, with the aim of constructing a more comprehensive quality control model for this preparation. Attached Figure Description

[0060] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exceeding the scope of protection claimed by the present invention.

[0061] Figure 1 This is a schematic diagram showing the selection results of the detection wavelength in an embodiment of the present invention. Wherein, A is a 3D high-performance liquid chromatography (HPLC) scan; B is a chromatogram at different wavelengths.

[0062] Figure 2 The images show chromatograms at wavelengths of 210 nm and 270 nm, respectively, according to an embodiment of the present invention. Specifically, A is the chromatogram at 210 nm; and B is the chromatogram at 270 nm.

[0063] Figure 3 This is a schematic diagram illustrating the result of establishing a dual-wavelength fused fingerprint spectrum according to an embodiment of the present invention. In the diagram, A represents a fusion example; B represents the fused fingerprint spectrum.

[0064] Figure 4 This is a schematic diagram illustrating the identification results of common peaks in an embodiment of the present invention. Wherein, A is the sample fusion chromatogram; B is the reference standard chromatogram.

[0065] Figure 5 This is a clustering tree diagram of 15 batches of samples in an embodiment of the present invention.

[0066] Figure 6 This is a scree plot of principal component analysis in an embodiment of the present invention.

[0067] Figure 7 This is a score graph of principal component analysis in an embodiment of the present invention. Detailed Implementation

[0068] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0069] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0070] This invention is intended to cover all options, variations, and equivalents that may be included in the field of prior art as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances described herein that can be applied in the practice of this invention. This invention is by no means limited to the description of methods and substances.

[0071] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0072] In this invention, the term "comprising" and "including" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

[0073] As described in the background section, the quality control method and its application for the Shuangyi Qushi Huoluo Capsules claimed in this invention are not found in the prior art. To address the above problem, this invention provides a fingerprint pattern construction method for Shuangyi Qushi Huoluo Capsules, which includes the following steps: Preparation of the test solution of the traditional Chinese medicine compound: Take an appropriate amount of the intermediate of Shuangyi Qushi Huoluo Capsules, place it in a container, add solvent, seal tightly, weigh, extract for a period of time, cool and weigh again, use solvent to replenish the lost weight, shake well, centrifuge, take the supernatant, filter, take the filtrate to obtain the test solution of the traditional Chinese medicine compound; wherein, the traditional Chinese medicine compound includes black ants, black-winged termites, ginseng, astragalus, atractylodes macrocephala, angelica sinensis, salvia miltiorrhiza, processed aconite, chicken blood vine, morinda officinalis, epimedium, achyranthes bidentata, clematis chinensis, centipede, scorpion, safflower, wolfberry and coix seed; Preparation of reference solutions: Weigh appropriate amounts of protocatechuic acid, protocatechuic aldehyde, amygdalin, verrucoside glucoside, hyperoside, rosmarinic acid, salvianolic acid B, cuscutaidine A, cuscutaidine B, cuscutaidine C, icariin, and ginsenoside Rb1 reference standards, and add methanol to prepare reference solutions with concentrations of 10-50 μg / ml for each of the following: protocatechuic acid, protocatechuic aldehyde, amygdalin, verrucoside glucoside, hyperoside, rosmarinic acid, salvianolic acid B, cuscutaidine A, cuscutaidine B, cuscutaidine C, icariin, and ginsenoside Rb1. Based on the results of high performance liquid chromatography detection of the test solution and the reference solution of the traditional Chinese medicine compound, the full-time dual-wavelength fusion fingerprint spectrum was fitted by the complete addition method, and the average spectrum of the fusion spectrum was calculated by the average value method to obtain the fingerprint spectrum of the traditional Chinese medicine compound. The chromatographic conditions for this high-performance liquid chromatography (HPLC) detection are as follows: a column packed with octadecylsilane-bonded silica gel is used; mobile phase A is selected from one or more of acetonitrile, methanol, and tetrahydrofuran; mobile phase B is an aqueous acid solution, an aqueous alkaline solution, and / or an aqueous buffer solution; the gradient elution program is as follows: 0–20 min, 0%A→4%A; 20–45 min, 4%A→13%A; 45–60 min, 13%A→18%A; 60–80 min, 18%A→26%A; 80–95 min, 26%A→34%A; 95–120 min, 34%A→57%A; the flow rate is 0.5–1.2 ml / min; the column temperature is 25–35 °C; the detection wavelength is 200–400 nm; and the injection volume is 1–20 μl.

[0074] In this invention, when time, flow rate, column temperature, wavelength, injection volume, mass ratio, volume percentage concentration, ratio, frequency, power, rotation speed, concentration, macro-quantitative similarity, or other values ​​or parameters are expressed as ranges, preferred ranges, or a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “0.5~1.2” is disclosed, the described range should be interpreted as including ranges “0.5~1.2”, “0.5~1.0”, “0.5~0.8”, “0.5~0.6”, “0.6~1.2”, “0.6~1.0”, “0.6~0.8”, “0.8~1.2”, “0.8~1.0”, “1.0~1.2”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0075] In a preferred embodiment, the intermediate of the Shuangyi Qushi Huoluo capsule includes a traditional Chinese medicine extract, a first medicinal material powder, and a second medicinal material powder.

[0076] In a preferred embodiment, the first medicinal powder is ginseng powder.

[0077] In a preferred embodiment, the second medicinal powder is fine powder of Clematis chinensis.

[0078] The intermediate of the Shuangyi Qushi Huoluo capsule of the present invention can be prepared with reference to the embodiment of ZL200410012720.6, for example, the following embodiment.

[0079] (1) Take the raw materials by weight: 2850g black ants, 950g black-winged subterranean termites, 190g ginseng, 1900g astragalus root Atractylodes macrocephala 570g, Angelica sinensis 1520g, Salvia miltiorrhiza 380g, processed Aconitum carmichaelii 114g Chicken blood vine 570g, Euphorbia pekinensis 380g, Epimedium brevicornu 380g, Achyranthes bidentata 190g Clematis chinensis 1900g, Centipede 76g, Scorpion 114g, Safflower 380g 1140g of goji berries and 380g of Job's tears (2) Pre-treatment of raw materials: Black ants, centipedes, scorpions, astragalus, atractylodes macrocephala, angelica, epimedium, chicken blood vine, morinda officinalis, achyranthes bidentata, wolfberry, coix seed, processed aconite, clematis, safflower, salvia miltiorrhiza, ginseng and other 18 kinds of Chinese medicinal materials are selected to remove impurities and washed before feeding; among them, ginseng and clematis are washed and dried at 60℃ for later use.

[0080] (3) Pulverization: Take the pre-treated ginseng and Clematis chinensis and pulverize them into fine powder. Pass them through a 100-mesh sieve to obtain the fine powder shown in Table 1. Sterilize them under high pressure at 105℃ for 60 minutes and dry them at 80℃ for later use.

[0081] Table 1 Information on Ginseng Powder and Clematis chinensis Powder

[0082] (4) Extraction: Weigh out the four Chinese medicinal herbs, namely black ants, centipedes, scorpions, and black-winged termites, according to the above feed amount, crush them into coarse powder, soak them in 60% ethanol, and then reflux them at 80℃ three times. Add 5, 4, and 3 times the amount of 60% ethanol for extraction, and extract for 1 hour each time. Combine the extracts, let them stand, filter them, and keep the filtrate for later use.

[0083] (5) Weigh out the following twelve medicinal materials according to the above-mentioned amount: Astragalus membranaceus, Atractylodes macrocephala, Angelica sinensis, Epimedium brevicornu, Spatholobus suberectus, Morinda officinalis, Achyranthes bidentata, Lycium barbarum, Coix lacryma-jobi, Aconitum carmichaelii (processed), Carthamus tinctorius, and Salvia miltiorrhiza. Add water and decoct twice, adding 8 and 7 times the amount of water respectively, for 1 hour each time. Combine the decoctions, filter, and concentrate the filtrate to a clear paste with a relative density of about 1.10 (70°C). Add ethanol to make the alcohol content reach 60%, stir well, and let stand for 24 hours.

