Fingerprint construction and detection methods of traditional Chinese medicine preparations for treating eczema

Abstract

The present invention provides a method for constructing a fingerprint of a traditional Chinese medicine preparation for treating eczema, comprising the following steps: taking gallic acid, gentiopicroside, geniposide, paeoniflorin, ammonium glycyrrhizate, salvianolic acid B, baicalin, baicalein and wogonin reference substances to prepare a mixed reference substance solution; mixing the traditional Chinese medicine preparation for treating eczema with an organic solvent to prepare a test solution; the organic solvent is selected from one or more of methanol aqueous solution, ethanol aqueous solution and anhydrous ethanol; the mixed reference substance solution and the test solution are respectively subjected to high performance liquid chromatography determination; wherein the conditions adopted by the high performance liquid chromatography include: mobile phase A is selected from one or more of methanol and acetonitrile, and mobile phase B is selected from one or more of phosphoric acid aqueous solution, acetic acid aqueous solution and formic acid aqueous solution; and a gradient elution program is adopted.

Description

Technical Field

[0001] The present invention relates to the field of detection of traditional Chinese medicine preparations, and in particular to a fingerprint spectrum construction method and a detection method of a traditional Chinese medicine preparation for treating eczema. Background Art

[0002] Research on the substance-based efficacy of traditional Chinese medicine (TCM) compound formulas is a key and hot technology area in the modernization of TCM research and is fundamental to determining the safety, efficacy, and quality control of TCM. TCM fingerprint technology is currently considered an effective means of TCM quality control. Based on systematic research on the chemical components of TCM, it is primarily used to evaluate the authenticity, quality, and stability of TCM materials and semi-finished TCM preparations. Its notable characteristics are its "holistic" and "fuzzy" nature. TCM preparations are complex, multi-component systems, and their quality should be evaluated using appropriate detection methods that provide rich identification information. However, current methods such as microscopic identification, physicochemical identification, and content determination are insufficient to address this issue. Therefore, it is necessary to establish a TCM fingerprint that can more comprehensively reflect the types and quantities of chemical components contained in TCM and its preparations, thereby providing a holistic description and evaluation of drug quality. This is consistent with the holistic theory of TCM.

[0003] Beijing Silian Pharmaceutical Co., Ltd. has developed a new Chinese medicine preparation for treating eczema. This medicine is composed of 12 herbs: Rehmannia root, Gypsum, Scutellaria baicalensis, Gentiana scabra, Sanguisorba officinalis, Prunus mume, White Peony Root, Gardenia jasminoides, Rhizoma Atractylodis Macrocephalae, Herba Siegesbeckiae, Salvia miltiorrhizae, and Licorice root. It has the effects of clearing the liver, cooling blood, detoxifying, invigorating the spleen, removing dampness, and relieving itching. It is clinically used for acute and subacute eczema characterized by damp-heat accumulation in the skin, with symptoms such as skin erythema, papules, blisters, erosions, and exudate. As a new type of Chinese medicine for treating eczema, there is currently no research on the fingerprint of its composition. Most studies have focused on the determination of single herbs, only able to quantitatively analyze some index components, but unable to reflect the overall chemical characteristics of the Chinese medicine preparation for treating eczema, resulting in certain defects in quality control. Therefore, it is necessary to establish a fingerprint construction method and detection method for traditional Chinese medicine preparations for the treatment of eczema, in order to control the quality of the terminal product formula particles and ensure the safety of clinical medication. Summary of the Invention

[0004] Based on this, the present invention provides a fingerprint spectrum construction method for traditional Chinese medicine preparations for treating eczema. The fingerprint spectrum construction method is simple to operate, and has good precision, repeatability, stability and durability. The baseline in the constructed spectrum is stable, the separation between the peaks is good, and the amount of chromatographic peak information contained is the most comprehensive.

[0005] The present invention is achieved through the following technical solutions.

[0006] A method for constructing a fingerprint of a traditional Chinese medicine preparation for treating eczema, wherein the traditional Chinese medicine preparation for treating eczema is a compound traditional Chinese medicine preparation composed of rehmannia root, gypsum, scutellaria baicalensis, gentian, sanguisorba officinalis, plum blossom, white peony root, gardenia jasminoides, stir-fried atractylodes macrocephala, siegesbeckia officinalis, salvia miltiorrhiza, and licorice, comprising the following steps:

[0007] Prepare a mixed reference solution by taking gallic acid, gentiopicroside, gardenoside, paeoniflorin, ammonium glycyrrhizate, salvianolic acid B, baicalin, baicalein and wogonin reference substances;

[0008] The traditional Chinese medicine preparation for treating eczema is mixed with an organic solvent to prepare a test solution; the organic solvent is selected from one or more of methanol aqueous solution, ethanol aqueous solution and anhydrous ethanol;

[0009] The mixed reference solution and the test solution are respectively subjected to high performance liquid chromatography determination;

[0010] Wherein, the conditions adopted by the high performance liquid chromatography include: mobile phase A is selected from one or more of methanol and acetonitrile, and mobile phase B is selected from one or more of phosphoric acid aqueous solution, acetic acid aqueous solution and formic acid aqueous solution; a gradient elution program is adopted; the gradient elution program includes: from 0 min to 5 min, the volume percentage of mobile phase A changes from 5% to 12%; from 5 min to 30 min, the volume percentage of mobile phase A changes from 12% to 14%; from 30 min to 32 min, the volume percentage of mobile phase A changes from 14% to 16%; from 32 min to 40 min, the volume percentage of mobile phase A changes from 16% to 18%; from 40 min to 65 min, the volume percentage of mobile phase A changes from 18% to 35%; from 65 min to 75 min, the volume percentage of mobile phase A changes from 35% to 50%; from 75 min to 80 min, the volume percentage of mobile phase A changes from 50% to 5%; the detection wavelength is 220 nm to 260 nm.

[0011] In one embodiment, the conditions adopted by the high performance liquid chromatography also include: a flow rate of 0.8 mL / min to 1.2 mL / min; a column temperature of 20° C. to 35° C.; and an octadecylsilane bonded silica gel column.

[0012] In one embodiment, the dimensions of the chromatographic column used in the high performance liquid chromatography include: column length of 240 mm to 260 mm, inner diameter of 4 mm to 5 mm, and particle size of 4.5 μm to 5.5 μm.

[0013] In one embodiment, preparing the mixed reference solution comprises the following steps:

[0014] The gallic acid, gentiopicroside, geniposide, paeoniflorin, ammonium glycyrrhizate, salvianolic acid B, baicalin, baicalein, and wogonin reference substances are mixed with a methanol aqueous solution.

