A method for constructing a chelidonium majus sample characteristic chromatogram and application thereof

Abstract

This invention provides a method for constructing and applying characteristic chromatograms of Chelidonium majus samples. The method employs ultra-high performance liquid chromatography (UHPLC) to establish characteristic chromatograms of Chelidonium majus formulation granules or its upstream raw materials. Specific steps include: preparation of the test sample solution, preparation of the reference herb solution, preparation of the reference standard solution, and acquisition of the characteristic chromatogram. The characteristic chromatogram construction method proposed in this invention fills a gap in the study of water-soluble components of Chelidonium majus. Furthermore, this method is simple to operate, has a short analysis cycle, good resolution of common peaks, and a stable baseline, providing a new analytical tool for the quality control of Chelidonium majus formulation granules.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine quality control, and relates to a quality evaluation method for Chelidonium majus formula granules, specifically a method for constructing UPLC characteristic maps of Chelidonium majus formula granules. Background Technology

[0002] Chelidonium majus L., a plant belonging to the genus Chelidonium in the Papaveraceae family, is a dried whole herb. It is also known as Dihuanglian, Niujinhua, Tuhuanglian, Babujin, Duanchangcao, and Xionghuangcao. It has a bitter taste, is cool in nature, and is poisonous. The whole herb is used medicinally for its analgesic, antitussive, diuretic, and detoxifying effects. It is mainly used to treat stomach pain, abdominal pain, enteritis, dysentery, chronic bronchitis, whooping cough, cough, jaundice, edema, ascites, scabies, boils, and snake and insect bites. Studies on the chemical composition of Chelidonium majus show that its main chemical components are alkaloids, and it also contains flavonoids, triterpenoid saponins, volatile oils, and vitamin C. Its main active ingredient is isoquinoline alkaloids, which have anti-inflammatory, antibacterial, antitussive, antiasthmatic, and analgesic effects.

[0003] Chelidonium majus is included in the 2020 edition of the Chinese Pharmacopoeia, which only includes identification items (physicochemical identification with reference to chelidonium majus reference material and chelidonine) and content determination items (HPLC content determination with reference to chelidonine) for the control of the quality of traditional Chinese medicine. There are no relevant standards for fingerprint chromatograms or characteristic chromatograms.

[0004] Chelidonium majus formula granules are made by decocting chelidonium majus medicinal materials in water. The efficacy and quality are consistent with the clinical decoction. Under the guidance of traditional Chinese medicine theory, they can be flexibly prepared by clinicians and are easy to use.

[0005] In the existing technology, although there is a lot of research on celandine, most of it focuses on the processing of medicinal materials, preparation processes of related components, dosage forms, etc., while there is a gap in the research on celandine formulation granules or water-soluble extracts (especially quality control and quality evaluation research).

[0006] Reference 1 uses UPLC-UV and QTOF-MS to establish characteristic spectra of 10 batches of Chelidonium majus medicinal materials. It identifies protopine, chelidonine, berberine, berberine, chelidonine red, sanguisorbin, and tetrahydroberberine using reference standards. However, this method requires a large amount of sample, has a complex extraction method, some common peaks have small peak areas, poor separation, and unstable baselines, which increases the difficulty of identification. Furthermore, it is only applicable to the medicinal material itself and cannot take into account other derived forms.

[0007] References:

[0008] Reference 1: Gu Yue, Qian Dawei, Duan Jin'ao, Wang Zhenzhong, Sun Guoling, Ni Shumao, Guo Jianming. Establishment of UPLC characteristic spectrum of Chelidonium majus and QTOF-MS analysis of alkaloid components [J]. Journal of Pharmaceutical Analysis, 2010, 30(05):780-786. Summary of the Invention

[0009] The problem the invention aims to solve

[0010] The 2020 edition of the Chinese Pharmacopoeia specifies that the content index of celandine in celandine is celandine red alkaloid. However, previous studies have found that this component cannot be detected in the standard decoction, extract and formulation granules obtained by decocting celandine slices in water using traditional methods. Therefore, this analytical method cannot be used for quality control of celandine formulation granules.

[0011] Based on the above problems, it is of great significance to establish a quality control method for Celandine Extract granules.

[0012] Solution for solving the problem

[0013] The present invention believes that the above-mentioned technical problems can be solved by implementing the following technical solutions:

[0014] [1] The present invention provides a method for constructing a feature map of Chelidonium majus samples, comprising the following steps:

[0015] (1) Preparation of test solution: Accurately weigh the Chelidonium majus sample, accurately add the extraction solvent and weigh, extract, pre-treat the sample extract by column chromatography to obtain the test solution;

[0016] (2) Preparation of reference medicinal material solution: Take white celandine reference medicinal material, add extraction solvent, extract, and pretreat the reference medicinal material extract by column chromatography to obtain reference medicinal material solution;

[0017] (3) Preparation of reference solution: At least two of the following are reference standards: magnoflorine, berberine hydrochloride, tetrahydroberberine, protopine, chelidonine, dihydrochelidonine and dihydrosanthemin. Reference solvent is added to each to obtain reference solution.

[0018] (4) Acquisition of characteristic chromatograms: The test solution, the reference medicinal material solution and the reference solution were detected by ultra-high performance liquid chromatography to obtain the characteristic chromatograms of the Chelidonium majus sample;

[0019] The celandine sample is a celandine formulation granule or a celandine medicinal material, decoction piece, standard decoction or water extract used to prepare the formulation granule.

