A method for constructing HPLC-ELSD fingerprint of a traditional Chinese medicine compound containing epimedium and its application

Abstract

The invention discloses a method for constructing an HPLC-ELSD fingerprint spectrum of a traditional Chinese medicine compound containing epimedium. The method comprises the following steps: obtaining an HPLC-ELSD fingerprint spectrum of the traditional Chinese medicine compound according to the results of high-performance liquid phase detection of a test solution and a reference solution; the chromatographic conditions of the high-performance liquid phase detection are: a chromatographic column whose filler is octadecyl bonded silica gel, a mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, a mobile phase B is water, acid water, an alkaline aqueous solution and / or a buffered saline solution, and a gradient elution program is: 0min, 10%A; 60min, 45%A; a flow rate of 0.5-1.5mL / min, a column temperature of 25-45°C, an injection volume of 10-30μl, a drift tube temperature of 100-130°C, and a carrier gas flow rate of 2-4L / min. The liquid chromatography-evaporative light detection (HPLC-ELSD) characteristic spectrum established by the present invention has a total of 22 characteristic peaks, 9 of which are identified by reference substances, and the remaining 13 characteristic peaks are attributed by medicinal materials. The method can be used for quality control of traditional Chinese medicine compound containing epimedium.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for constructing an HPLC-ELSD fingerprint spectrum of a traditional Chinese medicine compound containing epimedium and an application thereof. Background Art

[0002] Evaporative light scattering detector (ELSD) is an efficient liquid and solid separation technology. It is widely used in analytical techniques such as high performance liquid chromatography (HPLC), supercritical fluid chromatography (SCF), capillary electrophoresis (MCE) and thin layer chromatography (TLC). ELSD is a general detector that can detect most compounds that do not have UV absorption properties, such as sugars, fatty acids, amino acids, drugs, natural products and polymers. The basic idea of ​​ELSD principle is to convert the sample into fine particles by separating it, dissolving it in the mobile phase, and spraying and evaporating the sample through a high-speed airflow. These particles will be scattered in a special scatterer. Unlike traditional HPLC detectors, in ELSD, the components of the sample after separation are not detected directly, but converted into scattered particles. Since different components in the sample have different optical properties, they will produce different scattering angles and intensities in the scatterer. The ELSD detector detects the components by converting the scattering angles and intensities of these particles into electrical signals.

[0003] In summary, ELSD is a very useful analytical technique that is often used in separation techniques such as HPLC. It can detect compounds that do not have UV absorbing properties and has high sensitivity, linear response, and throughput stability.

[0004] There is no report on the method for constructing the HPLC-ELSD fingerprint of a traditional Chinese medicine compound containing Epimedium in the prior art. Summary of the invention

[0005] Based on this, the present invention provides a method for constructing an HPLC-ELSD fingerprint of a traditional Chinese medicine compound containing Epimedium, and the construction method comprises the following steps:

[0006] Preparation of the test solution: weigh an appropriate amount of the Chinese medicine compound preparation, place it in a first container of appropriate volume, add a first solvent and then perform ultrasonic extraction, take it out, cool it, weigh it, shake it well, filter it, and obtain a first filtrate; transfer the first filtrate to a second container, extract it with a second solvent, combine the extract layers, evaporate to dryness, add a third solvent to dissolve it and make it constant to the third container, shake it well, filter it, and obtain a second filtrate; transfer the second filtrate to obtain the test solution; wherein the Chinese medicine compound includes epimedium, radix dipsaci, rhizoma anemarrhenae, rhizoma rehmanniae, rhizoma sparganii, rhizoma curcumae, rhizoma ligustici, rhizoma rubrae and concha oyster;

[0007] Preparation of reference solution: weigh appropriate amounts of magnolidine, paeoniflorin, paeoniflorin, epimedin A, epimedin B, epimedin C, icariin, timosaponin BⅡ and dipsaponin VI reference substances, add methanol solution to prepare the reference solution with the concentration of magnolidine, paeoniflorin, paeoniflorin, epimedin A, epimedin B, epimedin C, icariin, timosaponin BⅡ and dipsaponin VI being 400-600 μg / ml respectively;

[0008] According to the results of high performance liquid chromatography detection of the test solution and the reference solution, a HPLC-ELSD fingerprint spectrum of the Chinese medicine compound is obtained;

[0009] The chromatographic conditions for high performance liquid phase detection are as follows: a chromatographic column with octadecyl bonded silica gel as filler is used, mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, mobile phase B is water, acid water, alkaline aqueous solution and / or buffered saline solution, and the gradient elution program is: 0 min, 10% A; 60 min, 45% A; flow rate is 0.5-1.5 mL / min, column temperature is 25-45°C, injection volume is 10-30 μl, drift tube temperature is 100-130°C, and carrier gas flow rate is 2-4 L / min.

[0010] Furthermore, in the preparation of the test solution, the first solvent is water, such as purified water or distilled water.

[0011] Furthermore, the second solvent is an alcohol, such as water-saturated n-butanol.

[0012] Furthermore, the third solvent is alcohol, such as methanol.

[0013] Furthermore, the first container is a conical flask.

[0014] Furthermore, the volume of the first container is 100 ml.

[0015] Furthermore, the second container is a funnel, such as a separatory funnel.

[0016] Furthermore, the third container is a measuring bottle.

[0017] Furthermore, the volume of the third container is 5 ml.

[0018] Furthermore, the mass / volume (g / ml) ratio between the Chinese medicine compound preparation and the first solvent is 0.01-0.5, for example, about 0.08.

[0019] Furthermore, the ultrasonic treatment time is 10 to 60 minutes, for example, about 30 minutes.

[0020] Furthermore, the volume of the first filtrate is 10-40 ml, for example 25 ml.

[0021] Furthermore, the volume / volume (ml / ml) ratio between the first filtrate and the first solvent is 0.1-1.0, such as about 0.5.

[0022] Furthermore, the extraction is performed 2 to 5 times, for example 3 times.

[0023] Furthermore, the volume of the second solvent is 20-60 ml / time, for example 40 ml / time.

[0024] Furthermore, the volume / volume (ml / ml) ratio between the second solvent and the first solvent is 0.5-1.5, for example, about 0.8.

[0025] Furthermore, the extract layer is an n-butanol layer.

[0026] Furthermore, the volume of the fixed volume is 5 ml.

[0027] Furthermore, the filtration is microporous membrane filtration.

[0028] Furthermore, the pore size of the microporous filter membrane is 0.45 μm.

[0029] Furthermore, the concentration of magnolamine in the reference solution is 450-550 μg / ml.

[0030] Furthermore, the concentration of albiflorin in the reference solution is 450-550 μg / ml.

[0031] Furthermore, the concentration of paeoniflorin in the reference solution is 450-550 μg / ml.

[0032] Furthermore, the concentration of epimedin A in the reference solution is 450-550 μg / ml.

[0033] Furthermore, the concentration of epimedin B in the reference solution is 450-550 μg / ml.

[0034] Furthermore, the concentration of epimedin C in the reference solution is 450-550 μg / ml.

[0035] Furthermore, the concentration of icariin in the reference solution is 450-550 μg / ml.

[0036] Furthermore, the concentration of Anemarrhena saponin BⅡ in the reference solution is 450-550 μg / ml.

[0037] Furthermore, the concentration of Dipsacus asper saponin VI in the reference solution is 450-550 μg / ml.

[0038] Furthermore, the reference substance solution is a mixed solution of about 500 μg / ml of magnolidine, about 500 μg / ml of paeoniflorin, about 500 μg / ml of paeoniflorin, about 500 μg / ml of epimedin A, about 500 μg / ml of epimedin B, about 500 μg / ml of epimedin C, about 500 μg / ml of icariin, about 500 μg / ml of anemarrhena saponin BⅡ, and about 500 μg / ml of dipsaponin VI.

[0039] Furthermore, the flow rate is 0.8-1.2 ml / min, for example, about 1.0 ml / min.

[0040] Furthermore, the column temperature is 30-40°C, for example, about 35°C.

[0041] Furthermore, the injection volume is 15-25 μl, for example, about 20 μl.

[0042] Furthermore, the temperature of the drift tube is 105-125°C, for example, about 115°C.

[0043] Furthermore, the carrier gas flow rate is 2.5 to 3.5 L / min, for example, about 3.1 L / min.

[0044] Furthermore, the theoretical plate number of the chromatographic peak corresponding to the reference substance is 6000.

[0045] Furthermore, the separation degree of the chromatographic peaks corresponding to the reference substance is greater than 1.5.

[0046] Furthermore, the chromatographic column is an Ultimate C18 chromatographic column, a YMC C18 chromatographic column, a Caprisil C18 AQ chromatographic column or a Spursil C18 chromatographic column.

[0047] Furthermore, the chromatographic column is a Spursil C18 chromatographic column.

[0048] Furthermore, the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, particle size 5 μm.

[0049] Furthermore, the mobile phase A is acetonitrile.

[0050] Furthermore, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from one or more of weak acids and their salts, weak bases and their salts of different concentrations.

[0051] Further, the acid aqueous solution, alkali aqueous solution and / or buffered saline 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 in different concentrations.

[0052] Furthermore, the acid aqueous solution is a 0.08% to 0.12% acid aqueous solution.