[0084] (6) Combine the refluxed alcohol solution and the alcohol precipitate, recover the ethanol, and concentrate the drug solution to a thick paste with a relative density of 1.30 (measured at 70℃) to obtain a thick paste for later use.

[0085] (7) Weigh the sterilized ginseng powder and clematis powder according to the above feed amount, mix them evenly, add the above thick paste and mix evenly to obtain the intermediate of Shuangyi Qushi Huoluo Capsules.

[0086] In a preferred embodiment, the container is a stoppered conical flask.

[0087] In a preferred embodiment, the solvent is an alcohol, such as methanol.

[0088] In a preferred embodiment, the volume percentage concentration of methanol is 40% to 60%, for example, about 50%.

[0089] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 50%" includes 50% ± 5%, or from 47.5% to 52.5%.

[0090] In a preferred embodiment, the mass / volume (g / ml) ratio of the intermediate of the Shuangyi Qushi Huoluo capsule to the solvent is 0.01 to 0.05, for example, about 0.02.

[0091] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.02" includes ±5% of 0.02, or from 0.019 to 0.021.

[0092] In a preferred embodiment, the extraction is ultrasonic extraction or reflux extraction, for example, ultrasonic extraction.

[0093] In a preferred embodiment, the frequency of the ultrasonic extraction is 20-60 kHz, for example, about 40 kHz.

[0094] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 40" includes ±5% of 40, or from 38 to 42.

[0095] In a preferred embodiment, the power of the ultrasonic extraction is 100~300 W, for example, about 200 W.

[0096] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 200" includes ±5% of 200, or from 190 to 210.

[0097] In a preferred embodiment, the ultrasonic extraction time is 25 to 35 minutes, for example, about 30 minutes.

[0098] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes 30 ±5%, or from 28.5 to 31.5.

[0099] In a preferred embodiment, the centrifugation speed is 3000~5000 r / min, for example, about 4000 r / min.

[0100] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 4000" includes ±5% of 4000, or from 3800 to 4200.

[0101] In a preferred embodiment, the centrifugation time is 5 to 15 minutes, for example, about 10 minutes.

[0102] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0103] In a preferred embodiment, the centrifugation is performed at room temperature.

[0104] In a preferred embodiment, the filtration is microporous membrane filtration.

[0105] In a preferred embodiment, the microporous filter membrane has a pore size of 0.22 μm.

[0106] In a preferred embodiment, the preparation of the reference solution is as follows: Appropriate amounts of protocatechuic acid, protocatechuic aldehyde, amygdalin, verrucoside glucoside, hyperoside, rosmarinic acid, salvianolic acid B, cuscutaidine A, cuscutaidine B, cuscutaidine C, icariin, and ginsenoside Rb1 reference standards are weighed and placed in a first volumetric flask, dissolved in methanol and diluted to the mark, and shaken well to obtain a stock solution of each of the above reference standards. Accurately pipette the stock solutions and place them in a second volumetric flask, dilute to the mark with methanol, and shaken well to prepare a reference solution with concentrations of 10-50 μg / ml for each of the following: protocatechuic acid, protocatechuic aldehyde, amygdalin, verrucoside glucoside, hyperoside, rosmarinic acid, salvianolic acid B, cuscutaidine A, cuscutaidine B, cuscutaidine C, icariin, and ginsenoside Rb1.

[0107] In a preferred embodiment, the first volumetric flask is a 5 mL volumetric flask.

[0108] In a preferred embodiment, the second volumetric flask is a 10 mL volumetric flask.

[0109] In a preferred embodiment, the volume of the stock solution is 0.1 to 1 mL, for example, about 0.5 mL.

[0110] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.5" includes ±5% of 0.5, or from 0.475 to 0.525.

[0111] In a preferred embodiment, the concentration of protocatechuic acid in the reference solution is about 18.1 μg / ml.

[0112] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 18.1" includes ±5% of 18.1, or from 17.195 to 19.005.

[0113] In a preferred embodiment, the concentration of protocatechuic aldehyde in the reference solution is about 18.5 μg / ml.

[0114] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 18.5" includes ±5% of 18.5, or from 17.575 to 19.425.

[0115] In a preferred embodiment, the concentration of amygdalin in the reference solution is about 17.1 μg / ml.

[0116] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 17.1" includes ±5% of 17.1, or from 16.245 to 17.955.

[0117] In a preferred embodiment, the concentration of verbascoside in the reference solution is about 23.0 μg / ml.

[0118] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 23.0" includes ±5% of 23.0, or from 21.85 to 24.15.

[0119] In a preferred embodiment, the concentration of hyperoside in the reference solution is about 16.3 μg / ml.

[0120] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 16.3" includes ±5% of 16.3, or from 15.485 to 17.115.

[0121] In a preferred embodiment, the concentration of rosmarinic acid in the reference solution is about 18.2 μg / ml.

[0122] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 18.2" includes ±5% of 18.2, or from 17.29 to 19.11.

[0123] In a preferred embodiment, the concentration of salvianolic acid B in the reference solution is about 20.4 μg / ml.

[0124] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20.4" includes ±5% of 20.4, or from 19.38 to 21.42.

[0125] In a preferred embodiment, the concentration of astragaloside A in the reference solution is about 19.1 μg / ml.

[0126] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 19.1" includes ±5% of 19.1, or from 18.145 to 20.055.

[0127] In a preferred embodiment, the concentration of astragaloside B in the reference solution is about 20.2 μg / ml.

[0128] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20.2" includes ±5% of 20.2, or from 19.19 to 21.21.

[0129] In a preferred embodiment, the concentration of astragaloside C in the reference solution is about 32.3 μg / ml.

[0130] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 32.3" includes ±5% of 32.3, or from 30.685 to 33.915.

[0131] In a preferred embodiment, the concentration of icariin in the reference solution is about 19.4 μg / ml.

[0132] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 19.4" includes ±5% of 19.4, or from 18.43 to 20.37.

[0133] In a preferred embodiment, the concentration of ginsenoside Rb1 in the reference solution is about 22.1 μg / ml.

[0134] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 22.1" includes ±5% of 22.1, or from 20.995 to 23.205.

[0135] In a preferred embodiment, the flow rate is 0.6 to 1.0 ml / min, for example, about 0.8 ml / min.

[0136] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.8" includes ±5% of 0.8, or from 0.76 to 0.84.

[0137] In a preferred embodiment, the column temperature is 28~32°C, for example, about 30°C.

[0138] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes 30 ±5%, or from 28.5 to 31.5.

[0139] In a preferred embodiment, the single wavelengths of the detection wavelengths are 210 nm and 270 nm, respectively.

[0140] In a preferred embodiment, the injection volume is 5 to 15 μl, for example, about 10 μl.

[0141] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0142] In a preferred embodiment, the chromatographic column is a Waters XSelect HSS T3 column.

[0143] In a preferred embodiment, the chromatographic column has the following specifications: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

[0144] In a preferred embodiment, the mobile phase A is acetonitrile.

[0145] In a preferred embodiment, the acid aqueous solution, alkaline aqueous solution, and / or buffer salt aqueous solution are selected from one or more weak acids and their salts, and weak bases and their salts of different concentrations.

[0146] In a preferred embodiment, the acidic aqueous solution, alkaline aqueous solution, and / or buffer salt aqueous solution is selected from formic acid, glacial acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid.

[0147] In a preferred embodiment, the acidic aqueous solution is a 0.01% to 1% acidic aqueous solution.

[0148] In a preferred embodiment, the acidic aqueous solution is a 0.01% to 0.2% phosphoric acid aqueous solution.

[0149] In a preferred embodiment, the acidic aqueous solution is an aqueous solution of about 0.1% phosphoric acid.

[0150] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.1%" includes 0.1% ± 5%, or from 0.095% to 0.105%.

[0151] In a preferred embodiment, the buffer salt solution is a phosphate solution and / or a phosphate solution.

[0152] In a preferred embodiment, the pH value of the buffer saline solution is not greater than 7.0.