[0015] In one embodiment, preparing the test solution comprises the following steps:

[0016] The traditional Chinese medicine preparation for treating eczema is mixed with the organic solvent, and then ultrasonic extraction or heating reflux extraction is performed, followed by filtration to obtain a filtrate.

[0017] In one embodiment, the number of common peaks in the fingerprint of the traditional Chinese medicine preparation for treating eczema is 26;

[0018] Among them, peak 3 is the chromatographic peak of gallic acid; peak 10 is the chromatographic peak of gardeniaside; peak 11 is the chromatographic peak of gentiopicroside; peak 13 is the chromatographic peak of paeoniflorin; peak 16 is the chromatographic peak of baicalin; peak 17 is the chromatographic peak of salvianolic acid B; peak 23 is the chromatographic peak of baicalein; peak 25 is the chromatographic peak of glycyrrhizic acid; and peak 26 is the chromatographic peak of wogonin.

[0019] The present invention also provides a method for detecting a Chinese medicine preparation for treating eczema, comprising the following steps:

[0020] Mixing the test substance with an organic solvent to prepare a test solution; the organic solvent is selected from one or more of methanol aqueous solution, ethanol aqueous solution and anhydrous ethanol;

[0021] The test solution is subjected to high performance liquid chromatography determination;

[0022] Wherein, the conditions adopted by the high performance liquid chromatography include: mobile phase A is selected from one or more of methanol and acetonitrile, and mobile phase B is selected from one or more of phosphoric acid aqueous solution, acetic acid aqueous solution and formic acid aqueous solution; a gradient elution program is adopted; the gradient elution program includes: from 0 min to 5 min, the volume percentage of mobile phase A changes from 5% to 12%; from 5 min to 30 min, the volume percentage of mobile phase A changes from 12% to 14%; from 30 min to 32 min, the volume percentage of mobile phase A changes from 14% to 16%; from 32 min to 40 min, the volume percentage of mobile phase A changes from 16% to 18%; from 40 min to 65 min, the volume percentage of mobile phase A changes from 18% to 35%; from 65 min to 75 min, the volume percentage of mobile phase A changes from 35% to 50%; from 75 min to 80 min, the volume percentage of mobile phase A changes from 50% to 5%; the detection wavelength is 220 nm to 260 nm.

[0023] In one embodiment, the conditions adopted by the high performance liquid chromatography also include: a flow rate of 0.8 mL / min to 1.2 mL / min; a column temperature of 20° C. to 35° C.; and an octadecylsilane bonded silica gel column.

[0024] In one embodiment, the dimensions of the chromatographic column used in the high performance liquid chromatography include: column length of 240 mm to 260 mm, inner diameter of 4 mm to 5 mm, and particle size of 4.5 μm to 5.5 μm.

[0025] In one embodiment, preparing the test solution comprises the following steps:

[0026] The analyte is mixed with the organic solvent, and then ultrasonic extraction or heating reflux extraction is performed, followed by filtration to obtain a filtrate.

[0027] Compared with the prior art, the fingerprint construction method of the traditional Chinese medicine preparation for treating eczema of the present invention has the following beneficial effects:

[0028] The present invention selects one or more of methanol aqueous solution, ethanol aqueous solution and anhydrous ethanol to pre-treat the traditional Chinese medicine preparation, and extracts the effective chemical components into the test solution to the greatest extent. The present invention also utilizes high performance liquid chromatography technology, limits the mobile phase to one or more of methanol and acetonitrile, and one or more of phosphoric acid aqueous solution, acetic acid aqueous solution and formic acid aqueous solution, adopts gradient elution, and further limits the gradient elution program, and limits the detection wavelength to 220nm~260nm. The fingerprint of the traditional Chinese medicine preparation for treating eczema finally obtained has a stable baseline, good separation effect of each chromatographic peak, all main peaks are in a baseline separation state, the chromatographic peak absorption is large, and the number of common peaks is large, which can be as high as 26, which can fully reflect the chemical information of most active ingredients, and show the chemical composition characteristics of the traditional Chinese medicine preparation for treating eczema, and can be directly applied to the qualitative and quantitative control of the traditional Chinese medicine preparation for treating eczema. At the same time, the fingerprint spectrum construction method of the traditional Chinese medicine preparation for treating eczema described in the present invention has strong specificity, good precision, repeatability, stability and durability, and can be used as an exclusive identification method and quality control means for each production process.

[0029] Furthermore, the fingerprint construction method of the traditional Chinese medicine preparation for treating eczema described in the present invention is simple to operate, greatly reduces the detection cost, and is conducive to promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Fingerprints of 10 batches of granules provided by the present invention;

[0031] Figure 2 The reference fingerprint provided by the present invention;

[0032] Figure 3 The mixed reference substance chromatogram provided by the present invention; wherein, chromatographic peak 3 is gallic acid, chromatographic peak 10 is geniposide, chromatographic peak 11 is gentiopicroside, chromatographic peak 13 is paeoniflorin, chromatographic peak 16 is baicalin, chromatographic peak 17 is salvianolic acid B, chromatographic peak 23 is baicalein, chromatographic peak 25 is glycyrrhizic acid, and chromatographic peak 26 is wogonin;

[0033] Figure 4 The precision investigation map provided by the present invention;

[0034] Figure 5 The repeatability survey map provided by the present invention;

[0035] Figure 6 The stability test graph provided by the present invention;

[0036] Figure 7 The column temperature durability test graph provided by the present invention;

[0037] Figure 8 The flow rate durability investigation graph provided by the present invention;

[0038] Figure 9 The wavelength durability inspection graph provided by the present invention;

[0039] Figure 10 The different extraction methods provided by the present invention are investigated in the graph;

[0040] Figure 11 A survey chart of different extraction solvent types provided by the present invention;

[0041] Figure 12 The present invention provides different extraction solvent volume survey patterns;

[0042] Figure 13 The different extraction time survey patterns provided by the present invention;

[0043] Figure 14 A diagram of the methanol-0.1% phosphoric acid aqueous solution mobile phase system provided by the present invention;

[0044] Figure 15 The survey spectrum of mobile phase B composed of different acid types provided by the present invention;

[0045] Figure 16 The spectrum obtained by the mobile phase elution program in Table 1 provided by the present invention;

[0046] Figure 17 The spectrum obtained by the mobile phase elution program in Table 5 provided by the present invention;

[0047] Figure 18 The spectrum provided for Comparative Example 1;

[0048] Figure 19 The spectrum provided for Comparative Example 2;

[0049] Figure 20 The spectrum provided for Comparative Example 3;

[0050] Figure 21 This is the spectrum provided for Comparative Example 3. DETAILED DESCRIPTION