[0053] Furthermore, the acid aqueous solution is a 0.08% to 0.12% phosphoric acid aqueous solution.

[0054] Furthermore, the acid aqueous solution is about 0.1% phosphoric acid aqueous solution.

[0055] Furthermore, the buffered saline solution is a phosphate aqueous solution and / or an acetate aqueous solution.

[0056] Furthermore, the pH value of the buffered saline solution is not greater than 7.0.

[0057] Furthermore, the mobile phase B is water.

[0058] Furthermore, the preparation method of the Chinese medicine compound preparation comprises: (1) weighing an appropriate amount of oysters and crushing them into oyster fine powder; and (2) weighing an appropriate amount of epimedium, radix dipsaci, rhizoma anemarrhenae, rhizoma rehmanniae, rhizoma sparganii, rhizoma curcumae, rhizoma ligustici and rhizoma peonyii, decocting them with water, filtering, concentrating the filtrate into a clear paste, adding the oyster fine powder, mixing, drying, crushing into fine powder, preparing the preparation, and drying.

[0059] Furthermore, in step (2), appropriate amounts of starch and sucrose are added simultaneously with the oyster powder.

[0060] Furthermore, the mass ratio between the epimedium, the dipsoma, the rhizoma anemarrhenae, the rehmannia root, the trifoliate green, the zedoaria, the rhizome of ligusticum, the rhizome of chuanxiong, the red peony root and the oyster is (500-1000):(20-100):(20-100):(20-100):(20-100):(20-100):(20-100):(20-100):(5-20).

[0061] Further, the mass ratio of the epimedium, the Dipsacus asper, the Anemarrhena asphodeloides, the Rehmannia glutinosa, the Trillium, the Curcuma zedoaria, the Ligusticum chuanxiong, the Paeonia lactiflora and the Oyster is about 650: about 50: about 50: about 40: about 50: about 50: about 50: about 50: about 50: about 10.

[0062] Furthermore, the oyster is a calcined oyster.

[0063] Furthermore, the decocting is performed 1 to 5 times, for example 3 times.

[0064] Furthermore, the decocting time is 0.5 to 3 hours, for example 1 to 2 hours.

[0065] Furthermore, the relative density of the clear paste at 50° C. is 1.2 to 1.40, for example, about 1.35.

[0066] Furthermore, the dosage form of the Chinese medicine compound preparation is granules or capsules.

[0067] Furthermore, the particles are sugar-containing particles or sugar-free particles.

[0068] Furthermore, the fingerprint spectrum includes peaks 1-22, among which peak 3 is magnolidine, peak 5 is paeoniflorin, peak 6 is paeoniflorin, peak 12 is anemarrhena saponin BII, peak 13 is epimedin A, peak 14 is epimedin B, peak 15 is epimedin C, peak 17 is icariin, and peak 18 is dipsaponin VI.

[0069] Furthermore, the fingerprint spectrum includes peaks 1-22, among which peaks 3, 13, 14, 15, 17 and 21 belong to the medicinal flavor of Epimedium; peaks 5 and 6 belong to the medicinal flavor of Red Peony Root; peaks 12 and 19 belong to the medicinal flavor of Anemarrhena Rhizoma; and peaks 1 and 18 belong to the medicinal flavor of Dipsacus Asper.

[0070] Furthermore, the fingerprint spectrum includes 22 characteristic peaks, with the chromatographic peak of paeoniflorin No. 6 as the reference peak, and the relative retention times of the other 21 characteristic peaks are 0.628±10% for chromatographic peak No. 1, 0.748±10% for chromatographic peak No. 2, 0.801±10% for chromatographic peak No. 3, 0.822±10% for chromatographic peak No. 4, 0.894±10% for chromatographic peak No. 5, 1.247±10% for chromatographic peak No. 7, 1.489±10% for chromatographic peak No. 8, 1.727±10% for chromatographic peak No. 9, 1.815±10% for chromatographic peak No. 10, and 11 Chromatographic peak No. 1 is 1.848±10%, chromatographic peak No. 12 is 1.993±10%, chromatographic peak No. 13 is 2.186±10%, chromatographic peak No. 14 is 2.241±10%, chromatographic peak No. 15 is 2.279±10%, chromatographic peak No. 16 is 2.316±10%, chromatographic peak No. 17 is 2.357±10%, chromatographic peak No. 18 is 2.585±10%, chromatographic peak No. 19 is 2.614±10%, chromatographic peak No. 20 is 2.773±10%, chromatographic peak No. 21 is 2.919±10%, and chromatographic peak No. 22 is 3.438±10%.

[0071] Furthermore, the fingerprint spectrum includes 22 characteristic peaks, with the chromatographic peak of paeoniflorin No. 6 as the reference peak, and the relative peak areas of the other 21 characteristic peaks are 0.106±10% for chromatographic peak No. 1, 0.047±10% for chromatographic peak No. 2, 0.153±10% for chromatographic peak No. 3, 0.042±10% for chromatographic peak No. 4, 1.137±10% for chromatographic peak No. 5, 0.065±10% for chromatographic peak No. 7, 0.142±10% for chromatographic peak No. 8, 0.991±10% for chromatographic peak No. 9, 0.170±10% for chromatographic peak No. 10, and 0.117±10% for chromatographic peak No. 11. Chromatographic peak No. 1 is 0.147±10%, chromatographic peak No. 12 is 0.326±10%, chromatographic peak No. 13 is 0.107±10%, chromatographic peak No. 14 is 0.121±10%, chromatographic peak No. 15 is 2.494±10%, chromatographic peak No. 16 is 0.557±10%, chromatographic peak No. 17 is 0.652±10%, chromatographic peak No. 18 is 0.654±10%, chromatographic peak No. 19 is 0.182±10%, chromatographic peak No. 20 is 0.285±10%, chromatographic peak No. 21 is 0.062±10%, and chromatographic peak No. 22 is 0.306±10%.

[0072] According to another aspect of the present invention, a quality control method for a Chinese medicinal compound containing Epimedium is provided, the quality control method comprising the following steps:

[0073] (1) Establishing a standard HPLC-ELSD fingerprint of a Chinese medicine compound reference sample according to the above-mentioned HPLC-ELSD fingerprint construction method;

[0074] (2) taking a solution of a Chinese medicine compound test sample, and performing detection according to the chromatographic conditions in the above-mentioned HPLC-ELSD fingerprint spectrum construction method to obtain an HPLC-ELSD fingerprint spectrum of the Chinese medicine compound test sample; and

[0075] (3) Compare the HPLC-ELSD fingerprint of the Chinese medicine compound sample obtained in step (2) with the standard HPLC-ELSD fingerprint of the Chinese medicine compound reference sample obtained in step (1). If it meets the requirements, it is a qualified product; if it does not meet the requirements, it is an unqualified product.

[0076] Furthermore, the compliance requirement includes one or more of the following:

[0077] (1) The HPLC-ELSD fingerprint of the Chinese medicine compound sample to be tested exhibits 22 characteristic chromatographic peaks, and the retention time of each characteristic chromatographic peak is within ±10% of the retention time value of the corresponding reference chromatographic peak in the standard HPLC-ELSD fingerprint of the Chinese medicine compound reference sample;

[0078] (2) With the paeoniflorin peak as the S peak, the relative retention time of each characteristic chromatographic peak in the HPLC-ELSD fingerprint of the Chinese medicine compound sample to be tested and the S peak is within ±10% of the relative retention time value of each characteristic chromatographic peak in the standard HPLC-ELSD fingerprint of the Chinese medicine compound reference sample; and

[0079] (3) According to the Chinese medicine chromatographic HPLC-ELSD fingerprint similarity evaluation system, the similarity between the HPLC-ELSD fingerprint of the Chinese medicine compound sample to be tested and the standard HPLC-ELSD fingerprint of the Chinese medicine compound reference sample shall be calculated, and the similarity shall not be less than 0.90.

[0080] According to another aspect of the present invention, there is provided a use of the above-mentioned construction method or the above-mentioned quality control method in quality detection, quality evaluation or quality control of a traditional Chinese medicine compound containing Epimedium.

[0081] Beneficial effects of the present invention:

[0082] The liquid chromatography-evaporative light detection (HPLC-ELSD) characteristic spectrum established by the present invention has a total of 22 characteristic peaks, 9 characteristic peaks of which are identified by using a reference substance, and the remaining 13 characteristic peaks are attributed by using medicinal materials. The method can be used for quality control of a traditional Chinese medicine compound containing epimedium. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0084] Figure 1 This is the HPLC-ELSD chromatogram of the traditional Chinese medicine compound containing Epimedium under chromatographic condition 1.

[0085] Figure 2 This is the HPLC-ELSD chromatogram of the traditional Chinese medicine compound containing Epimedium under chromatographic condition 2.

[0086] Figure 3 This is the HPLC-ELSD chromatogram of the traditional Chinese medicine compound containing Epimedium under chromatographic condition 3.

[0087] Figure 4 This is the HPLC-ELSD chromatogram of the traditional Chinese medicine compound containing Epimedium under chromatographic condition 4.

[0088] Figure 5 This is the HPLC-ELSD chromatogram of the traditional Chinese medicine compound containing Epimedium under chromatographic condition 5.

[0089] Figure 6 This is the HPLC-ELSD chromatogram of the traditional Chinese medicine compound containing Epimedium under chromatographic condition 6.