[0153] In a preferred embodiment, the single wavelengths of the detection wavelength are 210 nm and 270 nm, respectively. At nm, the fingerprint spectrum includes peaks 1-38, with peak 22 serving as a reference peak. The average relative retention times of peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38 are approximately 0.070, 0.075, and 0.08, respectively. 5. Approximately 0.103, approximately 0.126, approximately 0.183, approximately 0.209, approximately 0.225, approximately 0.242, approximately 0.257, approximately 0.269, approximately 0.301, approximately 0.327, approximately 0.365, approximately 0.384, approximately 0.561, approximately 0.622, approximately 0.643, approximately 0.814, approximately 0.860, approximately 0.978, approximately 1.026, approximately 1.041, approximately 1.151, approximately 1.180, approximately 1.261, approximately 1.368, approximately 1.428, approximately 1.649, approximately 1.675, approximately 1.708, approximately 1.792, approximately 1.807, approximately 1.828, approximately 1.876, approximately 1.886, and approximately 1.907.

[0154] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.070" includes ±5% of 0.070, or from 0.0665 to 0.0735; "about 0.075" includes ±5% of 0.075, or from 0.07125 to 0.07875; "about 0.085" includes ±5% of 0.085, or from 0.08075 to 0.08925; "about 0.103" includes ±5% of 0.103, or from 0.09785 to 0.10815; "about 0.126" includes ±5% of 0.126, or from 0.1197 to 0.1323; "about 0.183" includes ±5% of 0.183, or from 0.17385 to 0.19215; "about 0.209" includes 0.209 "About 0.225" includes ±5% of 0.225, or from 0.21375 to 0.23625; "About 0.242" includes ±5% of 0.242, or from 0.2299 to 0.2541; "About 0.257" includes ±5% of 0.257, or from 0.24415 to 0.26985; "About 0.269" includes ±5% of 0.269, or from 0.25555 to 0.28245; "About 0.301" includes ±5% of 0.301, or from 0.28595 to 0.31605; "About 0.327" includes ±5% of 0.327, or from 0.31065 to 0. 34335; “about 0.365” includes 0.365 ± 5%, or from 0.34675 to 0.38325; “about 0.384” includes 0.384 ± 5%, or from 0.3648 to 0.4032; “about 0.561” includes 0.561 ± 5%, or from 0.53295 to 0.58905; “about 0.622” includes 0.622 ± 5%, or from 0.5909 to 0.6531; “about 0.643” includes 0.643 ± 5%, or from 0.61085 to 0.67515; “about 0.814” includes 0.814 ± 5%, or from 0.7733 to 0.8547; “about 0.860” includes 0.860 "About 0.978" includes ±5% of 0.978, or from 0.9291 to 1.0269; "About 1.026" includes ±5% of 1.026, or from 0.9747 to 1.0773; "About 1.041" includes ±5% of 1.041, or from 0.98895 to 1.09305; "About 1.151" includes ±5% of 1.151, or from 1.09345 to 1.20855; "About 1.180" includes ±5% of 1.180, or from 1.121 to 1.239; "About 1.261" includes ±5% of 1.261, or from 1.19795 to 1.32405; "About 1."368" includes 1.368 ± 5%, or from 1.2996 to 1.4364; "about 1.428" includes 1.428 ± 5%, or from 1.3566 to 1.4994; "about 1.649" includes 1.649 ± 5%, or from 1.56655 to 1.73145; "about 1.675" includes 1.675 ± 5%, or from 1.59125 to 1.75875; "about 1.708" includes 1.708 ± 5%, or from 1.6226 to 1.7934; "about 1.792" includes 1.792 ± 5%. Or from 1.7024 to 1.8816; "about 1.807" includes ±5% of 1.807, or from 1.71665 to 1.89735; "about 1.828" includes ±5% of 1.828, or from 1.7366 to 1.9194; "about 1.876" includes ±5% of 1.876, or from 1.7822 to 1.9698; "about 1.886" includes ±5% of 1.886, or from 1.7917 to 1.9803; "about 1.907" includes ±5% of 1.907, or from 1.81165 to 2.00235.

[0155] In a preferred embodiment, the single wavelengths of the detection wavelength are 210 nm and 270 nm, respectively. At nm, the fingerprint spectrum includes peaks 1-38, with peak 22 serving as a reference peak. The average relative peak areas of peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38 are approximately 5.454, 19.242, and 15.56, respectively. 9. Approximately 1.820, approximately 4.310, approximately 1.265, approximately 6.609, approximately 3.713, approximately 3.074, approximately 2.877, approximately 1.764, approximately 2.257, approximately 1.554, approximately 1.064, approximately 1.187, approximately 1.250, approximately 3.034, approximately 1.121, approximately 1.223, approximately 0.565, approximately 1.069, approximately 1.642, approximately 0.812, approximately 0.961, approximately 0.403, approximately 0.641, approximately 1.123, approximately 0.483, approximately 1.436, approximately 0.615, approximately 0.449, approximately 3.748, approximately 0.370, approximately 0.686, approximately 0.851, approximately 0.703, and approximately 0.495.

[0156] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5.454" includes ±5% of 5.454, or from 5.1813 to 5.7267; "about 19.242" includes ±5% of 19.242, or from 18.2799 to 20.2041; "about 15.569" includes ±5% of 15.569, or from 14.79055 to 16.34745; "about 1.820" includes ±5% of 1.820, or from 1.729 to 1.911; "about 4.310" includes ±5% of 4.310, or from 4.0945 to 4.5255; "about 1.265" includes ±5% of 1.265, or from 1.20175 to 1.32825; "about 6.609" includes 6. 609 ± 5%, or from 6.27855 to 6.93945; “about 3.713” includes 3.713 ± 5%, or from 3.52735 to 3.89865; “about 3.074” includes 3.074 ± 5%, or from 2.9203 to 3.2277; “about 2.877” includes 2.877 ± 5%, or from 2.73315 to 3.02085; “about 1.764” includes 1.764 ± 5%, or from 1.6758 to 1.8522; “about 2.257” includes 2.257 ± 5%, or from 2.14415 to 2.36985; “about 1.554” includes 1.554 ± 5%, or from 1.4763 to 1. 0.6317; “about 1.064” includes 1.064 ± 5%, or from 1.0108 to 1.1172; “about 1.187” includes 1.187 ± 5%, or from 1.12765 to 1.24635; “about 1.250” includes 1.250 ± 5%, or from 1.1875 to 1.3125; “about 3.034” includes 3.034 ± 5%, or from 2.8823 to 3.1857; “about 1.121” includes 1.121 ± 5%, or from 1.06495 to 1.17705; “about 1.223” includes 1.223 ± 5%, or from 1.16185 to 1.28415; “about 0.565” includes 0.56 5 ± 5%, or from 0.53675 to 0.59325; “about 1.069” includes 1.069 ± 5%, or from 1.01555 to 1.12245; “about 1.642” includes 1.642 ± 5%, or from 1.5599 to 1.7241; “about 0.812” includes 0.812 ± 5%, or from 0.7714 to 0.8526; “about 0.961” includes 0.961 ± 5%, or from 0.91295 to 1.00905; “about 0.403” includes 0.403 ± 5%, or from 0.38285 to 0.42315; “about 0.641” includes 0.641 ± 5%, or from 0.60895 to 0.67305; “about 1.123” includes 1.123 ± 5%, or from 1.06685 to 1.17915; “about 0.483” includes 0.483 ± 5%, or from 0.45885 to 0.50715; “about 1.436” includes 1.436 ± 5%, or from 1.3642 to 1.5078; “about 0.615” includes 0.615 ± 5%, or from 0.58425 to 0.64575; “about 0.449” includes 0.449 ± 5%, or from 0.42655 to 0.47145; “about 3.748” includes 3.74 "About 0.370" includes ±5% of 0.370, or from 0.3515 to 0.3885; "About 0.686" includes ±5% of 0.686, or from 0.6517 to 0.7203; "About 0.851" includes ±5% of 0.851, or from 0.80845 to 0.89355; "About 0.703" includes ±5% of 0.703, or from 0.66785 to 0.73815; "About 0.495" includes ±5% of 0.495, or from 0.47025 to 0.51975.