[0051] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present invention. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present disclosure.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0053] The present invention provides a method for constructing a fingerprint spectrum of a traditional Chinese medicine preparation for treating eczema, wherein the traditional Chinese medicine preparation for treating eczema is a compound traditional Chinese medicine preparation composed of rehmannia root, gypsum, scutellaria baicalensis, gentian, sanguisorba officinalis, plum blossom, white peony root, gardenia jasminoides, stir-fried atractylodes macrocephala, siegesbeckia officinalis, salvia miltiorrhiza and licorice, comprising the following steps:

[0054] Prepare a mixed reference solution by taking gallic acid, gentiopicroside, gardenoside, paeoniflorin, ammonium glycyrrhizate, salvianolic acid B, baicalin, baicalein and wogonin reference substances;

[0055] The traditional Chinese medicine preparation for treating eczema is mixed with an organic solvent to prepare a test solution; the organic solvent is selected from one or more of methanol aqueous solution, ethanol aqueous solution and anhydrous ethanol;

[0056] The mixed reference solution and the test solution were subjected to high performance liquid chromatography determination respectively;

[0057] The conditions adopted in the high performance liquid chromatography include: mobile phase A is selected from one or more of methanol and acetonitrile, and mobile phase B is selected from one or more of aqueous phosphoric acid solution, aqueous acetic acid solution and aqueous formic acid solution; a gradient elution program is adopted; the gradient elution program includes: from 0 min to 5 min, the volume percentage of mobile phase A changes from 5% to 12%; from 5 min to 30 min, the volume percentage of mobile phase A changes from 12% to 14%; from 30 min to 32 min, the volume percentage of mobile phase A changes from 14% to 16%; from 32 min to 40 min, the volume percentage of mobile phase A changes from 16% to 18%; from 40 min to 65 min, the volume percentage of mobile phase A changes from 18% to 35%; from 65 min to 75 min, the volume percentage of mobile phase A changes from 35% to 50%; from 75 min to 80 min, the volume percentage of mobile phase A changes from 50% to 5%; the detection wavelength is 220 nm to 260 nm.

[0058] It is understood that the detection wavelength includes but is not limited to 220nm, 225nm, 230nm, 235nm, 236nm, 237nm, 238nm, 239nm, 240nm, 245nm, 250nm, 255nm, and 260nm. Preferably, the detection wavelength is 238nm±2nm. The chromatogram at 238nm±2nm contains the most comprehensive chromatographic peak information and a stable baseline. The chromatographic peak absorption is large, the chromatographic peaks are numerous, and the separation between the peaks is good. The chromatogram can obtain the most chromatographic component information and reflect the overall composition of the system as much as possible.

[0059] The present invention found that the chromatogram obtained by extracting the test sample with methanol aqueous solution or ethanol aqueous solution covers more chromatographic peak information and has a better peak shape.

[0060] In a specific example, the volume fraction of methanol in the methanol-water solution is 30% to 90%. It is understood that in the present invention, the volume fraction of methanol in the methanol-water solution includes, but is not limited to, 30%, 40%, 50%, 60%, 70%, 80%, and 90%. Considering the conservation of organic solvents and the fact that the test solution extracted with 50% methanol is easier to filter than with 30% methanol, preferably, a 50% volume fraction of methanol-water solution is used as the extraction solvent.

[0061] In a specific example, the volume fraction of ethanol in the ethanol aqueous solution is 30% to 99%. It is understood that in the present invention, the volume fraction of ethanol in the ethanol aqueous solution includes but is not limited to 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 99%.

[0062] In a specific example, mobile phase A is acetonitrile, and mobile phase B is aqueous phosphoric acid solution.

[0063] More specifically, the mobile phase B is a phosphoric acid aqueous solution with a volume fraction of 0.1% to 0.2%.

[0064] It is understood that in the present invention, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution includes, but is not limited to, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, and 0.2%. Taking into account the tolerance and service life of the chromatographic column, mobile phase B is more preferably a 0.1% volume fraction of phosphoric acid aqueous solution.

[0065] In a specific example, the conditions used in high performance liquid chromatography also include: a flow rate of 0.8 mL / min to 1.2 mL / min; a column temperature of 20° C. to 35° C.; and an octadecylsilane bonded silica gel column.

[0066] It is understood that in the present invention, flow rates include but are not limited to 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, and 1.2 mL / min. In the fingerprint obtained with flow rates within this range, the peak shapes of the chromatographic peaks and the overall resolution are both better. Preferably, the flow rate is 1.0 mL / min.

[0067] It can be understood that in the present invention, the column temperature includes but is not limited to 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., and 35° C. Preferably, the column temperature is 25° C.±5° C.

[0068] In one specific example, the dimensions of the chromatographic column used in HPLC include: a column length of 240 mm to 260 mm, an inner diameter of 4 mm to 5 mm, and a particle size of 4.5 μm to 5.5 μm. Preferably, the dimensions of the chromatographic column used in HPLC include: a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm. More specifically, the chromatographic column used in HPLC is Ultimate AQ-C18.

[0069] In a specific example, the conditions used in the high performance liquid chromatography further include: an injection volume of 5 μL to 20 μL, more specifically, an injection volume of 10 μL.

[0070] In a specific example, preparing a mixed reference solution includes the following steps:

[0071] Gallic acid, gentiopicroside, geniposide, paeoniflorin, ammonium glycyrrhizate, salvianolic acid B, baicalin, baicalein, and wogonin reference substances were mixed with methanol-water solution.

[0072] In a specific example, preparing the test solution includes the following steps:

[0073] The traditional Chinese medicine preparation for treating eczema is mixed with an organic solvent and then subjected to ultrasonic extraction or heating reflux extraction, and then filtered to obtain a filtrate.

[0074] In a specific example, the conditions for ultrasonic extraction include: power of 400W to 600W and frequency of 35kHz to 45kHz. More specifically, the conditions for ultrasonic extraction include: power of 500W and frequency of 40kHz.

[0075] In a specific example, the time for ultrasonic extraction or heating reflux extraction is 15 minutes to 60 minutes. It is understandable that in the present invention, the time for ultrasonic extraction or heating reflux extraction includes but is not limited to 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, and 60 minutes. Preferably, the time for ultrasonic extraction or heating reflux extraction is 30 minutes to 45 minutes. Taking into account the convenience of operation, more preferably, the time for ultrasonic extraction or heating reflux extraction is 30 minutes.

[0076] In a specific example, the volume of the organic solvent corresponding to 1g of the traditional Chinese medicine preparation for treating eczema is 25mL to 100mL. Preferably, the volume of the organic solvent corresponding to 1g of the traditional Chinese medicine preparation for treating eczema is 25mL.