[0090] Figure 7 This is the HPLC-ELSD chromatogram of the traditional Chinese medicine compound containing Epimedium under chromatographic condition 7.

[0091] Figure 8 The HPLC spectrum of the Chinese herbal compound containing Epimedium under chromatographic condition 7. Among them, from top to bottom are the HPLC spectrum of the Chinese herbal compound granules of batches GSBWT005, GSBWT004, GSBWT003, GSBWT002, and GSBWT001.

[0092] Fig. 9 This is a statistical chart of the number of chromatographic peaks in chromatographic columns with different fillers.

[0093] Fig.10 The HPLC chromatograms of the Chinese herbal compound granules containing Epimedium using different filler columns. From top to bottom, they are the HPLC spectra of the Chinese herbal compound granules corresponding to four different filler columns: Ultimate C18, YMC C18, Caprisil C18AQ and Spursil C18.

[0094] Fig.11 This is a statistical chart of the number of chromatographic peaks for different extraction solvents.

[0095] Fig.12 The peak area statistics of the chromatographic peaks of the Chinese herbal compound granule samples containing Epimedium with different extraction solvents.

[0096] Fig.13 The HPLC chromatograms of the Chinese herbal compound granules samples containing Epimedium with different extraction solvents are shown in Figure 1. From top to bottom, they are the HPLC spectra of the Chinese herbal compound granules corresponding to water, 80% methanol, ethanol, 90% methanol, 70% methanol, and 50% methanol as the extraction solvents.

[0097] Fig.14 This is a statistical chart of the number of chromatographic peaks of Chinese herbal compound granules containing Epimedium with different preparation methods (unextracted and extracted).

[0098] Fig.15 The HPLC chromatograms of different preparation methods (extraction and non-extraction) of Chinese herbal compound granules containing Epimedium. Among them, the first two from top to bottom are the HPLC spectra of the Chinese herbal compound granules corresponding to the extraction of the sample with water and then with n-butanol solution, and the last two are the HPLC spectra of the Chinese herbal compound granules corresponding to the extraction of the sample with water and without extraction.

[0099] Fig.16This is a statistical chart of the number of chromatographic peaks of Chinese herbal compound granules containing Epimedium with different preparation methods (ammonia washing and no ammonia washing).

[0100] Fig.17 The HPLC chromatograms of different preparation methods (ammonia washing and non-ammonia washing) of Chinese herbal compound granules containing Epimedium. Among them, the first two from top to bottom are the HPLC spectra of the Chinese herbal compound granules corresponding to the water extraction and ammonia washing of the samples, and the last two are the HPLC spectra of the Chinese herbal compound granules corresponding to the water extraction and non-ammonia washing of the samples.

[0101] Fig.18 HPLC chromatograms of the samples of Chinese herbal compound granules containing epimedium and the reference substance. A is the HPLC spectrum of the sample of the Chinese herbal compound sugar-free granules containing epimedium, which from top to bottom are the HPLC spectrum of the batches of Chinese herbal compound granules GSBWT005, GSBWT004, GSBWT003, GSBWT002, and GSBWT001. B is the HPLC spectrum of the sample of the Chinese herbal compound sugar-containing granules containing epimedium, which from top to bottom are the HPLC spectrum of the batches of Chinese herbal compound granules GSBYT005, GSBYT004, GSBYT003, GSBYT002, and GSBYT001.

[0102] Fig.19 HPLC chromatograms of the Chinese herbal compound granule sample containing Epimedium and the reference substance.

[0103] Among them, top-bottom: Chinese herbal compound granules containing epimedium-mixed standard-mangiferin-anemasaponin BII. Among them, 3. magnolamine, 5. paeoniflorin, 6. paeoniflorin (S), 12. anemarrhenasaponin BII, 13. epimedin A, 14. epimedin B, 15. epimedin C, 17. icariin, 18. chuanxiong saponin VI.

[0104] Fig. 20 HPLC chromatograms of Chinese herbal compound granules containing epimedium and control medicinal materials. Among them, A is the HPLC spectrum of the sample and Trillium and Anemarrhena (upper-lower: Chinese herbal compound granules containing epimedium-Anemarrhena-Trillium), B is the HPLC spectrum of the sample and Chuanxiong, Curcuma, and Radix Rehmanniae (upper-lower: Chinese herbal compound granules containing epimedium-Chuanxiong-Curcuma-Radix Rehmanniae), C is the HPLC spectrum of the sample and Dipsacus, epimedium, and red peony (upper-lower: Chinese herbal compound granules containing epimedium-Dipsacus-Epimedium-Red peony). DETAILED DESCRIPTION

[0105] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0106] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0107] The present invention is intended to encompass all alternatives, variations and equivalents that may be included in the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0108] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0109] In the present invention, the term "comprising" is synonymous with "including". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0110] As described in the background technology section, there is no report on the construction method of HPLC-ELSD fingerprint spectrum of a Chinese herbal compound containing Epimedium in the prior art. In order to solve the above problem, the present invention provides a method for constructing HPLC-ELSD fingerprint spectrum of a Chinese herbal compound containing Epimedium, and the construction method comprises the following steps:

[0111] Preparation of the test solution: weigh an appropriate amount of the Chinese medicine compound preparation, place it in a first container of appropriate volume, add a first solvent and then perform ultrasonic extraction, take it out, cool it, weigh it, shake it well, filter it, and obtain a first filtrate; transfer the first filtrate to a second container, extract it with a second solvent, combine the extract layers, evaporate to dryness, add a third solvent to dissolve it and make it constant to the third container, shake it well, filter it, and obtain a second filtrate; transfer the second filtrate to obtain the test solution; wherein the Chinese medicine compound includes epimedium, radix dipsaci, rhizoma anemarrhenae, rhizoma rehmanniae, rhizoma sparganii, rhizoma curcumae, rhizoma ligustici, rhizoma rubrae and concha oyster;

[0112] Preparation of reference solution: weigh appropriate amounts of magnolidine, paeoniflorin, paeoniflorin, epimedin A, epimedin B, epimedin C, icariin, timosaponin BⅡ and dipsaponin VI reference substances, add methanol solution to prepare the reference solution with the concentration of magnolidine, paeoniflorin, paeoniflorin, epimedin A, epimedin B, epimedin C, icariin, timosaponin BⅡ and dipsaponin VI being 400-600 μg / ml respectively;

[0113] According to the results of high performance liquid chromatography detection of the test solution and the reference solution, a HPLC-ELSD fingerprint spectrum of the Chinese medicine compound is obtained;

[0114] The chromatographic conditions for high performance liquid phase detection are as follows: a chromatographic column with octadecyl bonded silica gel as filler is used, mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, mobile phase B is water, acid water, alkaline aqueous solution and / or buffered saline solution, and the gradient elution program is: 0 min, 10% A; 60 min, 45% A; flow rate is 0.5-1.5 mL / min, column temperature is 25-45°C, injection volume is 10-30 μl, drift tube temperature is 100-130°C, and carrier gas flow rate is 2-4 L / min.

[0115] In the present invention, when concentration, flow rate, column temperature, injection volume, temperature, ratio, time, volume, number of times, or other values ​​or parameters are expressed as ranges, preferred ranges, or a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when the range "10-30" is disclosed, the described range should be interpreted as including the range "10-30", "10-25", "10-20", "10-15", "15-30", "15-25", "15-20", "20-30", "20-25", "25-30", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range.

[0116] In a preferred embodiment, in the preparation of the test solution, the first solvent is water, such as purified water or distilled water.

[0117] In a preferred embodiment, the second solvent is an alcohol, such as water-saturated n-butanol.

[0118] In a preferred embodiment, the third solvent is an alcohol, such as methanol.

[0119] In a preferred embodiment, the first container is a conical flask.

[0120] In a preferred embodiment, the volume of the first container is 100 ml.

[0121] In a preferred embodiment, the second container is a funnel, such as a separatory funnel.

[0122] In a preferred embodiment, the third container is a measuring bottle.

[0123] In a preferred embodiment, the volume of the third container is 5 ml.

[0124] In a preferred embodiment, the mass / volume (g / ml) ratio between the Chinese medicine compound preparation and the first solvent is 0.01-0.5, such as about 0.08.

[0125] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.08" includes ±5% of 0.08, or from 0.076 to 0.084.

[0126] In a preferred embodiment, the ultrasonic treatment time is 10 to 60 minutes, such as about 30 minutes.

[0127] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes ±5% of 30, or from 28.5 to 31.5.

[0128] In a preferred embodiment, the volume of the first filtrate is 10-40 ml, for example 25 ml.

[0129] In a preferred embodiment, the volume / volume (ml / ml) ratio between the first filtrate and the first solvent is 0.1-1.0, such as about 0.5.

[0130] In the present 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.

[0131] In a preferred embodiment, the extraction is performed 2 to 5 times, for example 3 times.

[0132] In a preferred embodiment, the volume of the second solvent is 20-60 ml / time, for example 40 ml / time.

[0133] In a preferred embodiment, the volume / volume (ml / ml) ratio between the second solvent and the first solvent is 0.5-1.5, such as about 0.8.

[0134] In the present 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.

[0135] In a preferred embodiment, the extract layer is an n-butanol layer.

[0136] In a preferred embodiment, the fixed volume is 5 ml.