[0157] In a preferred embodiment, when the single wavelengths of the detection wavelengths are 210 nm and 270 nm, peak 16 is protocatechuic acid, peak 17 is protocatechuic aldehyde, peak 19 is amygdalin, peak 24 is isoflavone glucoside, peak 25 is hyperoside, peak 28 is rosmarinic acid, peak 30 is salvianolic acid B, peak 33 is asparagine A, peak 34 is asparagine B, peak 35 is asparagine C, peak 36 is icariin, and peak 38 is ginsenoside Rb1.

[0158] According to another aspect of the present invention, a quality control method for Shuangyi Qushi Huoluo Capsules is provided, the quality control method comprising the following steps: (1) Establish the standard fingerprint spectrum of the reference sample of Shuangyi Qushi Huoluo Capsules according to the above fingerprint spectrum construction method; (2) Take the test solution of the traditional Chinese medicine compound and perform detection according to the chromatographic conditions in the above fingerprint chromatogram construction method to obtain the fingerprint chromatogram of the traditional Chinese medicine compound sample; and (3) Compare the fingerprint spectrum of the Chinese herbal compound sample to be tested obtained in step (2) with the standard fingerprint spectrum of the reference sample of the Shuangyi Qushi Huoluo capsule obtained in step (1), and evaluate it in combination with the system fingerprint quantitative method and / or chemical pattern recognition analysis. If it meets the requirements, it is a qualified product; if it does not meet the requirements, it is an unqualified product.

[0159] In a preferred embodiment, the chemical pattern recognition analysis includes cluster analysis and principal component analysis.

[0160] In a preferred embodiment, the evaluation index of the fingerprint quantification method of this system includes macro-qualitative similarity S. m Macro-quantitative similarity P m The relative deviation α between % and the sample fingerprint signal homogenization coefficient.

[0161] In a preferred embodiment, the compliance requirement includes: when using the system fingerprint quantitative method for evaluation, the evaluation indicators of the system fingerprint quantitative method simultaneously meet the following three conditions: (1) Macro-qualitative similarity S m Not less than 0.70; (2) Macro-quantitative similarity P m % ranges from 70% to 130%; and (3) The relative deviation α of the sample fingerprint signal homogenization coefficient is not higher than 0.30.

[0162] According to another aspect of the present invention, the above-described construction method or the above-described quality control method is provided for use in the quality testing and / or quality evaluation and / or quality control of Shuangyi Qushi Huoluo Capsules.

[0163] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0164] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0165] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this patent specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0166] Example 1. Experimental materials 1.1 Experimental Apparatus The instruments used in this experiment are shown in Table 2.

[0167] Table 2 Experimental Instruments

[0168] 1.2 Reagents and Chemicals The reagents used in this experiment are shown in Table 3.

[0169] Table 3 Experimental Reagents

[0170] 1.3 Experimental Samples The test sample used in this study was a capsule intermediate, which was a mixture of a thick extract and two fine powders of medicinal materials (the specific ratio was: thick extract: ginseng fine powder: Clematis chinensis fine powder = 410g: 22.8g: 228g), provided by Guangxi Shuangyi Pharmaceutical Co., Ltd., and the batch number information is shown in Table 4.

[0171] Table 4 Sample Information Table

[0172] 2. Experimental Methods 2.1 Solution Preparation 2.1.1 Preparation of the test solution Take the thick paste and the two fine powders, and mix them thoroughly according to the prescription ratio. Accurately weigh approximately 1 g of this mixture, place it in a stoppered conical flask, accurately add 50 mL of 50% methanol, seal tightly, and weigh. Sonicate (200 W, 40 kHz) for 30 min, remove, cool to room temperature, weigh again, and replenish the lost weight with 50% methanol. Mix well. Transfer to a centrifuge tube, centrifuge at 4000 r / min for 10 min (room temperature), collect the supernatant, filter through a 0.22 μm microporous membrane, and collect the filtrate.

[0173] 2.1.2 Preparation of reference solution Accurately weigh 1.81 mg of protocatechuic acid, 1.85 mg of protocatechuic aldehyde, 1.71 mg of amygdalin, 2.30 mg of verbascoside glucoside, 1.63 mg of hyperoside, 1.82 mg of rosmarinic acid, 2.04 mg of salvianolic acid B, 1.91 mg of aspartame A, 2.02 mg of aspartame B, 3.23 mg of aspartame C, 1.94 mg of icariin, and 2.21 mg of ginsenoside Rb1, and place them separately in 5 mL volumetric flasks. Dissolve them in methanol and dilute to the mark, then shake well to obtain the stock solutions of each reference standard. Accurately pipette 0.5 mL of each stock solution into a 10 mL volumetric flask, dilute to the mark with methanol, and shake well to obtain the mixed reference solution.

[0174] 2.2 Screening and Validation of Chromatographic Conditions 2.2.1 Screening of chromatographic conditions (1) Full gradient: 0~80 min, 5%~95% A; Under these conditions, all chromatographic peaks appeared within 0~70 min, but the baseline was severely unstable, the chromatographic peaks overlapped, and the resolution could not reach the requirement of greater than 1.5, which could not meet the requirements of fingerprint chromatogram. Subsequent gradient optimization was carried out based on (1).

[0175] (2) Gradient 2: 0~20 min, 5%~12% A; 20~60 min, 12%~45% A; 60~100 min, 45%~80% A; Under these conditions, the baseline drifts and multiple peaks overlap severely in the 20~60 min interval, the separation cannot reach the requirement of greater than 1.5, and the number of characteristic peaks cannot be determined.

[0176] (3) Gradient 3: 0~20 min, 0%~12% A; 20~60 min, 12%~45% A; 60~100 min, 45%~70% A; Under these conditions, the baseline improved in the 0~20 min period, but the overall baseline was still unstable. The total analysis time of 100 min was still insufficient to achieve a stable baseline by slowing down the gradient, and the resolution could not meet the requirement of greater than 1.5. The analysis time needed to be further extended.

[0177] (4) Gradient 4: 0~10 min, 0%~10% A; 10~40 min, 10%~25% A; 40~75 min, 25%~50% A; 70~120 min, 50%~70% A; Under this condition, the total time is extended to 120 min, but the baseline is still not stable overall, and the separation degree cannot reach the requirement of greater than 1.5, which cannot meet the requirements of fingerprint spectrum.

[0178] (5) Gradient 5: 0~20 min, 0%~4% A; 20~45 min, 4%~15% A; 45~60 min, 15%~20% A; 60~100 min, 20%~32% A; 100~120 min, 32%~53% A; Under these conditions, the baseline is mostly stable, but the chromatographic peak resolution cannot reach the requirement of greater than 1.5, and there are still peaks in the last 5 min (elution is not completely finished).

[0179] (6) Final gradient conditions: 0~20 min, 0~4% A; 20~45 min, 4%~13% A; 45~60 min, 13%~18% A; 60~80 min, 18%~26% A; 80~95 min, 26%~34% A; 95~120 min, 34%~57% A. Under the final gradient conditions, the baseline was stable, all chromatographic peaks appeared, the chromatographic peak resolution reached the requirement of greater than 1.5, and there were no chromatographic peaks in the last 5 minutes, which met the requirements of the compound fingerprint chromatogram.

[0180] 2.2.2 Validation of chromatographic conditions A Waters XSelect HSS T3 column (4.6 × 250 mm, 5 μm) was used at a column temperature of 30 °C. The mobile phase was acetonitrile (A) and 0.1% phosphoric acid aqueous solution (B), with a flow rate of 0.8 mL / min. The gradient elution program was as follows: 0–20 min, 0% A → 4% A; 20–45 min, 4% A → 13% A; 45–60 min, 13% A → 18% A; 60–80 min, 18% A → 26% A; 80–95 min, 26% A → 34% A; 95–120 min, 34% A → 57% A. Detection wavelengths were 210 nm and 270 nm. The injection volume was 10 μL.