[0077] More specifically, preparing the test solution comprises the following steps:

[0078] Take a traditional Chinese medicine preparation for treating eczema, grind it into powder, weigh 1g of each, accurately add 25ml of 50% methanol, weigh it, ultrasonically treat it (power 500W, frequency 40kHz) for 30 minutes, let it cool, make up the lost weight with 50% methanol, shake it well, and filter it.

[0079] In a specific example, the number of common peaks in the fingerprint of a traditional Chinese medicine preparation for treating eczema is 26;

[0080] Among them, peak 3 is the chromatographic peak of gallic acid; peak 10 is the chromatographic peak of gardeniaside; peak 11 is the chromatographic peak of gentiopicroside; peak 13 is the chromatographic peak of paeoniflorin; peak 16 is the chromatographic peak of baicalin; peak 17 is the chromatographic peak of salvianolic acid B; peak 23 is the chromatographic peak of baicalein; peak 25 is the chromatographic peak of glycyrrhizic acid; and peak 26 is the chromatographic peak of wogonin.

[0081] In a specific example, the dosage form of the traditional Chinese medicine preparation for treating eczema is granules.

[0082] In a specific example, the preparation of a traditional Chinese medicine preparation for treating eczema includes the following steps:

[0083] Add appropriate amount of water to 12 ingredients: Rehmannia root 10g, Gypsum 15g, Scutellaria baicalensis 10g, Gentiana 3g, Sanguisorba officinalis 10g, Prunus mume 5g, White Peony 10g, Gardenia 3g, Stir-fried Atractylodes macrocephala 10g, Siegesbeckia 10g, Salvia miltiorrhiza 10g, and Licorice 5g. Boil the above 12 ingredients twice for 1 hour each time. Filter the decoction, concentrate the filtrate to a relative density of about 1.2, spray-dry it into dry extract powder, add appropriate amount of dextrin, mix well, make into granules, and dry.

[0084] The present invention also provides a method for detecting a Chinese medicine preparation for treating eczema, comprising the following steps:

[0085] Mixing the test substance with an organic solvent to prepare a test solution; the organic solvent is selected from one or more of methanol aqueous solution, ethanol aqueous solution and anhydrous ethanol;

[0086] The solution of the test product is subjected to high performance liquid chromatography;

[0087] The conditions adopted in the high performance liquid chromatography include: mobile phase A is selected from one or more of methanol and acetonitrile, and mobile phase B is selected from one or more of aqueous phosphoric acid solution, aqueous acetic acid solution and aqueous formic acid solution; a gradient elution program is adopted; the gradient elution program includes: from 0 min to 5 min, the volume percentage of mobile phase A changes from 5% to 12%; from 5 min to 30 min, the volume percentage of mobile phase A changes from 12% to 14%; from 30 min to 32 min, the volume percentage of mobile phase A changes from 14% to 16%; from 32 min to 40 min, the volume percentage of mobile phase A changes from 16% to 18%; from 40 min to 65 min, the volume percentage of mobile phase A changes from 18% to 35%; from 65 min to 75 min, the volume percentage of mobile phase A changes from 35% to 50%; from 75 min to 80 min, the volume percentage of mobile phase A changes from 50% to 5%; the detection wavelength is 220 nm to 260 nm.

[0088] In a specific example, the conditions used in high performance liquid chromatography also include: a flow rate of 0.8 mL / min to 1.2 mL / min; a column temperature of 20° C. to 35° C.; and an octadecylsilane bonded silica gel column.

[0089] In a specific example, the dimensions of the chromatographic column used in high performance liquid chromatography include: a column length of 240 mm to 260 mm, an inner diameter of 4 mm to 5 mm, and a particle size of 4.5 μm to 5.5 μm.

[0090] In a specific example, preparing the test solution includes the following steps:

[0091] After mixing the analyte with the organic solvent, ultrasonic extraction or heating reflux extraction is performed, and then filtration is performed to obtain the filtrate.

[0092] The following is a further detailed description of the fingerprint construction method of the Chinese medicine preparation for treating eczema of the present invention in conjunction with specific examples. The raw materials used in the following examples, unless otherwise specified, are all commercially available products.

[0093] Example 1

[0094] This embodiment provides a method for constructing a fingerprint of a traditional Chinese medicine preparation for treating eczema, as follows:

[0095] 1. Instruments and Materials

[0096] 1.1 Instrument

[0097] High-performance liquid chromatography (HPLC) instrument (Waters e2695), Empower chromatography workstation; XSE205DU electronic analytical balance (Mettler-Toledo); Milli-Q Reference ultrapure water instrument (Merck Chemical Technology (Shanghai) Co., Ltd.); KQ-500D ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0098] 1.2 Materials

[0099] 1.2.1 Reagents

[0100] Acetonitrile (chromatographic grade, Honeywell), phosphoric acid (analytical grade, Shanghai MacLean Biochemical Technology Co., Ltd.), methanol (analytical grade, Fuchen (Tianjin) Chemical Reagent Co., Ltd.).

[0101] 1.2.2 Reference substances

[0102] Gallic acid (batch number: 110831-20201906, purity: 91.5%),

[0103] Gardenia jasminoides (Batch No.: 110831-20201906, Purity: 91.5%),

[0104] Gentianopicroside (batch number: 110770-201918, purity: 97.1%),

[0105] Paeoniflorin (batch number: 110736-202145, purity: 100%),

[0106] Ammonium glycyrrhizate (batch number: 110731-202122, purity: 100%),

[0107] Salvianolic acid B (111562-201917, purity: 96.6%);

[0108] Baicalin (110715-202122, purity: 94.2%),

[0109] Baicalein (111595-201808, purity: 97.9%),

[0110] Wogonin (111514-201709, purity: 100%),

[0111] All were purchased from China Food and Drug Inspection Institute.

[0112] 1.2.3 Test samples

[0113] Different batches of granules for treating eczema are all produced by Beijing Silian Pharmaceutical Co., Ltd.

[0114] 2 Methods and Results

[0115] 2.1 Chromatographic conditions

[0116] Octadecylsilane-bonded silica gel was used as the packing (Ultimate AQ-C18 column, dimensions 250 mm × 4.6 mm, 5 μm); acetonitrile was used as mobile phase A, and 0.1% aqueous phosphoric acid was used as mobile phase B. Gradient elution was performed; the detection wavelength was 238 nm; the flow rate was 1.0 ml / min; and the column temperature was 25°C. The gradient elution program is shown in Table 1.

[0117] Table 1 Mobile phase elution program

[0118]

[0119] 2.2 Preparation of mixed reference solution

[0120] Take appropriate amount of gallic acid, gentiopicroside, gardeniaside, paeoniflorin, ammonium glycyrrhizate, salvianolic acid B, baicalin, baicalein, and wogonin reference substances, weigh accurately, add 50% methanol to dissolve and dilute to a concentration of about 50 μg / ml reference substance solution.