[0137] In a preferred embodiment, the filtration is microporous membrane filtration.

[0138] In a preferred embodiment, the pore size of the microporous filter membrane is 0.45 μm.

[0139] In a preferred embodiment, the concentration of magnolidine in the reference solution is 450-550 μg / ml.

[0140] In a preferred embodiment, the concentration of albiflorin in the reference solution is 450-550 μg / ml.

[0141] In a preferred embodiment, the concentration of paeoniflorin in the reference solution is 450-550 μg / ml.

[0142] In a preferred embodiment, the concentration of epimedin A in the reference solution is 450-550 μg / ml.

[0143] In a preferred embodiment, the concentration of epimedin B in the reference solution is 450-550 μg / ml.

[0144] In a preferred embodiment, the concentration of epimedin C in the reference solution is 450-550 μg / ml.

[0145] In a preferred embodiment, the concentration of icariin in the reference solution is 450-550 μg / ml.

[0146] In a preferred embodiment, the concentration of Anemarrhena saponin BⅡ in the reference solution is 450-550 μg / ml.

[0147] In a preferred embodiment, the concentration of dipsaponin VI in the reference solution is 450-550 μg / ml.

[0148] In a preferred embodiment, the reference substance solution is a mixed solution of about 500 μg / ml of magnolidine, about 500 μg / ml of paeoniflorin, about 500 μg / ml of paeoniflorin, about 500 μg / ml of epimedin A, about 500 μg / ml of epimedin B, about 500 μg / ml of epimedin C, about 500 μg / ml of icariin, about 500 μg / ml of anemarrhena saponin BⅡ, and about 500 μg / ml of dipsaponin VI.

[0149] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 500" includes ±5% of 500, or from 475 to 525.

[0150] In a preferred embodiment, the flow rate is 0.8-1.2 ml / min, such as about 1.0 ml / min.

[0151] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1.0" includes ±5% of 1.0, or from 0.95 to 1.05.

[0152] In a preferred embodiment, the column temperature is 30-40°C, such as about 35°C.

[0153] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 35" includes ±5% of 35, or from 33.25 to 36.75.

[0154] In a preferred embodiment, the injection volume is 15-25 μl, for example, about 20 μl.

[0155] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20" includes ±5% of 20, or from 19 to 21.

[0156] In a preferred embodiment, the drift tube temperature is 105-125°C, such as about 115°C.

[0157] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 115" includes ±5% of 115, or from 109.25 to 120.75.

[0158] In a preferred embodiment, the carrier gas flow rate is 2.5 to 3.5 L / min, for example, about 3.1 L / min.

[0159] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3.1" includes ±5% of 3.1, or from 2.945 to 3.255.

[0160] In a preferred embodiment, the theoretical plate number of the chromatographic peak corresponding to the reference substance is 6000.

[0161] In a preferred embodiment, the separation degree of the chromatographic peaks corresponding to the reference substance is greater than 1.5.

[0162] In a preferred embodiment, the chromatographic column is an Ultimate C18 chromatographic column, a YMC C18 chromatographic column, a Caprisil C18 AQ chromatographic column or a Spursil C18 chromatographic column.

[0163] In a preferred embodiment, the chromatographic column is a Spursil C18 chromatographic column.

[0164] In a preferred embodiment, the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, particle size 5 μm.

[0165] In a preferred embodiment, the mobile phase A is acetonitrile.

[0166] In a preferred embodiment, the acid aqueous solution, base aqueous solution and / or buffered saline solution is selected from one or more of weak acids and their salts, weak bases and their salts of different concentrations.

[0167] In a preferred embodiment, the acid aqueous solution, alkali aqueous solution and / or buffered saline 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 in different concentrations.

[0168] In a preferred embodiment, the acid aqueous solution is a 0.08% to 0.12% acid aqueous solution.

[0169] In a preferred embodiment, the acid aqueous solution is a 0.08% to 0.12% phosphoric acid aqueous solution.

[0170] In a preferred embodiment, the aqueous acid solution is about 0.1% phosphoric acid aqueous solution.

[0171] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.1%" includes ±5% of 0.1%, or from 0.095% to 0.105%.

[0172] In a preferred embodiment, the buffered saline solution is a phosphate aqueous solution and / or an acetate aqueous solution.

[0173] In a preferred embodiment, the pH value of the buffered saline solution is not greater than 7.0.

[0174] In a preferred embodiment, the mobile phase B is water.

[0175] In a preferred embodiment, the preparation method of the Chinese medicine compound preparation comprises: (1) weighing an appropriate amount of oysters and crushing them into oyster fine powder; and (2) weighing an appropriate amount of epimedium, Dipsacus asper, Anemarrhena asphodeloides, Rehmannia root, Trillium, Curcuma zedoaria, Ligusticum chuanxiong and Paeonia lactiflora, decocting with water, filtering, concentrating the filtrate into a clear paste, adding the oyster fine powder, mixing, drying, crushing into fine powder, making a preparation, and drying.

[0176] In a preferred embodiment, in step (2), appropriate amounts of starch and sucrose are added simultaneously with the addition of the oyster powder.

[0177] In a preferred embodiment, the mass ratio of the epimedium, the dipsacus, the rhizoma anemarrhenae, the rehmannia root, the trifoliate green, the zedoaria, the rhizome of ligusticum, the rhizome of chuanxiong, the red peony root and the oyster is (500-1000):(20-100):(20-100):(20-100):(20-100):(20-100):(20-100):(20-100):(5-20).

[0178] In a preferred embodiment, the mass ratio of the Epimedium, the Dipsacus asper, the Anemarrhena asphodeloides, the Rehmannia glutinosa, the Trillium, the Curcuma, the Ligusticum chuanxiong, the Red Peony Root and the Oyster is about 650: about 50: about 50: about 40: about 50: about 50: about 50: about 50: about 50: about 10.

[0179] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 650" includes ±5% of 650, or from 617.5 to 682.5; "about 50" includes ±5% of 50, or from 47.5 to 52.5; "about 40" includes ±5% of 40, or from 38 to 42; "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0180] In a preferred embodiment, the oyster is calcined oyster.

[0181] In a preferred embodiment, the decocting is performed 1 to 5 times, for example 3 times.

[0182] In a preferred embodiment, the decocting time is 0.5 to 3 hours, such as 1 to 2 hours.

[0183] In a preferred embodiment, the relative density of the clear paste at 50° C. is 1.2-1.40, for example, about 1.35.

[0184] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1.35" includes ±5% of 1.35, or from 1.2825 to 1.4175.

[0185] In a preferred embodiment, the dosage form of the Chinese medicine compound preparation is granules or capsules.

[0186] In a preferred embodiment, the particles are sugar-containing particles or sugar-free particles.

[0187] In a preferred embodiment, the particles are Gusongbao particles.

[0188] In a preferred embodiment, the fingerprint spectrum includes peaks 1-22, wherein peak 3 is magnolidine, peak 5 is paeoniflorin, peak 6 is paeoniflorin, peak 12 is anemarrhena saponin BII, peak 13 is epimedin A, peak 14 is epimedin B, peak 15 is epimedin C, peak 17 is icariin, and peak 18 is dipsaponin VI.

[0189] In a preferred embodiment, the fingerprint spectrum includes peaks 1-22, among which peaks 3, 13, 14, 15, 17 and 21 belong to the medicinal flavor of Epimedium; peaks 5 and 6 belong to the medicinal flavor of Red Peony; peaks 12 and 19 belong to the medicinal flavor of Anemarrhena; and peaks 1 and 18 belong to the medicinal flavor of Dipsacus asper.

[0190] In a preferred embodiment, the fingerprint includes 22 characteristic peaks, with the chromatographic peak of paeoniflorin No. 6 as the reference peak, and the relative retention times of the other 21 characteristic peaks are 0.628±10% for chromatographic peak No. 1, 0.748±10% for chromatographic peak No. 2, 0.801±10% for chromatographic peak No. 3, 0.822±10% for chromatographic peak No. 4, 0.894±10% for chromatographic peak No. 5, 1.247±10% for chromatographic peak No. 7, 1.489±10% for chromatographic peak No. 8, 1.727±10% for chromatographic peak No. 9, and 1.815±10% for chromatographic peak No. 10. %, chromatographic peak No. 11 1.848±10%, chromatographic peak No. 12 1.993±10%, chromatographic peak No. 13 2.186±10%, chromatographic peak No. 14 2.241±10%, chromatographic peak No. 15 2.279±10%, chromatographic peak No. 16 2.316±10%, chromatographic peak No. 17 2.357±10%, chromatographic peak No. 18 2.585±10%, chromatographic peak No. 19 2.614±10%, chromatographic peak No. 20 2.773±10%, chromatographic peak No. 21 2.919±10%, chromatographic peak No. 22 3.438±10%.