[0181] 2.3 Establishment of Dual-Wavelength Fusion Fingerprint Fifteen batches of samples were collected, and test solutions were prepared according to the method described in section "2.1". The samples were then injected and analyzed under the chromatographic conditions described in section "2.2", and chromatograms at wavelengths of 210 nm and 270 nm were recorded. The chromatographic data at each wavelength were imported into the Traditional Chinese Medicine Chromatographic Fingerprint Super-Information Feature Digital Evaluation System 4.0. Data fusion was performed using the complete addition method to generate full-time dual-wavelength fused fingerprint chromatograms for the 15 batches of samples. The average chromatogram of the fused chromatogram was calculated using the averaging method and used as the reference fingerprint chromatogram (RFP) for subsequent systematic fingerprint quantitative evaluation.

[0182] 2.4 Methodological Examination 2.4.1 Precision Test Take the test solution from the same batch number, prepare it according to the method in section "2.1", inject it continuously 6 times under the chromatographic conditions in section "2.2", record the chromatograms at wavelengths of 210 nm and 270 nm respectively, and calculate the RSD of the relative retention time and relative peak area of ​​each common peak.

[0183] 2.4.2 Repeatability Test Take 6 samples from the same batch number, prepare test solutions in parallel according to the method in section "2.1", and analyze them under the chromatographic conditions in section "2.2". Record the chromatograms at wavelengths of 210 nm and 270 nm, and calculate the RSD of the relative retention time and relative peak area of ​​each common peak.

[0184] 2.4.3 Stability Test Take the test solution of the same batch number, prepare it according to the method under section "2.1", place it at room temperature, and inject it for analysis at 0, 3, 6, 9, 12 and 24 h according to the chromatographic conditions under section "2.2". Record the chromatograms at wavelengths of 210 nm and 270 nm respectively, and calculate the RSD of the relative retention time and relative peak area of ​​each common peak.

[0185] 2.5 Quantitative Evaluation of System Fingerprint 2.5.1 Principle of System Fingerprint Quantitative Method The sample fingerprint vector and the standard fingerprint vector are defined as follows: and x i and y i Calculate the area of ​​each fingerprint peak. and The cosine of the included angle is the qualitative similarity ( S F This reflects the similarity in the distribution ratio of chemical components between the sample and the standard fingerprint spectrum, but... S F There is a problem where large peaks mask smaller peaks, therefore the standard fingerprint vector... Do ,sample Do , and The cosine of the angle between them is the ratio qualitative similarity ( The fingerprint peaks are equally weighted. Combining the two qualitative properties, the dual qualitative similarity ( S F and The mean of ) is called the macro-qualitative similarity ( S m Formula 1) is used to monitor the quantity and distribution ratio of chemical fingerprints as a whole.

[0186] Projection content similarity ( C Quantitative similarity (QS) can reflect the similarity between the sample and the standard fingerprint spectrum in terms of overall content, but it is severely affected by large peaks and cannot reflect the loss of small peaks. P Treating all peak integral values ​​equally accurately reflects the content changes of the corresponding chemical components in smaller peaks, this approach combines two quantitative properties to achieve dual quantitative similarity. C and PThe mean of ) is called the macro-quantitative similarity ( P m Formula 2) can monitor the overall content of chemical fingerprints.

[0187] The fingerprint signal homogenization coefficient (γ) reflects the degree of homogenization of the chemical fingerprint signal distribution. The closer γ is to 1, the more similar the magnitudes of the fingerprint signals become. x and standard fingerprint γ y The closer the sample is to the standard fingerprint, the more similar it is to the standard fingerprint spectrum. Therefore, the sample γ is defined as... x The relative deviation α is the coefficient of variation of fingerprint homogenization (Formula 3), used S m , P m The method of evaluating the quality of traditional Chinese medicine (TCM) by combining it with α is called the systematic fingerprint quantitative method, which classifies the quality of TCM into 8 levels. The specific level classification is shown in Table 5.

[0188] (1) (2) (3) Table 5. Classification of Traditional Chinese Medicine Quality Grades Using Systematic Fingerprint Quantitative Method

[0189] 2.5.2 Quantitative Evaluation Method for System Fingerprint Based on the dual-wavelength fusion fingerprint chromatograms of 15 batches of samples and the control fingerprint chromatograms established under section "2.3", the digital evaluation system for super-information features of traditional Chinese medicine chromatographic fingerprint chromatograms 4.0 was used to calculate the fingerprint chromatograms of each sample and the control. S m , P m And α, determine the quality grade of each batch of samples according to the standards in Table 5.

[0190] 2.6 Chemical Pattern Recognition Analysis The common peak areas of the dual-wavelength fusion fingerprint spectra of 15 batches of samples were imported into SPSS 27.0 software. System cluster analysis was performed using the inter-group linkage method with Euclidean distance as the metric. At the same time, principal component analysis was performed to examine the clustering and dispersion trends of different batches of samples through principal component score plots, so as to evaluate the overall consistency of the samples and verify the reliability of the system fingerprint quantitative evaluation results.

[0191] 3. Experimental Results 3.1 Optimization of test sample solution preparation conditions 3.1.1 Selection of extraction solvent The extraction method was fixed as ultrasound, and the extraction time was 30 min. Test solutions were prepared using 30% methanol, 50% methanol, 70% methanol, and anhydrous methanol as extraction solvents, respectively. The solutions were injected and analyzed under the same chromatographic conditions, and the chromatograms were recorded. The extraction efficiency of different extraction solvents was investigated using the total peak area and the number of identifiable peaks under the same integral parameters as evaluation indicators. The results are shown in Table 6.

[0192] Table 6 shows that extraction with 50% methanol yielded the largest total peak area, with 50 identifiable peaks. Extraction with 30% methanol and 70% methanol yielded 49 and 51 peaks respectively, showing no significant difference from 50% methanol, but their total peak areas were lower. Extraction with anhydrous methanol resulted in significantly lower total peak area and peak count compared to other solvents. The overall chromatogram profiles of the test solutions obtained with different extraction solvents were basically consistent, with stable baselines. Extraction with 50% methanol showed higher response values ​​for each major chromatographic peak, consistent with the results in Table 6. Considering all factors, 50% methanol was selected as the extraction solvent.

[0193] Table 6 Comparison of extraction rates with different extraction solvents

[0194] 3.1.2 Selection of Extraction Method With 50% methanol as the fixed extraction solvent and an extraction time of 30 min, test solutions were prepared using both ultrasonic and reflux extraction methods. The solutions were then analyzed under the same chromatographic conditions, and the chromatograms were recorded. The extraction efficiency of different methods was evaluated using total peak area and the number of identifiable peaks as evaluation indicators. The results are shown in Table 7. Table 7 shows that the total peak area was greater with ultrasonic extraction than with reflux extraction, while the number of identifiable peaks was comparable. Considering all factors, ultrasonic extraction was selected as the extraction method.

[0195] Table 7 Comparison of extraction rates for different extraction methods

[0196] 3.1.3 Selection of extraction time The extraction solvent was fixed at 50% methanol, and the extraction method was ultrasonic extraction. Test solutions were prepared by extraction for 20, 30, and 40 min, respectively, and analyzed under the same chromatographic conditions. Chromatograms were recorded. The extraction efficiency at different extraction times was examined using total peak area and the number of identifiable peaks as evaluation indicators. The results are shown in Table 8. Table 8 shows that the total peak area was largest at 30 min; the number of peaks was slightly higher at 40 min than at 30 min, but the total peak area decreased; and all indicators were lower at 20 min than the other two groups. Considering all factors, 30 min was selected as the extraction time.

[0197] Table 8 Comparison of extraction rates at different extraction times

[0198] 3.2 Optimization of chromatographic conditions 3.2.1 Selection of detection wavelength The test solution was scanned across the entire wavelength range of 200–400 nm using a PDA detector to obtain a 3D chromatogram (results are shown in Figure 1). Figure 1 (As shown in A). The results showed that the UV absorption characteristics of various components in Shuangyi Qushi Tongluo Capsules varied considerably, with many chromatographic peaks responding near 210 nm, while some chromatographic peaks exhibited characteristic absorption at 270 nm. Chromatograms at different wavelengths were compared (results are shown in A). Figure 1 As shown in Figure B, the chromatographic peaks are most numerous and the response value is highest at a wavelength of 210 nm; the peak shape and resolution are better at a wavelength of 270 nm than at other wavelengths. Considering the number of chromatographic peaks, response value, and separation effect, 210 nm and 270 nm were selected as the detection wavelengths for the dual-wavelength fusion fingerprint spectrum.