[0121] 2.3 Preparation of test solution

[0122] Take 10 batches of granules of this product, grind them into powder, weigh 1g of each, accurately add 25ml of 50% methanol, weigh, and ultrasonically treat (power 500W, frequency 40kHz) for 30 minutes. Cool, make up the lost weight with 50% methanol, shake well, and filter to obtain 10 batches of test solution.

[0123] 2.4 Establishment of fingerprint

[0124] Accurately pipette 10 μL of the reference solution and the test solution respectively, inject them into the HPLC instrument, and measure them according to the chromatographic conditions under the above “2.1” to obtain the fingerprints of different batches of samples, such as Figure 1The fingerprints of 10 batches of samples were imported into the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition). After multi-point correction and data matching, the average value method was used to generate the superposition of the HPLC fingerprints of the granules, as shown in Figure 2. Figure 2 As shown, and the reference fingerprint (R) and determine the common peaks, such as Figure 3 The fingerprints of 10 batches of samples were compared with the control fingerprints, and the similarities were all greater than 0.90, as shown in Table 2.

[0125] Table 2 Similarity between the fingerprints of 10 batches of granules for treating eczema and the control fingerprint (R)

[0126]

[0127]

[0128] 2.5 Determination and Identification of Common Peaks A correlation study was conducted between the granules and the individual medicinal ingredients in the formula. The peak retention times and UV absorption spectra of the test sample chromatograms were compared with those of the individual medicinal ingredients and the reference sample chromatograms. The common peaks in the granules' fingerprints were assigned and identified. Furthermore, the peak retention times and UV absorption spectra of some components were identified by comparison with those of the reference sample. The results are shown in Table 3.

[0129] The fingerprints of 10 batches of the granules determined 26 common peaks and identified the components of 9 chromatographic peaks as follows: Peak 3 is gallic acid, Peak 10 is gardeniaside, Peak 11 is gentiopicroside, Peak 13 is paeoniflorin, Peak 16 is baicalin, Peak 17 is salvianolic acid B, Peak 23 is baicalein, Peak 25 is glycyrrhizic acid, and Peak 26 is wogonin.

[0130] The 26 common peaks are attributed as follows: Peak 1 comes from Rehmannia glutinosa, Peak 2 comes from Siegesbeckia scoparia, Peak 4 comes from Prunus mume, Peaks 5 and 11 come from Gentiana scabra, Peaks 3, 6, 8, 9, 14 and 24 come from Sanguisorba officinalis, Peaks 7 and 10 come from Gardenia jasminoides and stir-fried Atractylodes macrocephala, Peaks 3, 12 and 13 come from Paeonia lactiflora, Peaks 15 and 22 come from stir-fried Atractylodes macrocephala, Peaks 16, 18, 19, 20, 21, 23 and 26 come from Scutellaria baicalensis, Peak 17 comes from Salvia miltiorrhiza, and Peak 25 comes from Glycyrrhiza uralensis. Both Sanguisorba officinalis and White Peony Root contain gallic acid, so Peak 3 (gallic acid peak) is a chromatographic peak shared by Sanguisorba officinalis and White Peony Root.

[0131] Table 3 Common peak identification and medicinal flavor attribution results of fingerprints of traditional Chinese medicine preparations

[0132]

[0133] 3 Methodological Investigation

[0134] 3.1 Precision

[0135] Take the granules of this product, grind them into powder, prepare the test solution according to the method in "2.3", and inject 10μl of the sample six times continuously under the chromatographic conditions in "2.1". Record the chromatogram. Figure 4 The RSDs of retention time and peak area of ​​each common peak were 0.1% to 0.3% and 1.6% to 2.9%, respectively, indicating good precision.

[0136] 3.2 Repeatability

[0137] Take the granules of this product, grind them into powder, weigh 6 portions in parallel, prepare 6 portions of test solution according to the method under "2.3", inject them respectively under the chromatographic conditions under "2.1", and record the chromatogram, such as Figure 5 Taking baicalin peak as reference peak, the RSDs of relative retention time and relative peak area of ​​each common peak were 0.1% to 0.3% and 0.1% to 3.0%, respectively, indicating that the established fingerprint detection method has good repeatability.

[0138] 3.3 Stability

[0139] Take the granules of this product and prepare the test solution according to the method under "2.3". Under the chromatographic conditions under "2.1", inject the sample at 0h, 2h, 4h, 8h, 12h, and 24h respectively, and record the chromatogram, such as Figure 6 The RSDs of the retention time and peak area of ​​the common peaks were 0.1% to 0.3% and 0.8% to 2.8%, respectively, indicating that the test solution was stable within 24 hours.

[0140] 3.4 Durability

[0141] Take the granules of this product, grind them into powder, and prepare the test solution according to the method under "2.3". Based on the chromatographic conditions under "2.1", change one condition (column temperature, flow rate, detection wavelength, different types of chromatographic columns) within a certain range. Import the obtained chromatogram into the traditional Chinese medicine fingerprint similarity evaluation software for similarity comparison. Using baicalin as the reference peak, calculate the relative retention time and relative peak area of ​​the common peak to investigate the durability of this method.

[0142] 3.4.1 Column temperature durability investigation

[0143] Prepare the test solution according to the method under "2.3". Based on the chromatographic conditions under "2.1", examine the effect of column temperature 25±5℃ on the test results and record the chromatogram, such as Figure 7The results showed that the chromatograms at column temperatures of 20℃, 25℃, and 30℃ were compared with the reference fingerprint, and the similarities were ≥0.999. The relative retention times and relative peak areas of the common peaks were 0.1%-0.4% and 0.6%-2.7%, respectively, indicating that the established fingerprint detection method had good durability at a column temperature of 25±5℃.

[0144] 3.4.2 Durability investigation of flow rate

[0145] Prepare the test solution according to the method under "2.3". Based on the chromatographic conditions under "2.1", examine the effect of a flow rate of 1.0±0.2ml / min on the test results and record the chromatogram, such as Figure 8 The results showed that the chromatograms at a flow rate of 1.0±0.2ml / min were compared with the reference fingerprints, and the similarities were ≥0.998. The relative retention time and relative peak area of ​​the common peaks were 0.1%-0.3% and 1.2%-2.3%, respectively, indicating that the established fingerprint detection method had good durability at a flow rate of 1.0±0.2ml / min.