[0191] In a preferred embodiment, the fingerprint includes 22 characteristic peaks, with the chromatographic peak of paeoniflorin No. 6 as the reference peak, and the relative peak areas of the other 21 characteristic peaks are 0.106±10% for chromatographic peak No. 1, 0.047±10% for chromatographic peak No. 2, 0.153±10% for chromatographic peak No. 3, 0.042±10% for chromatographic peak No. 4, 1.137±10% for chromatographic peak No. 5, 0.065±10% for chromatographic peak No. 7, 0.142±10% for chromatographic peak No. 8, 0.991±10% for chromatographic peak No. 9, and 0.170±10% for chromatographic peak No. 10. , chromatographic peak No. 11 0.147±10%, chromatographic peak No. 12 0.326±10%, chromatographic peak No. 13 0.107±10%, chromatographic peak No. 14 0.121±10%, chromatographic peak No. 15 2.494±10%, chromatographic peak No. 16 0.557±10%, chromatographic peak No. 17 0.652±10%, chromatographic peak No. 18 0.654±10%, chromatographic peak No. 19 0.182±10%, chromatographic peak No. 20 0.285±10%, chromatographic peak No. 21 0.062±10%, chromatographic peak No. 22 0.306±10%.

[0192] According to another aspect of the present invention, a quality control method for a Chinese medicinal compound containing Epimedium is provided, the quality control method comprising the following steps:

[0193] (1) Establishing a standard HPLC-ELSD fingerprint of a Chinese medicine compound reference sample according to the above-mentioned HPLC-ELSD fingerprint construction method;

[0194] (2) taking a solution of a Chinese medicine compound test sample, and performing detection according to the chromatographic conditions in the above-mentioned HPLC-ELSD fingerprint spectrum construction method to obtain an HPLC-ELSD fingerprint spectrum of the Chinese medicine compound test sample; and

[0195] (3) Compare the HPLC-ELSD fingerprint of the Chinese medicine compound sample obtained in step (2) with the standard HPLC-ELSD fingerprint of the Chinese medicine compound reference sample obtained in step (1). If it meets the requirements, it is a qualified product; if it does not meet the requirements, it is an unqualified product.

[0196] In a preferred embodiment, the compliance requirements include one or more of the following:

[0197] (1) The HPLC-ELSD fingerprint of the Chinese medicine compound sample to be tested exhibits 22 characteristic chromatographic peaks, and the retention time of each characteristic chromatographic peak is within ±10% of the retention time value of the corresponding reference chromatographic peak in the standard HPLC-ELSD fingerprint of the Chinese medicine compound reference sample;

[0198] (2) With the paeoniflorin peak as the S peak, the relative retention time of each characteristic chromatographic peak in the HPLC-ELSD fingerprint of the Chinese medicine compound sample to be tested and the S peak is within ±10% of the relative retention time value of each characteristic chromatographic peak in the standard HPLC-ELSD fingerprint of the Chinese medicine compound reference sample; and

[0199] (3) According to the Chinese medicine chromatographic HPLC-ELSD fingerprint similarity evaluation system, the similarity between the HPLC-ELSD fingerprint of the Chinese medicine compound sample to be tested and the standard HPLC-ELSD fingerprint of the Chinese medicine compound reference sample shall be calculated, and the similarity shall not be less than 0.90.

[0200] According to another aspect of the present invention, there is provided a use of the above-mentioned construction method or the above-mentioned quality control method in quality detection, quality evaluation or quality control of a traditional Chinese medicine compound containing Epimedium.

[0201] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0202] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0203] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in this patent specification can be used in combination with any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0204] Example

[0205] 1. Experimental Materials

[0206] 1.1 Instruments

[0207] Shimadzu LC-20AT high performance liquid chromatograph (Shimadzu Corporation, Japan); Alltech ELSD2000ES evaporative light scattering detector (Alltech Corporation, USA), GCK3308 fully automatic air source (China, Beijing Zhonghuipu Analytical Technology Research Institute), XSE105DU electronic analytical balance (Mettler, Switzerland); KQ-250DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.);

[0208] 1.2 Reagents and drugs

[0209] Reference substances: magnolamine (batch number: RFS-M02602101009), mangiferin (batch number: RFS-M01802011021), paeoniflorin (batch number: RFS-S01102204022), epimedin A (batch number: RFS-C01311804027), epimedin B (batch number: RFS-C01211804027), epimedin C (batch number: RFS-C01111803 006), icariin (batch number: RFS-Y00411803005), and anemarrhena saponin BⅡ (batch number: RFS-Z00402207029). The above reference substances were purchased from Chengdu Ruifensidedan Biotechnology Co., Ltd., HPLC grade, and the contents were all>98% calculated by HPLC area normalization method. Paeoniflorin (batch number: 110736-201842) was purchased from China Food and Drug Biological Products Control Institute for content determination.

[0210] There are ten batches of Chinese herbal compound granules containing Epimedium (sugar-free: GSBWT001, GSBWT002, GSBWT003, GSBWT004, GSBWT005; sugar-containing: GSBYT001, GSBYT002, GSBYT003, GSBYT004, GSBYT005; methanol, acetonitrile, distilled water.

[0211] 2. Method Condition Optimization

[0212] 2.1. Investigation of different chromatographic conditions

[0213] Table 1 Chromatographic conditions 1

[0214] Time (min) A％ B％ 0 30 70 50 90 10

[0215] like Figure 1 As shown, under chromatographic condition 1 in Table 1, the chromatographic peaks are concentrated at the front end, and too few chromatographic peaks are obtained, which requires further optimization.

[0216] Table 2 Chromatographic conditions 2

[0217] Time (min) A％ B％ 0 30 70 30 70 30

[0218] like Figure 2 As shown, under chromatographic condition 2 in Table 2, the chromatographic peaks are concentrated at the front end, too few chromatographic peaks are obtained, and the chromatographic peak separation is poor, which needs further optimization.

[0219] Table 3 Chromatographic conditions 3

[0220] Time (min) A％ B％ 0 30 70 30 45 55

[0221] like Figure 3As shown, under chromatographic condition 3 in Table 3, the obtained chromatographic peaks are still too few and concentrated at the front end, which requires further optimization.

[0222] Table 4 Chromatographic conditions 4

[0223] Time (min) A％ B％ 0 10 90 50 45 55

[0224] like Figure 4 As shown, under chromatographic condition 4 in Table 4, the distribution of chromatographic peaks is relatively reasonable, but the separation of some chromatographic peaks is poor and needs further optimization.

[0225] Table 5 Chromatographic conditions 5

[0226] Time (min) A％ B％ 0 10 90 15 22 78 20 32 68 60 40 60

[0227] like Figure 5 As shown, under chromatographic condition 5 in Table 5, the elution gradient changes greatly, and there are fewer chromatographic peaks at the beginning and the end, which needs further optimization.

[0228] Table 6 Chromatographic conditions 6

[0229] Time (min) A％ B％ 0 10 90 15 22 78 16 30 70 35 30 70 60 45 55

[0230] like Figure 6 As shown, under chromatographic condition 6 in Table 6, the chromatographic peaks are still distributed in the middle part, and the separation of the middle chromatographic peaks is poor, which needs further optimization.

[0231] Table 7 Chromatographic conditions 7

[0232] time A％ B％ 0 10 90 60 45 55

[0233] like Figure 7 As shown in Table 7, under chromatographic condition 7, the chromatographic peaks are reasonably distributed and the separation of each chromatographic peak is good, so chromatographic condition 7 is adopted. The HPLC spectrum of the Chinese herbal compound granules containing epimedium under chromatographic condition 7 is as follows Figure 8 shown.

[0234] 2.2. Investigation of different chromatographic columns

[0235] This study compared the separation of four different filler columns, Ultimate C18, YMC C18, Caprisil C18 AQ and Spursil C18. Considering the chromatograms obtained by the four columns, Ultimate C18 obtained 23 chromatographic peaks, YMC C18 obtained 20 chromatographic peaks, Caprisil C18 AQ obtained 24 chromatographic peaks, and Spursil C18 obtained 26 chromatographic peaks. The number of chromatographic peaks (e.g. Fig. 9 ) and chromatographic peak separation (as shown Fig.10As shown in the figure, it was found that the Spursil C18 column had the largest number of chromatographic peaks, the largest amount of chromatographic peak information, and the best separation, so the Spursil C18 column was finally selected.

[0236] 2.3. Investigation of different extraction solvents

[0237] This study compared six extraction solvents: water, 50% methanol, 70% methanol, 80% methanol, 90% methanol, and ethanol. The results showed that (e.g. Fig.11 26 chromatographic peaks were obtained when water was extracted, 20 chromatographic peaks were obtained when 50% methanol was extracted, 22 chromatographic peaks were obtained when 70% methanol was extracted, 17 chromatographic peaks were obtained when 80% methanol was extracted, 20 chromatographic peaks were obtained when 90% methanol was extracted, and 13 chromatographic peaks were obtained when ethanol was extracted. This shows that the number of chromatographic peaks obtained when water was extracted is the largest, indicating that the most components were extracted, and the sum of all characteristic peak areas is the largest (as shown in Figure 2). Fig.12 As shown in the figure, water extraction has the best effect and the characteristic peak information is the richest. Therefore, water extraction is selected as the best extraction solvent. The specific chromatogram information of the six solvents is shown in the figure. Fig.13 shown.

[0238] 2.4. Investigation of the preparation method of test solution

[0239] 2.4.1. Sample extraction process investigation

[0240] A comparison was made on whether extraction was required during the preparation of the test products. The specific steps are as follows:

[0241] Process 1, water extraction without extraction: take about 4g of the Chinese medicine compound granules containing Epimedium, accurately weigh, put into a 100mL conical flask, accurately add 50mL of pure water, ultrasonically extract for 30min, take out, cool, weigh again, shake well, filter, and obtain.