[0199] 3.2.2 Selection of Chromatographic Column Under the same chromatographic conditions, the separation performance of four chromatographic columns—XB-C18, AQ-C18, PG-C18, and T3—on the samples was investigated. The total peak area and the number of identifiable peaks were used as evaluation indicators, and the results are shown in Table 9. Table 9 shows that the T3 column had the largest total peak area and the most identifiable peaks, significantly outperforming the other three columns. Therefore, the T3 column was selected for subsequent experiments.

[0200] Table 9 Comparison of separation effects of different chromatographic columns

[0201] 3.2.3 Selection of Mobile Phase System Under the same chromatographic conditions, the separation effects of four mobile phase systems—methanol-water, methanol-0.1% phosphoric acid solution, acetonitrile-water, and acetonitrile-0.1% phosphoric acid solution—on the samples were investigated. The total peak area, the number of identifiable peaks, and baseline stability were used as evaluation indicators. The results are shown in Table 10. Table 10 shows that the acetonitrile-0.1% phosphoric acid solution system had the highest number of identifiable peaks and the most stable baseline. Considering all factors, acetonitrile-0.1% phosphoric acid solution was selected as the mobile phase system.

[0202] Table 10 Comparison of separation performance of different chromatographic columns

[0203] 3.2.4 Investigation of column temperature With other chromatographic conditions fixed, column temperatures were set at 25, 30, and 35℃ to investigate the effect of different column temperatures on sample separation. The total peak area and the number of identifiable peaks were used as evaluation indicators. The results are shown in Table 11. Table 11 shows that the total peak area was largest and the number of identifiable peaks was highest at a column temperature of 30℃. Therefore, 30℃ was selected as the chromatographic column temperature.

[0204] Table 11 Comparison of separation effects at different column temperatures

[0205] 3.2.5. Examination of Flow Velocity With other chromatographic conditions fixed, flow rates of 0.8, 1.0, and 1.2 mL / min were set to investigate the effect of different flow rates on sample separation. The total peak area and the number of identifiable peaks were used as evaluation indicators, and the results are shown in Table 12. Table 12 shows that the total peak area was largest and the number of identifiable peaks was highest at a flow rate of 0.8 mL / min. Therefore, 0.8 mL / min was selected as the chromatographic flow rate.

[0206] Table 12 Comparison of separation effects at different flow rates

[0207] 3.2.6 Extend the washout time In establishing the fingerprinting method, to ensure that all analytes are effectively eluted and to avoid affecting column performance or subsequent analysis due to the residue of strongly retained substances, this experiment investigated the extension of elution time. When the elution time was extended to 150 min, no obvious chromatographic peak was observed after 120 min. This indicates that all components in the sample have been completely eluted, with no strongly adsorbed residues, and that these chromatographic conditions can accurately reflect the fingerprint information.

[0208] 3.3 Methodological Investigation Results 3.3.1 Precision Test Results The results of the precision test are shown in Tables 13 and 14. The RSD of the relative retention time of each common peak is less than 0.40%, and the RSD of the relative peak area is less than 4.70%, indicating that the instrument has good precision.

[0209] Table 13 Results of the relative retention time precision test (n=6)

[0210] Table 14 Results of the relative peak area precision test (n=6)

[0211] 3.3.2 Repeatability Test Results The repeatability test results showed that the RSD of the relative retention time of each common peak was less than 0.60%, and the RSD of the relative peak area was less than 4.80%, indicating that the method has good repeatability.

[0212] 3.3.3 Stability Test Results The stability test results showed that the RSD of the relative retention time of each common peak was less than 1.44%, and the RSD of the relative peak area was less than 4.80%, indicating that the test solution had good stability within 24 h.

[0213] 3.4 Establishment of Dual-Wavelength Fusion Fingerprint Fifteen batches of samples were analyzed according to the methods described in sections “2.1.1” and “2.2”, and chromatograms at wavelengths of 210 nm and 270 nm were recorded, respectively. Figure 2 A and Figure 2 As shown in B, the chromatographic data at each wavelength are imported into the Digital Evaluation System for Super-Information Features of Chromatographic Fingerprint of Traditional Chinese Medicine 4.0 for fusion. An example of the fusion process is shown below. Figure 3 As shown in A, the generated dual-wavelength fusion fingerprint spectra of 15 batches of samples are shown below. Figure 3 B. After calibration, a total of 38 common peaks were identified. The average spectrum of the fusion spectrum was calculated using the averaging method and used as the reference fingerprint spectrum RFP. Figure 3 The red pattern in B is used for subsequent quantitative evaluation of the system fingerprint.

[0214] 3.5. Common Peak Identification The retention time was used to identify some common peaks. The chromatograms of the sample fusion and the reference standard were compared as follows: Figure 4 As shown, a total of 12 chromatographic peaks were identified, namely: peak 16 protocatechuic acid, peak 17 protocatechuic aldehyde, peak 19 amygdalin, peak 24 verbascoside, peak 25 hyperoside, peak 28 rosmarinic acid, peak 30 salvianolic acid B, peak 33 ascorbic acid A, peak 34 ascorbic acid B, peak 35 ascorbic acid C, peak 36 icariin, and peak 38 ginsenoside Rb1.

[0215] 3.6 Quantitative Evaluation Results of System Fingerprint Based on the fingerprint spectra of 15 batches of samples under single-wavelength and dual-wavelength fusion conditions at 210 nm and 270 nm, the macro-qualitative similarity between each sample and the control fingerprint spectra was calculated using the Digital Evaluation System for Hyper-Information Features of Traditional Chinese Medicine Chromatographic Fingerprint Spectra 4.0. S m ), macro-quantitative similarity ( P m The fingerprint homogenization variation coefficient (α) and the quality grade of each batch of samples were determined according to the standards in Table 5.

[0216] The results showed that under dual-wavelength fusion conditions, 15 batches of samples...S m , P m The mean values ​​of α and σ were 0.983, 99.6%, and 0.024, respectively, with quality grades ranging from 1 to 4, including 8 batches of grade 1, 4 batches of grade 2, 2 batches of grade 3, and 1 batch of grade 4. The 15 batches of samples... S m All are greater than 0.7. P m All were within the range of 70% to 130%, indicating good consistency in quality among the batches of samples.

[0217] 3.7 Results of Chemical Pattern Recognition Analysis 3.7.1 Hierarchical Cluster Analysis The peak areas of 38 common peaks from the dual-wavelength fusion fingerprint spectra of 15 batches of samples were imported into SPSS 27.0 software. Hierarchical cluster analysis was performed using the inter-group linkage method with Euclidean distance as the metric. The results are as follows: Figure 5 As shown. By Figure 5 It can be seen that when the square Euclidean distance is 20, the 15 batches of samples are divided into 4 categories: S1 is a separate category, S2 is a separate category, S14 is a separate category, and the remaining 12 batches of samples are clustered into one category.

[0218] 3.7.2 Principal Component Analysis The peak areas of 38 common peaks from the dual-wavelength fusion fingerprint spectra of 15 batches of samples were imported into SPSS 27.0 software for principal component analysis. Using an eigenvalue greater than 2.5 as the extraction criterion, the first 6 principal components were extracted. Their eigenvalues, variance contribution rates, and cumulative variance contribution rates are shown in Table 15. The scree plot is shown below. Figure 6 As shown, the cumulative variance contribution rate of the first 6 principal components is 80.180%, which can explain most of the information of the 38 common peaks.

[0219] The loading matrices of each principal component are shown in Table 16. Principal component 1 is mainly highly correlated with peaks G31, G35, G24, and G5; principal component 2 is mainly highly correlated with peaks G21, G15, G12, and G17; principal component 3 is mainly highly correlated with peaks G36, G8, G33, and G18; principal component 4 is mainly highly correlated with peaks G13, G14, and G23; principal component 5 is mainly highly correlated with peaks G4, G11, and G5; and principal component 6 is mainly highly correlated with peaks G4, G26, and G20.