[0146] 3.4.3 Durability of detection wavelength

[0147] Prepare the test solution according to the method under "2.3". Based on the chromatographic conditions under "2.1", examine the effect of the detection wavelength fluctuation at 238nm±2nm on the test results and record the chromatogram, such as Figure 9 The results show that the chromatogram similarity is ≥0.999 when the wavelength is 238nm±2nm, and the relative retention time and relative peak area of ​​the common peaks are 0.1%-0.3% and 0.8%-2.8%, respectively, indicating that the fluctuation of the detection wavelength within the range of 238nm±2nm has little effect on the detection results.

[0148] 4 Comparative analysis of extraction conditions

[0149] 4.1 Extraction method

[0150] Take the granules of this product, grind them into powder, accurately weigh 1g, weigh and accurately add 25ml of 50% methanol, weigh, ultrasonically treat (power 500W, frequency 40kHz), heat and reflux for 60 minutes, let cool, add 50% methanol to make up the loss, filter, and take the filtrate. Accurately take 10μl of the test solution respectively, inject it into the liquid chromatograph for detection, and record the chromatogram, such as Figure 10 shown.

[0151] The results showed that the chromatographic similarity of the sample solution prepared by ultrasonication and reflux was 0.999, and there was no significant difference in the number of chromatographic peaks or peak areas. Therefore, either ultrasonication or reflux extraction can be used as the extraction method, but ultrasonication is preferred for its ease of operation.

[0152] 4.2 Types of extraction solvents

[0153] Take the granules of this product, grind them into powder, accurately weigh 1g, accurately add 30% methanol, 50% methanol, 90% methanol, 30% ethanol, 50% ethanol, and 25ml of ethanol respectively, weigh, and treat with ultrasound (power 500W, frequency 40KHz) for 30 minutes, let cool, make up the weight loss with the corresponding extraction solvent, filter, and take the filtrate to obtain the test solution prepared with different concentrations of methanol and ethanol. Accurately draw 10μl of each test solution, inject it into the liquid chromatograph for detection, and record the chromatogram, such as Figure 11 shown.

[0154] The results showed that the chromatogram similarity of the test solution extracted by the six solvents was above 0.998, and the chromatographic peak shape and symmetry of the test solution extracted by methanol solution of different concentrations were better. Considering the saving of organic solvents and the fact that the test solution extracted by 50% methanol is easier to filter than that extracted by 30% methanol, 50% methanol was selected as the extraction solvent.

[0155] 4.3 Extraction solvent volume

[0156] Take the granules of this product, grind them into powder, accurately weigh 1g, accurately add 25ml, 50ml and 100ml of 50% methanol, weigh, treat with ultrasound (power 500W, frequency 40KHz) for 30 minutes, let cool, make up the weight loss with 50% methanol, filter, and take the filtrate to obtain the test solution prepared with different concentrations of methanol and ethanol. Accurately take 10μl of each test solution, inject it into the liquid chromatograph for detection, and record the chromatogram, such as Figure 12 shown.

[0157] The results showed that the three solvent volumes had little effect on the number of chromatographic peaks of the test sample, but when the solvent volume was 50 ml and 100 ml, the peak area of ​​the chromatographic peak was smaller, and when the solvent volume was 25 ml, the peak area of ​​the chromatographic peak was more moderate, so the extraction solvent volume was preferably 25 ml.

[0158] 4.4 Extraction time

[0159] Take the granules of this product, grind them into powder, accurately weigh 1g, accurately add 25ml of 50% methanol, weigh, and treat with ultrasound (power 500W, frequency 40KHz) for 15, 30, 45, and 60 minutes. Let it cool, make up the weight loss with 50% methanol, filter, and take the filtrate to obtain the test solution prepared with different concentrations of methanol and ethanol. Accurately draw 10μl of each test solution, inject it into the liquid chromatograph for detection, and record the chromatogram, such as Figure 13 shown.

[0160] The results showed that the similarity of the chromatograms of the test sample after ultrasonic treatment for 15, 30, 45, and 60 minutes was greater than 0.999. To ensure that the active ingredients of the drug can be fully extracted, the extraction time is set to be more than 15 minutes, such as 15 to 60 minutes, preferably 30 to 45 minutes. Considering the convenience of operation, the extraction time is preferably selected to be 30 minutes.

[0161] In summary, the preferred conditions for preparing the test solution are:

[0162] Take the granules of this product, grind them into powder, weigh 1g, accurately add 25ml of 50% methanol, weigh, ultrasonically treat (power 500W, frequency 40kHz) for 30 minutes, let cool, make up the lost weight with 50% methanol, shake well, filter, and obtain.

[0163] 5 Comparative analysis of mobile phases

[0164] Preparation of test solution: Take the granules of this product, grind them into powder, weigh 1g, accurately add 25ml of 50% methanol, weigh, ultrasonically treat (power 500W, frequency 40kHz) for 30 minutes, cool, make up the loss with 50% methanol, shake well, filter, and obtain.

[0165] 5.1 Investigation of the Methanol-0.1% Phosphoric Acid Mobile Phase System

[0166] Chromatographic conditions: The chromatographic column was Ultimate AQ-C18, 250 mm × 4.6 mm, 5 μm; methanol was used as mobile phase A, and 0.1% phosphoric acid aqueous solution was used as mobile phase B, with gradient elution; the detection wavelength was 238 nm; the flow rate was 1.0 ml / min; and the column temperature was 25°C. The gradient elution program is shown in Table 4. The resulting chromatogram is shown in Figure 14 shown.

[0167] Table 4 Gradient elution program

[0168]

[0169] The main components of the test sample can be reflected in the methanol-0.1% phosphoric acid aqueous solution mobile phase system, and the chromatographic peak separation is good. However, the analysis time required when using methanol-0.1% phosphoric acid aqueous solution as the mobile phase is long and the consumption of organic phase methanol is large. Therefore, acetonitrile-0.1% phosphoric acid aqueous solution is preferred as the mobile phase system.

[0170] 5.2 Investigation of different acid types in mobile phase B

[0171] Chromatographic conditions: The chromatographic column was Ultimate AQ-C18, 250 mm × 4.6 mm, 5 μm; acetonitrile was used as mobile phase A, and gradient elution was performed with mobile phases B consisting of 0.1% aqueous phosphoric acid, 0.1% aqueous formic acid, and 0.1% aqueous glacial acetic acid (gradient elution program as in Table 1). The differences in mobile phase B compositions with different acid types were compared, and the resulting chromatograms were as follows: Figure 15 The results showed that the similarity of the chromatograms obtained by the mobile phase systems consisting of acetonitrile-0.1% phosphoric acid aqueous solution, acetonitrile-0.1% formic acid aqueous solution, and acetonitrile-0.1% glacial acetic acid aqueous solution was greater than 0.98. The chromatographic peak shape of the chromatogram of the acetonitrile-0.1% phosphoric acid aqueous solution system was better, so acetonitrile-0.1% phosphoric acid aqueous solution was preferred as the mobile phase system.