[0242] Process 2, extract with n-butanol solution 3 times after water extraction: take about 4g of Chinese medicine compound granules containing epimedium, accurately weigh, place in a 100mL conical flask, accurately add 50mL of purified water, ultrasonically extract for 30min, take out, cool, weigh again, shake well, and filter. Accurately draw 25mL of the filtrate and place in a separatory funnel, extract 3 times with n-butanol saturated with water, 40mL each time, combine the n-butanol layers, evaporate to dryness, dissolve with methanol and dilute to 5mL volumetric flask, shake well, filter with a 0.45μm microporous filter membrane, and take the filtrate.

[0243] The results showed that after extraction with n-butanol, the number of chromatographic peaks increased significantly, so the sample was extracted with n-butanol three times. The statistical results of the number of characteristic peaks obtained by the two preparation methods are shown in Fig.14 The specific chromatogram information is shown in Fig.15 shown.

[0244] 2.4.2. Ammonia washing process investigation

[0245] Investigate whether ammonia water is used for washing during the preparation of the test solution. The specific steps are as follows:

[0246] Process 1, ammonia washing: take about 4g of Chinese medicine compound granules containing epimedium, accurately weigh, place in a 100mL conical flask, accurately add 50mL of purified water, ultrasonically extract for 30min, take out, cool, weigh again, shake well, and filter. Accurately absorb 25mL of the filtrate and place in a separatory funnel, extract 3 times with water-saturated n-butanol, 40mL each time, combine the n-butanol layers, wash the n-butanol solution three times with ammonia solution, evaporate the n-butanol solution, dissolve it with methanol and make it up to 5mL volumetric flask, shake well, filter with a 0.45μm microporous filter membrane, and take the filtrate.

[0247] Process 2, no ammonia washing: take about 4g of Chinese medicine compound granules containing epimedium, accurately weigh, place in a 100mL conical flask, accurately add 50mL of purified water, ultrasonically extract for 30min, take out, cool, weigh again, shake well, and filter. Accurately draw 25mL of the filtrate and place in a separatory funnel, extract 3 times with water-saturated n-butanol, 40mL each time, combine the n-butanol layers, evaporate to dryness, dissolve with methanol and make up to 5mL volumetric flask, shake well, filter with a 0.45μm microporous filter membrane, and take the filtrate.

[0248] The results showed that the number of chromatographic peaks was greatly lost after washing with ammonia water, so the step of washing with ammonia water was omitted. The statistical results of the number of characteristic peaks obtained by the two preparation methods are shown in Figure 2. Fig.16 The specific chromatogram information is shown in Fig.17 shown.

[0249] 3. Establishment of feature map

[0250] 3.1. Preparation of test solution

[0251] Take about 4g of this product, weigh it accurately, put it in a 100mL conical flask, accurately add 50mL of pure water, ultrasonically extract for 30min, take it out, cool it, weigh it again, shake it well, and filter it. Accurately take 25mL of the filtrate and put it in a separatory funnel, extract it 3 times with water-saturated n-butanol, 40mL each time, combine the n-butanol layers, evaporate to dryness, dissolve it with methanol and make it up to 5mL volumetric flask, shake it well, filter it with a 0.45μm microporous filter membrane, and take the filtrate to obtain it.

[0252] 3.2 Preparation of reference solution

[0253] Accurately weigh appropriate amounts of magnolidine, paeoniflorin, paeoniflorin, epimedin A, epimedin B, epimedin C, icariin, anemarrhena saponin BⅡ, and dipsaponin VI reference substances, and dissolve them in methanol to obtain the product.

[0254] 3.3 Chromatographic conditions

[0255] Octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A, water was used as mobile phase B, and gradient elution was performed according to the elution conditions in Table 7. Among them, the flow rate was 1.0 mL / min, the column temperature was 35°C, the injection volume was 20 μl, the drift tube temperature was 115°C, and the carrier gas flow rate was 3.1 L / min.

[0256] Among them, the HPLC chromatograms of the Chinese medicine compound granule sample containing Epimedium and the reference substance are as follows: Fig.18 shown.

[0257] 3.4 Methodological Review

[0258] 3.4.1 Precision investigation

[0259] Take the same batch of test solution (batch number 20231210), inject continuously 6 times according to "3.3, chromatographic conditions", take peak 6 as reference peak S, and record the chromatogram. Record and calculate the relative retention time and relative peak area of ​​22 characteristic peaks of the traditional Chinese medicine compound granule sample containing epimedium. The results are shown in Tables 8 and 9.

[0260] Table 8 Precision investigation results of characteristic spectra of Chinese herbal compound granules containing Epimedium (relative peak area)

[0261]

[0262]

[0263] Table 9 Precision investigation results of characteristic spectra of Chinese herbal compound granules containing Epimedium (relative retention time)

[0264]

[0265]

[0266] 3.4.2 Stability Study

[0267] Take the same batch of Chinese medicine compound granules containing Epimedium (batch number 20231210), grind it into powder, and inject it at 0, 80, 160, 240, 320, 400min, 8, 24, and 48h according to "3.1, Preparation of test solution" and "3.3, Chromatographic conditions", take peak 6 as the reference peak S, and record the chromatogram. Record and calculate the retention time and relative peak area of ​​22 characteristic peaks, and the results are shown in Tables 10 and 11.

[0268] Table 10: Results of stability study of characteristic spectra of Chinese herbal compound granules containing Epimedium (relative peak area)

[0269]

[0270]

[0271] Table 11: Results of stability study on characteristic spectra of Chinese herbal compound granules containing Epimedium (relative retention time)

[0272]

[0273]

[0274] 3.4.3 Repeatability study

[0275] Take the same batch of Chinese medicine compound granules containing Epimedium (batch number 20231210), grind them into powder, and prepare 6 parallel portions. According to "3.1, Preparation of test solution" and "3.3, Chromatographic conditions", take peak 6 as the reference peak S, and record the chromatogram. Record and calculate the retention time and relative peak area of ​​22 characteristic peaks, and the results are shown in Tables 12 and 13.

[0276] Table 12 Repeatability test results of characteristic spectra of Chinese herbal compound granules containing Epimedium (relative peak area)

[0277]

[0278] Table 13 Repeatability test results of characteristic spectra of Chinese herbal compound granules containing Epimedium (relative retention time)

[0279]

[0280] 3.5. Calibration of characteristic spectra and common characteristic peaks

[0281] According to the relevant parameters given by the HPLC spectra of 10 batches of test samples, the chromatographic peaks of all components of the Chinese herbal compound granules containing Epimedium appeared within 60 minutes, and all spectral peaks were well separated.

[0282] Five batches of sugar-containing granules and sugar-free granules were analyzed, and a total of 22 common characteristic peaks were identified in the present invention. After identification with the reference substance, 8 components were identified by retention time and ultraviolet absorption characteristics, among which peak 3 was magnolidine, peak 5 was paeoniflorin, peak 6 was paeoniflorin, peak 12 was anemarrhena saponin BII, peak 13 was epimedin A, peak 14 was epimedin B, peak 15 was epimedin C, peak 17 was icariin, and peak 18 was dipsaponin VI. The results are as follows Fig.19 shown.

[0283] Taking chromatographic peak No. 6 with a retention time of about 16.6 min as the reference peak (the reference peak is labeled S, and other characteristic peaks are calibrated with Arabic numerals 1, 2, 3...N in sequence), the relative peak area ratios (Table 14) and relative retention times (Table 15) of 22 chromatographic peaks in 10 batches of samples were calculated. The values ​​were relatively fixed, so they were calibrated as common fingerprint peaks.

[0284] Table 14 Calculation results of relative peak areas of common fingerprint peaks of 10 batches of Chinese herbal compound granules containing Epimedium

[0285]

[0286]

[0287] Table 15 Calculation results of relative retention time of common fingerprint peaks of 10 batches of Chinese herbal compound granules containing Epimedium

[0288]

[0289]

[0290] 3.6 Similarity Evaluation

[0291] The similarity evaluation system of Chinese medicine chromatographic characteristic patterns was used to evaluate the similarity of 9 batches of Chinese medicine compound granules containing epimedium (5 batches without sugar and 4 batches with sugar). The results showed that the quality of the 9 batches of Chinese medicine compound granules containing epimedium was uniform and stable.

[0292] 3.7. Similarity calculation

[0293] The similarity results are shown in Table 16.

[0294] Table 16 Similarity of characteristic spectra of Chinese herbal compound granules containing epimedium with sugar and Chinese herbal compound granules containing epimedium without sugar

[0295]

[0296]

[0297] 3.8. Using medicinal materials to attribute chromatographic peaks

[0298] Take the medicinal materials of Epimedium, Dipsacus asper, Anemarrhena asphodeloides, Rehmannia root, Trillium, Curcuma zedoaria, Ligusticum chuanxiong, and Paeonia lactiflora, refer to the preparation method of the test solution to obtain a single medicinal material control solution, analyze it according to the above conditions, and obtain the HPLC chromatogram of the medicinal material control solution and the Chinese medicine compound granules containing Epimedium. By comparing the retention time of each chromatographic peak of the single medicinal material control solution and the sample chromatogram, it is identified that Peak 1 and Peak 18 are Dipsacus asper saponin VI from Dipsacus asper; Peak 3 is Magnolia alkaloids, Peak 13 is Epimedin A, Peak 14 is Epimedin B, Peak 15 is Epimedin C, Peak 17 is Icariin, and Peak 21 is from Epimedium; Peak 5 is Paeoniflorin and Peak 6 is Paeoniflorin from Paeonia lactiflora; Peak 12 is Anemarrhena saponin BII and Peak 19 is from Anemarrhena asphodeloides. Fig. 20 shown.