[0220] The scores of the 15 batches of samples on each principal component and their overall scores are shown in Table 17. The overall scores of the 15 batches of samples range from -0.616 to 0.774, with S1 having the highest overall score (0.774) and S8 having the lowest (-0.616), indicating that there are certain differences in the overall chemical characteristics among different batches of samples. A three-dimensional score plot was plotted based on the scores of the first three principal components, as shown in the principal component score plot below. Figure 7 As shown. From Figure 7 As can be seen, S1, S2, and S14 are relatively dispersed in three-dimensional space, while the other 12 batches of samples are relatively concentrated.

[0221] Table 15 Eigenvalues ​​and Variance Contribution Rates

[0222] Table 16 Sample Composition Matrix

[0223] Table 17 Component Scores and Overall Score

[0224] 4. Summary This study established a dual-wavelength fusion fingerprint analysis method for Shuangyi Qushi Tongluo Capsules, and combined it with systematic fingerprint quantification and chemical pattern recognition technology to evaluate the quality consistency of 15 batches of samples.

[0225] System fingerprint quantitative method uses macro-qualitative similarity S m Macro-quantitative similarity P m Combining these methods allows for a comprehensive evaluation of the quality of traditional Chinese medicine (TCM) from both qualitative and quantitative dimensions. The results of this study show that under the combined conditions, no batches of the 15 batches were classified as grade 5 or lower, and the quality grade distribution was relatively concentrated, indicating good overall consistency in sample quality. It is worth noting that some batches showed lower quality at a single wavelength of 270 nm due to… P m The samples that deviated from the recommended level and were initially classified as level 5 or 6 returned to the normal range after fusion. This phenomenon demonstrates that dual-wavelength fusion can effectively correct evaluation biases caused by abnormal responses of some components under a single wavelength, making the results closer to the true quality of the samples.

[0226] Cluster analysis grouped S1, S2, and S14 into separate clusters, while the remaining 12 batches were grouped into another. Principal component analysis (PCA) also showed that S1, S2, and S14 were spatially dispersed, while the remaining 12 batches were relatively clustered. This classification result is largely consistent with the grading determination of the systematic fingerprinting method: S14, as the only 4-level batch under the fusion condition, has... P mThe value was 117.2%, exceeding the Level 1 range, indicating a significant difference in the overall chemical composition quantification characteristics compared to other batches. While S1 and S2 were Level 2 in the systematic fingerprint quantification method, they were separated in PCA, suggesting that unsupervised pattern recognition methods are more sensitive to subtle differences between samples. This complementary multi-method evaluation model helps to more comprehensively identify potential quality risks.

[0227] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, any changes or modifications made by those skilled in the art based on the ideas of the present invention, its specific implementation methods, and its application scope, are all within the scope of protection of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for constructing a fingerprint spectrum of Shuangyi Qushi Huoluo Capsules, characterized in that, The construction method includes the following steps: Preparation of the test solution of the traditional Chinese medicine compound: Take an appropriate amount of the intermediate of Shuangyi Qushi Huoluo Capsules, place it in a container, add methanol, seal tightly, weigh, extract for a period of time, cool and weigh again, use methanol to make up for the lost weight, shake well, centrifuge, take the supernatant, filter, take the filtrate to obtain the test solution of the traditional Chinese medicine compound; wherein, the traditional Chinese medicine compound includes black ants, black-winged termites, ginseng, astragalus, atractylodes macrocephala, angelica sinensis, salvia miltiorrhiza, processed aconite, chicken blood vine, morinda officinalis, epimedium, achyranthes bidentata, clematis chinensis, centipede, scorpion, safflower, wolfberry and coix seed; Preparation of reference solutions: Weigh appropriate amounts of protocatechuic acid, protocatechuic aldehyde, amygdalin, vermouth isoflavone glucoside, hyperoside, rosmarinic acid, salvianolic acid B, choleretic acid A, choleretic acid B, choleretic acid C, icariin, and ginsenoside Rb1 reference standards, and add methanol to prepare reference solutions with concentrations of 10-50 μg / ml for each of the following: protocatechuic acid, protocatechuic aldehyde, amygdalin, vermouth isoflavone glucoside, hyperoside, rosmarinic acid, salvianolic acid B, choleretic acid A, choleretic acid B, choleretic acid C, icariin, and ginsenoside Rb1. Based on the results of high performance liquid chromatography detection of the test solution of the traditional Chinese medicine compound and the reference solution, the full-time dual-wavelength fusion fingerprint spectrum was fitted by the complete addition method, and the average spectrum of the fusion spectrum was calculated by the average value method to obtain the fingerprint spectrum of the traditional Chinese medicine compound. The chromatographic conditions for high-performance liquid chromatography (HPLC) detection were as follows: a column packed with octadecylsilane-bonded silica gel was used; mobile phase A was acetonitrile; mobile phase B was approximately 0.1% aqueous phosphoric acid solution; and the gradient elution program was as follows: 0–20 min, 0%A → 4%A; 20–45 min, 4%A → 13%A; 45–60 min, 13%A → 18%A; 60–80 min, 18%A → 26%A; 80–95 min, 26%A → 34%A; 95–120 min, 34%A → 57%A; the flow rate was 0.5–1.2 ml / min; the column temperature was 25–35 °C; the detection wavelength was 200–400 nm; and the injection volume was 1–20 μl. When the single wavelengths of the detection wavelengths are 210 nm and 270 nm, respectively, the fingerprint spectrum includes peaks 1-38, with peak 22 serving as a reference peak. The average relative retention times of peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38 are approximately 0.070, 0.075, and 0.08, respectively.

5. Approximately 0.103, approximately 0.126, approximately 0.183, approximately 0.209, approximately 0.225, approximately 0.242, approximately 0.257, approximately 0.269, approximately 0.301, approximately 0.327, approximately 0.365, approximately 0.384, approximately 0.561, approximately 0.622, approximately 0.643, approximately 0.814, approximately 0.860, approximately 0.978, approximately 1.026, approximately 1.041, approximately 1.151, approximately 1.180, approximately 1.261, approximately 1.368, approximately 1.428, approximately 1.649, approximately 1.675, approximately 1.708, approximately 1.792, approximately 1.807, approximately 1.828, approximately 1.876, approximately 1.886, and approximately 1.907; When the single wavelengths of the detection wavelengths are 210 nm and 270 nm, respectively, peak 16 is protocatechuic acid, peak 17 is protocatechuic aldehyde, peak 19 is amygdalin, peak 24 is isoflavone glucoside, peak 25 is hyperoside, peak 28 is rosmarinic acid, peak 30 is salvianolic acid B, peak 33 is ascorbic acid A, peak 34 is ascorbic acid B, peak 35 is ascorbic acid C, peak 36 is icariin, and peak 38 is ginsenoside Rb1.

2. The construction method according to claim 1, characterized in that, The intermediate of the Shuangyi Qushi Huoluo Capsule includes a traditional Chinese medicine extract, a first medicinal material powder, and a second medicinal material powder.

3. The construction method according to claim 2, characterized in that, The first medicinal powder is ginseng fine powder.

4. The construction method according to claim 2, characterized in that, The second medicinal powder is fine powder of Clematis chinensis.

5. The construction method according to claim 1, characterized in that, The container is a stoppered conical flask.

6. The construction method according to claim 1, characterized in that, In the preparation of the traditional Chinese medicine compound test solution, the volume percentage concentration of methanol is 40%~60%.

7. The construction method according to claim 6, characterized in that, The volume percentage concentration of methanol is approximately 50%.

8. The construction method according to claim 1, characterized in that, In the preparation of the traditional Chinese medicine compound test solution, the mass / volume ratio of the Shuangyi Qushi Huoluo capsule intermediate to the methanol is 0.01~0.05, with units of g / ml.