[0172] 5.3 Investigation of mobile phase elution procedure

[0173] Chromatographic conditions: The chromatographic column was Ultimate AQ-C18, 250 mm × 4.6 mm, 5 μm; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid aqueous solution was used as mobile phase B, with gradient elution; the detection wavelength was 238 nm; the flow rate was 1.0 ml / min; and the column temperature was 25°C. The gradient elution program is shown in Tables 1 and 5, respectively, and the results are shown in Tables 1 and 5, respectively. Figure 16 and Figure 17 shown.

[0174] Table 5 Mobile phase elution program

[0175]

[0176] Depend on Figure 17 As shown in Table 5, some of the main chromatographic peaks in the spectrum obtained under the gradient elution program overlap and are not separated, and the chromatographic peak separation is poor. The chromatographic peaks of the main components in the spectrum under the gradient elution program in Table 1 have good separation, so the gradient elution program in Table 1 is preferred.

[0177] Example 2

[0178] This embodiment provides a method for detecting a Chinese medicine preparation for treating eczema, which is as follows:

[0179] 1. Preparation of test solution

[0180] Take the sample to be tested, grind it into powder, weigh 1g respectively, accurately add 25ml of 50% methanol, weigh it, and ultrasonically treat it (power 500W, frequency 40kHz) for 30 minutes. Let it cool, make up the lost weight with 50% methanol, shake it well, filter it, and you are done.

[0181] 2. Detection

[0182] The test sample solution was accurately drawn and injected into a high performance liquid chromatograph for determination. The conditions of the high performance liquid chromatograph were the same as those under "2.1" in Example 1.

[0183] 26 characteristic peaks can be detected in all the samples to be tested, among which 9 peaks including gallic acid, gardeniaside, gentiopicroside, paeoniflorin, baicalin, salvianolic acid B, baicalein, glycyrrhizic acid and wogonin correspond to the retention times of the corresponding reference peaks. It can be considered that the quality of the samples to be tested is stable and meets the quality requirements.

[0184] Comparative Example 1

[0185] This comparative example provides a method for attempting to construct a fingerprint of a traditional Chinese medicine preparation for treating eczema. The steps are roughly the same as those in Example 1, except that the extraction solvent of the test sample is changed in Comparative Example 1, as follows:

[0186] Preparation of test solution: Take the granules of this product, grind them into powder, weigh 1g, add 25ml of water, ultrasonically treat (power 500W, frequency 40kHz) for 20 minutes, filter, and extract the filtrate twice with ethyl acetate by shaking, 25ml each time, combine the ethyl acetate, evaporate to dryness, dissolve the residue in methanol and transfer it to a 25ml volumetric flask, dilute to the scale, shake well, filter, and take the filtrate.

[0187] Chromatographic conditions: Octadecylsilane bonded silica gel as the packing (Ultimate AQ-C18 column, dimensions 250 mm × 4.6 mm, 5 μm); acetonitrile as mobile phase A, 0.1% aqueous phosphoric acid as mobile phase B, gradient elution; detection wavelength at 238 nm; flow rate at 1.0 ml / min; column temperature at 25°C. The gradient elution program was consistent with that shown in Table 1.

[0188] The fingerprint spectrum obtained in this comparative example is as follows Figure 18 It can be seen that the number of chromatographic peaks in the spectrum obtained in this comparative example is significantly reduced compared to that in Example 1, indicating that most components were not extracted by ethyl acetate.

[0189] Comparative Example 2

[0190] This comparative example provides a method for attempting to construct a fingerprint of a traditional Chinese medicine preparation for treating eczema. The steps are roughly the same as those in Example 1, except that the type of mobile phase is changed in Comparative Example 2, as follows:

[0191] Preparation of test solution: Take the granules of this product, grind them into powder, weigh 1g, accurately add 25ml of 50% methanol, weigh, ultrasonically treat (power 500W, frequency 40kHz) for 30 minutes, cool, make up the loss with 50% methanol, shake well, filter, and obtain.

[0192] Chromatographic conditions: Octadecylsilane bonded silica gel as the packing (Ultimate AQ-C18 column, dimensions 250 mm × 4.6 mm, 5 μm); acetonitrile as mobile phase A, water as mobile phase B, gradient elution; detection wavelength at 238 nm; flow rate at 1.0 ml / min; column temperature at 25°C. The gradient elution program was consistent with that shown in Table 1.

[0193] The fingerprint spectrum obtained in this comparative example is as follows Figure 19 It can be seen that some of the main components in the spectrum obtained in this comparative example did not show peaks under the acetonitrile-water mobile phase system, and the chromatographic peak information obtained by eluting with the acetonitrile-water mobile phase system was incomplete.

[0194] Comparative Example 3

[0195] This comparative example provides a method for attempting to construct a fingerprint of a traditional Chinese medicine preparation for treating eczema. The steps are roughly the same as those in Example 1, except that the detection wavelength is changed in Comparative Example 4, as follows:

[0196] Preparation of test solution: Take the granules of this product, grind them into powder, weigh 1g, accurately add 25ml of 50% methanol, weigh, ultrasonically treat (power 500W, frequency 40kHz) for 30 minutes, cool, make up the loss with 50% methanol, shake well, filter, and obtain.

[0197] Chromatographic conditions: Octadecylsilane bonded silica gel as the packing (Ultimate AQ-C18 column, dimensions 250 mm × 4.6 mm, 5 μm); acetonitrile as mobile phase A, 0.1% aqueous phosphoric acid as mobile phase B, gradient elution; detection wavelengths at 210 nm and 275 nm, respectively; flow rate at 1.0 ml / min; column temperature at 25°C. The gradient elution program was consistent with that shown in Table 1.

[0198] The fingerprint spectrum with a detection wavelength of 210nm is as follows Figure 20 As shown, the fingerprint spectrum with a detection wavelength of 275nm is as follows Figure 21 As shown, it can be seen that the spectrum obtained in this comparative example is compared with that in Example 1. The chromatogram with a detection wavelength of 238 nm contains more comprehensive chromatographic peak information, and there are fewer absorption peaks at detection wavelengths of 210 nm and 275 nm, or the absorption peak response values ​​are lower and the peak areas are smaller.