[0299] In summary, the liquid chromatography-evaporative light detection (HPLC-ELSD) characteristic spectrum established in this study has a total of 22 characteristic peaks, 9 of which were identified using reference substances, and the remaining 13 characteristic peaks were attributed to medicinal materials. This method can be used for the quality control of two specifications of preparations of Chinese herbal compound granules containing Epimedium, both sugar-type and sugar-free.

[0300] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for constructing an HPLC-ELSD fingerprint of a Chinese medicinal compound containing Epimedium, characterized in that: The construction method comprises the following steps: Preparation of the test solution: weigh an appropriate amount of the Chinese medicine compound preparation, place it in a first container of appropriate volume, add a first solvent and then perform ultrasonic extraction, take it out, cool it, weigh it, shake it well, filter it, and obtain a first filtrate; transfer the first filtrate to a second container, extract it with a second solvent, combine the extract layers, evaporate to dryness, add a third solvent to dissolve it and make it to a fixed volume in a third container, shake it well, filter it, and obtain a second filtrate; transfer the second filtrate to obtain the test solution; wherein the Chinese medicine compound The prescription comprises epimedium, radix dipsaci, rhizoma anemarrhenae, rhizoma rehmanniae, rhizoma sparganii, rhizoma curcumae, rhizoma chuanxiong, rhizoma rubra and oyster; wherein the mass ratio of epimedium, rhizoma dipsaci, rhizoma anemarrhenae, rhizoma rehmanniae, rhizoma sparganii, rhizoma curcumae, rhizoma chuanxiong, rhizoma rubra and oyster is (500-1000):(20-100):(20-100):(20-100):(20-100):(20-100):(20-100):(20-100):(5-20; Wherein, the first solvent is water, and the mass / volume ratio between the Chinese medicine compound preparation and the first solvent is 0.076-0.084, in units of g / ml; Wherein, the second solvent is water-saturated n-butanol, and the volume / volume ratio between the second solvent and the first solvent is 0.76-0.84, in ml / ml; Wherein, the third solvent is methanol; Preparation of reference solution: weigh appropriate amounts of magnolidine, paeoniflorin, paeoniflorin, epimedin A, epimedin B, epimedin C, icariin, timosaponin BⅡ and dipsaponin VI reference substances, add methanol solution to prepare the reference solution with a concentration of magnolidine, paeoniflorin, paeoniflorin, epimedin A, epimedin B, epimedin C, icariin, timosaponin BⅡ and dipsaponin VI of 400-600 μg / ml respectively; According to the results of high performance liquid phase detection of the test solution and the reference solution, a HPLC-ELSD fingerprint of the Chinese medicine compound is obtained; The chromatographic conditions for high performance liquid phase detection are as follows: a chromatographic column with octadecyl bonded silica gel as filler is used, mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, mobile phase B is water, acid water, alkaline aqueous solution and / or buffered saline solution, and the gradient elution program is: 0 min, 10% A; 60 min, 45% A; flow rate is 0.5-1.5 mL / min, column temperature is 25-45°C, injection volume is 10-30 μl, drift tube temperature is 100-130°C, and carrier gas flow rate is 2-4 L / min.

2. The construction method according to claim 1, characterized in that: In the preparation of the test solution, the first solvent is purified water or distilled water.

3. The construction method according to claim 1, characterized in that: The first container is a conical flask.

4. The construction method according to claim 1, characterized in that: The volume of the first container is 100 ml.

5. The construction method according to claim 1, characterized in that: The second container is a funnel.

6. The construction method according to claim 5, characterized in that: The second container is a separatory funnel.

7. The construction method according to claim 1, characterized in that: The third container is a measuring bottle.

8. The construction method according to claim 1, characterized in that: The volume of the third container is 5 ml.

9. The construction method according to claim 1, characterized in that: The ultrasonic extraction time is 10 to 60 minutes.

10. The construction method according to claim 9, characterized in that: The ultrasonic treatment time is 28.5 to 31.5 minutes.

11. The construction method according to claim 1, characterized in that: The volume of the first filtrate is 10-40 ml.

12. The construction method according to claim 11, characterized in that: The volume of the first filtrate was 25 ml.

13. The construction method according to claim 1, characterized in that: The volume / volume (ml / ml) ratio between the first filtrate and the first solvent is 0.1 to 1.

0.

14. The construction method according to claim 13, characterized in that: The volume / volume (ml / ml) ratio between the first filtrate and the first solvent is 0.475 to 0.

525.

15. The construction method according to claim 1, characterized in that: The extraction is performed 2 to 5 times.

16. The construction method according to claim 15, characterized in that: The extraction was performed 3 times.

17. The construction method according to claim 1, characterized in that: The volume of the second solvent is 20-60 ml / time.

18. The construction method according to claim 17, characterized in that: The volume of the second solvent is 40 ml / time.

19. The construction method according to claim 1, characterized in that: The extract layer is an n-butanol layer.

20. The construction method according to claim 1, characterized in that: The volume of the fixed volume is 5 ml.

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

22. The construction method according to claim 21, characterized in that: The pore size of the microporous filter membrane is 0.45 μm.

23. The construction method according to claim 1, characterized in that: The concentration of magnolidine in the reference solution is 450-550 μg / ml.

24. The construction method according to claim 1, characterized in that: The concentration of albiflorin in the reference solution is 450-550 μg / ml.

25. The construction method according to claim 1, characterized in that: The concentration of paeoniflorin in the reference solution is 450-550 μg / ml.

26. The construction method according to claim 1, characterized in that: The concentration of epimedin A in the reference solution is 450-550 μg / ml.

27. The construction method according to claim 1, characterized in that: The concentration of epimedin B in the reference solution is 450-550 μg / ml.

28. The construction method according to claim 1, characterized in that: The concentration of epimedin C in the reference solution is 450-550 μg / ml.

29. The construction method according to claim 1, characterized in that: The concentration of icariin in the reference solution is 450-550 μg / ml.

30. The construction method according to claim 1, characterized in that: The concentration of Anemarrhena saponin BⅡ in the reference solution is 450-550 μg / ml.

31. The construction method according to claim 1, characterized in that: The concentration of Dipsacus asper saponin VI in the reference solution is 450-550 μg / ml.

32. The construction method according to claim 1, characterized in that: The reference substance solution is a mixed solution of magnoline 475-525 μg / ml, paeoniflorin 475-525 μg / ml, paeoniflorin 475-525 μg / ml, epimedin A 475-525 μg / ml, epimedin B 475-525 μg / ml, epimedin C 475-525 μg / ml, icariin 475-525 μg / ml, anemarrhena saponin BⅡ 475-525 μg / ml, and dipsaponin VI 475-525 μg / ml.

33. The construction method according to claim 1, characterized in that: The flow rate is 0.8-1.2 ml / min.

34. The construction method according to claim 33, characterized in that: The flow rate is 0.95-1.05 ml / min.

35. The construction method according to claim 1, characterized in that: The column temperature is 30-40°C.

36. The construction method according to claim 35, characterized in that: The column temperature is 33.25-36.75°C.

37. The construction method according to claim 1, characterized in that: The injection volume is 15 to 25 μl.

38. The construction method according to claim 37, characterized in that: The injection volume is 19-21 μl.

39. The construction method according to claim 1, characterized in that: The temperature of the drift tube is 105-125°C.

40. The construction method according to claim 39, characterized in that: The temperature of the drift tube is 109.25-120.75°C.

41. The construction method according to claim 1, characterized in that: The carrier gas flow rate is 2.5 to 3.5 L / min.

42. The construction method according to claim 41, characterized in that: The carrier gas flow rate is 2.945-3.255 L / min.

43. The construction method according to claim 1, characterized in that: The theoretical plate number of the chromatographic peak corresponding to the reference substance is 6000.

44. The construction method according to claim 1, characterized in that: The separation degree of the chromatographic peaks corresponding to the reference substance is greater than 1.

5.

45. The construction method according to claim 1, characterized in that: The chromatographic column is an Ultimate C18 chromatographic column, a YMC C18 chromatographic column, a Caprisil C18AQ chromatographic column or a Spursil C18 chromatographic column.

46. ​​The construction method according to claim 45, characterized in that The chromatographic column is a Spursil C18 chromatographic column.

47. The construction method according to claim 1, characterized in that The specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

48. The construction method according to claim 1, characterized in that: The mobile phase A is acetonitrile.

49. The construction method according to claim 1, characterized in that: The acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from one or more of weak acids and their salts, weak bases and their salts of different concentrations.

50. The construction method according to claim 49, characterized in that: The acid aqueous solution, alkali aqueous solution and / or buffered saline solution are 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 in different concentrations.

51. The construction method according to claim 50, characterized in that: The acid aqueous solution is 0.08% to 0.12% acid aqueous solution.

52. The construction method according to claim 51, characterized in that: The acid aqueous solution is a 0.08% to 0.12% phosphoric acid aqueous solution.