9. The construction method according to claim 8, characterized in that, The mass / volume ratio of the intermediate of the Shuangyi Qushi Huoluo Capsule to the methanol is approximately 0.02, in g / ml.

10. The construction method according to claim 1, characterized in that, The extraction is performed by ultrasonic extraction or reflux extraction.

11. The construction method according to claim 10, characterized in that, The extraction method is ultrasonic extraction.

12. The construction method according to claim 11, characterized in that, The frequency of the ultrasonic extraction is 20~60 kHz.

13. The construction method according to claim 12, characterized in that, The frequency of the ultrasonic extraction is approximately 40 kHz.

14. The construction method according to claim 11, characterized in that, The power of the ultrasonic extraction is 100~300 W.

15. The construction method according to claim 14, characterized in that, The power of the ultrasonic extraction is approximately 200 W.

16. The construction method according to claim 11, characterized in that, The ultrasonic extraction time is 25-35 minutes.

17. The construction method according to claim 16, characterized in that, The ultrasonic extraction time is approximately 30 minutes.

18. The construction method according to claim 1, characterized in that, The centrifuge speed is 3000~5000 r / min.

19. The construction method according to claim 18, characterized in that, The centrifuge speed is approximately 4000 r / min.

20. The construction method according to claim 1, characterized in that, The centrifugation time is 5-15 minutes.

21. The construction method according to claim 20, characterized in that, The centrifugation time is approximately 10 minutes.

22. The construction method according to claim 1, characterized in that, The centrifugation was performed at room temperature.

23. The construction method according to claim 1, characterized in that, The filtration is microporous membrane filtration.

24. The construction method according to claim 23, characterized in that, The microporous filter membrane has a pore size of 0.22 μm.

25. The construction method according to claim 1, characterized in that, Preparation of the reference solution: Weigh appropriate amounts of protocatechuic acid, protocatechuic aldehyde, amygdalin, verrucoside glucoside, hyperoside, rosmarinic acid, salvianolic acid B, cuscuta A, cuscuta B, cuscuta C, icariin, and ginsenoside Rb1 reference standards, and place them separately in a first volumetric flask. Add methanol to dissolve and dilute to the mark, shake well, and use as the stock solution of the above reference standards. Accurately pipette the above stock solutions and place them in a second volumetric flask. Add methanol to dilute to the mark, shake well, and prepare the reference solution with a concentration of 10~50 μg / ml for each of the following: protocatechuic acid, protocatechuic aldehyde, amygdalin, verrucoside glucoside, hyperoside, rosmarinic acid, salvianolic acid B, cuscuta A, cuscuta B, cuscuta C, icariin, and ginsenoside Rb1.

26. The construction method according to claim 25, characterized in that, The first volumetric flask is a 5 mL volumetric flask.

27. The construction method according to claim 25, characterized in that, The second volumetric flask is a 10 mL volumetric flask.

28. The construction method according to claim 25, characterized in that, The volume of the stock solution is 0.1~1 mL.

29. The construction method according to claim 28, characterized in that, The volume of the stock solution is approximately 0.5 mL.

30. The construction method according to claim 25, characterized in that, The concentration of protocatechuic acid in the reference solution was approximately 18.1 μg / ml.

31. The construction method according to claim 25, characterized in that, The concentration of protocatechuic aldehyde in the reference solution was approximately 18.5 μg / ml.

32. The construction method according to claim 25, characterized in that, The concentration of amygdalin in the reference solution was approximately 17.1 μg / ml.

33. The construction method according to claim 25, characterized in that, The concentration of verbascoside in the reference solution was approximately 23.0 μg / ml.

34. The construction method according to claim 25, characterized in that, The concentration of hyperoside in the reference solution was approximately 16.3 μg / ml.

35. The construction method according to claim 25, characterized in that, The concentration of rosmarinic acid in the reference solution was approximately 18.2 μg / ml.

36. The construction method according to claim 25, characterized in that, The concentration of salvianolic acid B in the reference solution was approximately 20.4 μg / ml.

37. The construction method according to claim 25, characterized in that, The concentration of astragaloside A in the reference solution was approximately 19.1 μg / ml.

38. The construction method according to claim 25, characterized in that, The concentration of astragaloside B in the reference solution was approximately 20.2 μg / ml.

39. The construction method according to claim 25, characterized in that, The concentration of astragaloside C in the reference solution was approximately 32.3 μg / ml.

40. The construction method according to claim 25, characterized in that, The concentration of icariin in the reference solution was approximately 19.4 μg / ml.

41. The construction method according to claim 25, characterized in that, The concentration of ginsenoside Rb1 in the reference solution was approximately 22.1 μg / ml.

42. The construction method according to claim 1, characterized in that, The flow rate is 0.6~1.0 ml / min.

43. The construction method according to claim 42, characterized in that, The flow rate is approximately 0.8 ml / min.

44. The construction method according to claim 1, characterized in that, The column temperature is 28~32℃.

45. The construction method according to claim 44, characterized in that, The column temperature is approximately 30°C.

46. ​​The construction method according to claim 1, characterized in that, The injection volume is 5~15 μl.

47. The construction method according to claim 46, characterized in that, The injection volume is approximately 10 μl.

48. The construction method according to claim 1, characterized in that, The chromatographic column used was a Waters XSelect HSST3 column.

49. The construction method according to claim 48, characterized in that, The specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

50. The construction method according to claim 1, characterized in that, When the single wavelengths of the detection wavelengths are 210 nm and 270 nm, respectively, the fingerprint spectrum includes peaks 1-38, with peak 22 serving as a reference peak. The average relative peak areas of peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38 are approximately 5.454, 19.242, and 15.5, respectively. 69, about 1.820, about 4.310, about 1.265, about 6.609, about 3.713, about 3.074, about 2.877, about 1.764, about 2.257, about 1.554, about 1.064, about 1.187, about 1.250, about 3.034, about 1.121, about 1.223, about 0.565, about 1.069, about 1.642, about 0.812, about 0.961, about 0.403, about 0.641, about 1.123, about 0.483, about 1.436, about 0.615, about 0.449, about 3.748, about 0.370, about 0.686, about 0.851, about 0.703 and about 0.

495.

51. A quality control method for a Shuangyi Qushi Huoluo capsule, characterized in that, The quality control method includes the following steps: (1) Establish a standard fingerprint spectrum of the reference sample of Shuangyi Qushi Huoluo Capsules according to the fingerprint spectrum construction method according to any one of claims 1 to 50; (2) Take the test solution of the traditional Chinese medicine compound and detect it under the chromatographic conditions in the fingerprint spectrum construction method according to any one of claims 1 to 50 to obtain the fingerprint spectrum of the traditional Chinese medicine compound sample to be tested; as well as (3) Compare the fingerprint spectrum of the Chinese herbal compound sample to be tested obtained in step (2) with the standard fingerprint spectrum of the reference sample of the Shuangyi Qushi Huoluo Capsule obtained in step (1), and evaluate it in combination with the system fingerprint quantitative method and / or chemical pattern recognition analysis. If it meets the requirements, it is a qualified product; if it does not meet the requirements, it is an unqualified product.

52. The quality control method according to claim 51, characterized in that, The chemical pattern recognition analysis includes cluster analysis and principal component analysis.

53. The quality control method according to claim 51, characterized in that, The evaluation metrics for the system fingerprint quantification method include macro-qualitative similarity S. m Macro-quantitative similarity P m The relative deviation α between % and the sample fingerprint signal homogenization coefficient.

54. The quality control method according to claim 51, characterized in that, The requirements include: when using the system fingerprint quantitative method for evaluation, the evaluation indicators of the system fingerprint quantitative method must simultaneously meet the following three conditions: (1) Macro-qualitative similarity S m Not less than 0.70; (2) Macro-quantitative similarity P m % ranges from 70% to 130%; and (3) The relative deviation α of the sample fingerprint signal homogenization coefficient is not higher than 0.

30.

55. Use of the construction method according to any one of claims 1 to 50 or the quality control method according to any one of claims 51 to 54 in the quality testing and / or quality evaluation and / or quality control of Shuangyi Qushi Huoluo Capsules.

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