[0199] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0200] The embodiments described above only express several implementation methods of the present invention, which are convenient for understanding the technical solutions of the present invention in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, several variations and improvements can be made, which all fall within the scope of protection of the present invention. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the scope of protection of the claims attached to the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A method for constructing a fingerprint of a traditional Chinese medicine preparation for treating eczema, wherein the traditional Chinese medicine preparation for treating eczema is a compound traditional Chinese medicine preparation consisting of Rehmannia root, Gypsum Fibrosum, Scutellaria baicalensis, Gentiana scabra, Sanguisorba officinalis, Prunus mume, White Peony Root, Gardenia jasminoides, Rhizoma Atractylodis Macrocephalae, Herba Siegesbeckiae, Salvia miltiorrhizae and Radix Glycyrrhizae, characterized in that: The steps include: Prepare a mixed reference solution by taking gallic acid, gentiopicroside, geniposide, paeoniflorin, ammonium glycyrrhizate, salvianolic acid B, baicalin, baicalein and wogonin reference substances; The traditional Chinese medicine preparation for treating eczema is mixed with an organic solvent to prepare a test solution; the organic solvent is selected from methanol aqueous solution, ethanol aqueous solution or anhydrous ethanol; The mixed reference solution and the test solution are respectively subjected to high performance liquid chromatography determination; Wherein, the conditions adopted by the high performance liquid chromatography include: mobile phase A is selected from methanol or acetonitrile, and mobile phase B is selected from aqueous phosphoric acid, aqueous acetic acid or aqueous formic acid; a gradient elution program is adopted; the gradient elution program includes: from 0 min to 5 min, the volume percentage of mobile phase A changes from 5% to 12%; from 5 min to 30 min, the volume percentage of mobile phase A changes from 12% to 14%; from 30 min to 32 min, the volume percentage of mobile phase A changes from 14% to 16%; from 32 min to 40 min, the volume percentage of mobile phase A changes from 16% to 18%; from 40 min to 65 min, the volume percentage of mobile phase A changes from 18% to 35%; from 65 min to 75 min, the volume percentage of mobile phase A changes from 35% to 50%; from 75 min to 80 min, the volume percentage of mobile phase A changes from 50% to 5%; the detection wavelength is 236 nm to 240 nm; the chromatographic column is an octadecylsilane bonded silica column.

2. The method for constructing a fingerprint of a Chinese medicine preparation for treating eczema according to claim 1, wherein: The conditions adopted by the high performance liquid chromatography also include: a flow rate of 0.8 mL / min to 1.2 mL / min; and a column temperature of 20° C. to 35° C.

3. The method for constructing a fingerprint of a Chinese medicine preparation for treating eczema according to claim 1, characterized in that: The dimensions of the chromatographic column used in the high performance liquid chromatography include: a column length of 240 mm to 260 mm, an inner diameter of 4 mm to 5 mm, and a particle size of 4.5 μm to 5.5 μm.

4. The method for constructing a fingerprint of a Chinese medicine preparation for treating eczema according to claim 1, wherein: The preparation of the mixed reference solution includes the following steps: The gallic acid, gentiopicroside, geniposide, paeoniflorin, ammonium glycyrrhizate, salvianolic acid B, baicalin, baicalein, and wogonin reference substances were mixed with a methanol aqueous solution.

5. The method for constructing a fingerprint of a Chinese medicine preparation for treating eczema according to claim 1, wherein: The preparation of the test solution includes the following steps: The traditional Chinese medicine preparation for treating eczema is mixed with the organic solvent, and then ultrasonic extraction or heating reflux extraction is performed, followed by filtration to obtain a filtrate.

6. The method for constructing a fingerprint of a traditional Chinese medicine preparation for treating eczema according to any one of claims 1 to 5, characterized in that: The number of common peaks in the fingerprint of the Chinese herbal preparation for treating eczema is 26; Among them, peak 3 is the chromatographic peak of gallic acid; peak 10 is the chromatographic peak of gardeniaside; peak 11 is the chromatographic peak of gentiopicroside; peak 13 is the chromatographic peak of paeoniflorin; peak 16 is the chromatographic peak of baicalin; peak 17 is the chromatographic peak of salvianolic acid B; peak 23 is the chromatographic peak of baicalein; peak 25 is the chromatographic peak of glycyrrhizic acid; and peak 26 is the chromatographic peak of wogonin.

7. A method for detecting a Chinese medicine preparation for treating eczema, characterized in that: The Chinese medicine preparation for treating eczema is a compound Chinese medicine preparation composed of Rehmannia root, Gypsum, Scutellaria baicalensis, Gentiana scabra, Sanguisorba officinalis, Prunus mume, White Peony Root, Gardenia jasminoides, Rhizoma Atractylodis Macrocephalae, Herba Siegesbeckiae, Salvia miltiorrhizae and Licorice root, and the detection method comprises the following steps: Prepare a mixed reference solution by taking gallic acid, gentiopicroside, geniposide, paeoniflorin, ammonium glycyrrhizate, salvianolic acid B, baicalin, baicalein and wogonin reference substances; Mixing the test substance with an organic solvent to prepare a test solution; the organic solvent is selected from methanol aqueous solution, ethanol aqueous solution or anhydrous ethanol; Performing high performance liquid chromatography on the mixed reference solution and the test solution respectively; Wherein, the conditions adopted by the high performance liquid chromatography include: mobile phase A is selected from methanol or acetonitrile, and mobile phase B is selected from aqueous phosphoric acid, aqueous acetic acid or aqueous formic acid; a gradient elution program is adopted; the gradient elution program includes: from 0 min to 5 min, the volume percentage of mobile phase A changes from 5% to 12%; from 5 min to 30 min, the volume percentage of mobile phase A changes from 12% to 14%; from 30 min to 32 min, the volume percentage of mobile phase A changes from 14% to 16%; from 32 min to 40 min, the volume percentage of mobile phase A changes from 16% to 18%; from 40 min to 65 min, the volume percentage of mobile phase A changes from 18% to 35%; from 65 min to 75 min, the volume percentage of mobile phase A changes from 35% to 50%; from 75 min to 80 min, the volume percentage of mobile phase A changes from 50% to 5%; the detection wavelength is 236 nm to 240 nm; the chromatographic column is an octadecylsilane bonded silica column.

8. The method for detecting a Chinese medicinal preparation for treating eczema according to claim 7, wherein: The conditions adopted by the high performance liquid chromatography also include: a flow rate of 0.8 mL / min to 1.2 mL / min; and a column temperature of 20° C. to 35° C.

9. The method for detecting a Chinese medicinal preparation for treating eczema according to claim 7, wherein: The dimensions of the chromatographic column used in the high performance liquid chromatography include: a column length of 240 mm to 260 mm, an inner diameter of 4 mm to 5 mm, and a particle size of 4.5 μm to 5.5 μm.

10. The method for detecting a traditional Chinese medicine preparation for treating eczema according to any one of claims 7 to 9, characterized in that: The preparation of the test solution includes the following steps: The analyte is mixed with the organic solvent, and then ultrasonic extraction or heating reflux extraction is performed, followed by filtration to obtain a filtrate.

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