53. The construction method according to claim 52, characterized in that: The acid aqueous solution is a 0.095% to 0.105% phosphoric acid aqueous solution.

54. The construction method according to claim 50, characterized in that: The buffered saline solution is a phosphate aqueous solution and / or an acetate aqueous solution.

55. The construction method according to claim 54, characterized in that: The pH value of the buffered saline solution is not greater than 7.

0.

56. The construction method according to claim 1, characterized in that: The mobile phase B is water.

57. The construction method according to claim 1, characterized in that: The preparation method of the traditional Chinese medicine compound preparation comprises: (1) weighing an appropriate amount of oysters and crushing them into oyster fine powder; and (2) weighing an appropriate amount of epimedium, radix scutellariae, rhizoma anemarrhenae, rhizoma rehmanniae, rhizoma sparganii, rhizoma curcumae, rhizoma ligustici and rhizoma peonyii, decocting them with water, filtering, concentrating the filtrate into a clear paste, adding the oyster fine powder, mixing, drying, crushing into fine powder, preparing the preparation, and drying.

58. The construction method according to claim 57, characterized in that: In step (2), appropriate amounts of starch and sucrose are added together with the oyster powder.

59. The construction method according to claim 1, characterized in that: The mass ratios of the epimedium, the Dipsacus asper, the Anemarrhena asphodeloides, the Rehmannia root, the Trillium, the Curcuma zedoaria, the Ligusticum chuanxiong, the Paeonia lactiflora and the Concha Ostreae are (617.5-682.5):(47.5-52.5):(47.5-52.5):(38-42):(47.5-52.5):(47.5-52.5):(47.5-52.5):(47.5-52.5):(9.5-10.5).

60. The construction method according to claim 57, characterized in that: The oyster is calcined oyster.

61. The construction method according to claim 57, characterized in that: The decoction is performed 1 to 5 times.

62. The construction method according to claim 61, characterized in that: The decoction was performed 3 times.

63. The construction method according to claim 57, characterized in that: The decoction time is 0.5 to 3 hours.

64. The construction method according to claim 63, characterized in that: The decocting time is 1 to 2 hours.

65. The construction method according to claim 57, characterized in that: The relative density of the clear paste at 50° C. is 1.2 to 1.

40.

66. The construction method according to claim 57, characterized in that: The relative density of the clear paste at 50° C. is 1.2825 to 1.4175.

67. The construction method according to claim 57, characterized in that: The dosage form of the Chinese medicine compound preparation is granules or capsules.

68. The construction method according to claim 67, characterized in that: The particles are sugar-containing particles or sugar-free particles.

69. The construction method according to claim 1, characterized in that: The fingerprint spectrum includes peaks 1-22, among which peak 3 is magnolidine, peak 5 is paeoniflorin, peak 6 is paeoniflorin, peak 12 is anemarrhena saponin BII, peak 13 is epimedin A, peak 14 is epimedin B, peak 15 is epimedin C, peak 17 is icariin, and peak 18 is dipsaponin VI.

70. The construction method according to claim 1, characterized in that: The fingerprint spectrum includes peaks 1-22, among which peaks 3, 13, 14, 15, 17 and 21 belong to the medicinal flavor of Epimedium; peaks 5 and 6 belong to the medicinal flavor of Red Peony Root; peaks 12 and 19 belong to the medicinal flavor of Anemarrhena Rhizoma; and peaks 1 and 18 belong to the medicinal flavor of Dipsacus Asper.

71. The construction method according to claim 1, characterized in that The fingerprint spectrum includes 22 characteristic peaks, with the chromatographic peak of paeoniflorin No. 6 as the reference peak, and the relative retention times of the other 21 characteristic peaks are: No. 1 chromatographic peak 0.628±10%, No. 2 chromatographic peak 0.748±10%, No. 3 chromatographic peak 0.801±10%, No. 4 chromatographic peak 0.822±10%, No. 5 chromatographic peak 0.894±10%, No. 7 chromatographic peak 1.247±10%, No. 8 chromatographic peak 1.489±10%, No. 9 chromatographic peak 1.727±10%, No. 10 chromatographic peak 1.815±10%, No. 11 chromatographic peak 1.840±10%, No. 12 chromatographic peak 1.830±10%, No. 13 chromatographic peak 1.820±10%, No. 14 chromatographic peak 1.830±10%, No. 15 chromatographic peak 1.840±10%, No. 16 chromatographic peak 1.850±10%, No. 17 chromatographic peak 1.860±10%, No. 18 chromatographic peak 1.870±10%, No. 19 chromatographic peak 1.880±10%, No. 20 chromatographic peak 1.860±10%, No. 21 chromatographic peak 1.860±10%, No. 22 The chromatographic peak No. 12 is 1.848±10%, the chromatographic peak No. 12 is 1.993±10%, the chromatographic peak No. 13 is 2.186±10%, the chromatographic peak No. 14 is 2.241±10%, the chromatographic peak No. 15 is 2.279±10%, the chromatographic peak No. 16 is 2.316±10%, the chromatographic peak No. 17 is 2.357±10%, the chromatographic peak No. 18 is 2.585±10%, the chromatographic peak No. 19 is 2.614±10%, the chromatographic peak No. 20 is 2.773±10%, the chromatographic peak No. 21 is 2.919±10%, and the chromatographic peak No. 22 is 3.438±10%.

72. The construction method according to claim 1, characterized in that The fingerprint spectrum includes 22 characteristic peaks, with the chromatographic peak of paeoniflorin No. 6 as the reference peak, and the relative peak areas of the other 21 characteristic peaks are as follows: No. 1 chromatographic peak 0.106±10%, No. 2 chromatographic peak 0.047±10%, No. 3 chromatographic peak 0.153±10%, No. 4 chromatographic peak 0.042±10%, No. 5 chromatographic peak 1.137±10%, No. 7 chromatographic peak 0.065±10%, No. 8 chromatographic peak 0.142±10%, No. 9 chromatographic peak 0.991±10%, No. 10 chromatographic peak 0.170±10%, No. 11 chromatographic peak 0.180±10%, No. 12 chromatographic peak 0.206±10%, No. 13 chromatographic peak 0.213±10%, No. 14 chromatographic peak 0.236±10%, No. 15 chromatographic peak 0.244±10%, No. 16 chromatographic peak 0.263±10%, No. 17 chromatographic peak 0.270±10%, No. 18 chromatographic peak 0.286±10%, No. 19 chromatographic peak 0.304±10%, No. 20 chromatographic peak 0.313±10%, No. 21 The chromatographic peak No. 12 is 0.147±10%, the chromatographic peak No. 13 is 0.107±10%, the chromatographic peak No. 14 is 0.121±10%, the chromatographic peak No. 15 is 2.494±10%, the chromatographic peak No. 16 is 0.557±10%, the chromatographic peak No. 17 is 0.652±10%, the chromatographic peak No. 18 is 0.654±10%, the chromatographic peak No. 19 is 0.182±10%, the chromatographic peak No. 20 is 0.285±10%, the chromatographic peak No. 21 is 0.062±10%, and the chromatographic peak No. 22 is 0.306±10%.

73. A quality control method for a Chinese medicinal compound containing Epimedium, characterized in that: The quality control method comprises the following steps: (1) Establishing a standard HPLC-ELSD fingerprint of a Chinese medicine compound reference sample according to the HPLC-ELSD fingerprint construction method described in any one of claims 1 to 72; (2) taking a solution of a Chinese medicine compound test sample, and performing detection according to the chromatographic conditions in the HPLC-ELSD fingerprint construction method according to any one of claims 1 to 72, to obtain an HPLC-ELSD fingerprint of the Chinese medicine compound test sample; as well as (3) Compare the HPLC-ELSD fingerprint of the Chinese medicine compound sample to be tested obtained in step (2) with the HPLC-ELSD fingerprint of the Chinese medicine compound reference sample standard obtained in step (1). The product that meets the requirements is qualified, and the product that does not meet the requirements is unqualified.

74. The quality control method according to claim 73, characterized in that: The compliance requirements include one or more of the following: (1) The HPLC-ELSD fingerprint of the Chinese medicine compound sample to be tested exhibits 22 characteristic chromatographic peaks, and the retention time of each characteristic chromatographic peak is within ±10% of the retention time value of the corresponding reference chromatographic peak in the standard HPLC-ELSD fingerprint of the Chinese medicine compound reference sample; (2) With the paeoniflorin peak as the S peak, the relative retention time of each characteristic chromatographic peak in the HPLC-ELSD fingerprint of the Chinese medicine compound sample to be tested and the S peak is within ±10% of the relative retention time value of each characteristic chromatographic peak in the standard HPLC-ELSD fingerprint of the Chinese medicine compound reference sample; and (3) According to the Chinese medicine chromatographic HPLC-ELSD fingerprint similarity evaluation system, the similarity between the HPLC-ELSD fingerprint of the Chinese medicine compound sample to be tested and the standard HPLC-ELSD fingerprint of the Chinese medicine compound reference sample is calculated, and the similarity shall not be less than 0.

90.

75. Use of the construction method according to any one of claims 1 to 72 or the quality control method according to claim 73 or 74 in quality detection, quality evaluation or quality control of a traditional Chinese medicine compound containing Epimedium.

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