[0020] [2] According to the method described in [1], the chromatographic conditions of the column chromatography include: using alumina as the stationary phase and using alcohol or a mixture of alcohol and haloalkanes as the eluent;

[0021] Preferably, the stationary phase is neutral alumina; and / or, the amount of the stationary phase is 4-8 g / g of celandine sample;

[0022] And / or,

[0023] Preferably, the eluent is methanol or a mixture of methanol and dichloromethane; and / or, the amount of the eluent is 20-80 mL / g of Chelidonium majus sample.

[0024] [3] According to the method described in [1] or [2], the chromatographic conditions of the ultra-high performance liquid chromatography method include: using octadecyl endolaminated amide group bonded silica gel as the stationary phase, using a binary system composed of nitriles and phosphate buffer as the mobile phase, using gradient elution as the elution method, and the detection wavelength is 250-300 nm.

[0025] Preferably, the chromatographic column using the stationary phase is an Acclaim PA2 column;

[0026] And / or,

[0027] Preferably, the nitrile is acetonitrile; and / or, the phosphate buffer solution is an aqueous solution of potassium dihydrogen phosphate with a pH of 2.5 to 4.5, preferably with a concentration of 0.005-0.01 mol / L.

[0028] [4] The method according to any one of [1] to [3], wherein at least one of berberine hydrochloride and dihydrosanthemine, as well as magnoflorine, tetrahydroberberine, hydroproopyrine, chelidonine and dihydrochelidonine, is used as a reference standard;

[0029] Preferably, magnoflorine, berberine hydrochloride, tetrahydroberberine, protopine, chelidonine, dihydrochelidonine, and dihydrosanthemin are used as reference standards.

[0030] [5] The method according to any one of [1] to [4], wherein the extraction solvent is water or a 10% to 100% v / v methanol aqueous solution.

[0031] [6] The method according to any one of [1] to [5], wherein the extraction is one of ultrasonic treatment, heating reflux or shaking extraction, preferably ultrasonic treatment.

[0032] [7] The method according to any one of [1] to [6], wherein the gradient elution procedure is as follows: 0 to 10 min, the volume fraction of mobile phase A increases from 15% to 20%, and the volume fraction of mobile phase B decreases from 85% to 80%; 10 to 18 min, the volume fraction of mobile phase A increases from 20% to 70%, and the volume fraction of mobile phase B decreases from 80% to 30%; 18 to 22 min, the volume fraction of mobile phase A is 70%, and the volume fraction of mobile phase B is 30%; 22 to 22.5 min, the volume fraction of mobile phase A decreases from 70% to 15%, and the volume fraction of mobile phase B increases from 30% to 85%; 22.5 to 27 min, the volume fraction of mobile phase A is 15%, and the volume fraction of mobile phase B is 85%.

[0033] [8] According to the method described in [3], the chromatographic conditions of the ultra-high performance liquid chromatography method further include: a flow rate of 0.2 to 0.4 mL / min and a column temperature of 15 to 50 °C.

[0034] [9] The method according to any one of [1] to [8], wherein the feature spectrum has at least ten feature peaks;

[0035] Preferably, peak 4, corresponding to the chromatographic peak of berberine hydrochloride reference standard, is designated as peak S1, and the relative retention times of peaks 1, 2, 3, and 5 with peak S1 are calculated; peak 10, corresponding to the chromatographic peak of dihydrosandricaline reference standard, is designated as peak S2, and the relative retention times of peaks 6 to 9 with peak S2 are calculated. The relative retention times are all within ±10% of the specified values, which are: 0.28 (peak 1), 0.72 (peak 2), 0.90 (peak 3), 1.14 (peak 5), 0.69 (peak 6), 0.71 (peak 7), 0.85 (peak 8), and 0.97 (peak 9).

[0036]

[10] The present invention also provides the use of the celandine sample feature map constructed according to any one of [1] to [9] in the quality detection of celandine samples.

[0037]

[11] A method for quality testing of Celandine Formula Granules, comprising the following steps: taking the sample to be tested as the test sample, constructing a feature spectrum according to any one of the methods described in [1] to [9], comparing the obtained feature spectrum with the feature spectrum constructed according to any one of the methods described in [1] to [9], and obtaining the quality testing result.

[0038] The effects of the invention

[0039] (1) This invention uses ultra-high performance liquid chromatography to establish characteristic chromatograms of celandine formulation granules or its upstream raw materials. This method is simple to operate and has low detection time and cost, providing a new analytical means for the quality control of celandine formulation granules.

[0040] (2) The feature map construction method proposed in this invention studies the components in the decoction of Chelidonium majus, filling the gap in the study of water-soluble components of Chelidonium majus.

[0041] (3) The characteristic chromatographic method proposed in this invention uses neutral alumina to purify and enrich alkaloid components; the mobile phase is a buffer salt and phosphoric acid is used to adjust to a suitable pH. This method abandons the traditional method of analyzing alkaloids (such as using ion-pairing reagents), which greatly ensures the reproducibility of the characteristic chromatographic method and reduces the consumption of chromatographic columns.

[0042] (4) The characteristic spectrum method of the present invention, with the reference material of Chelidonium majus, established a characteristic spectrum containing 10 characteristic peaks and identified 7 common peaks, including magnoflorine, proopioline, chelidonine, berberine hydrochloride, tetrahydroberberine, dihydrochelidonine and dihydrosandriline, accounting for 70%.

[0043] (5) The present invention uses UPLC to establish characteristic spectral methods, which has a short analysis cycle, good separation of common peaks, and stable baseline. At the same time, the characteristic spectral studies of medicinal materials, standard decoctions, extracts, and formulation granules all have corresponding characteristic peaks, which can better reflect the consistency between formulation granules and pharmaceutical intermediates. This provides a basis for tracing the characteristic components in white celandine formulation granules and upstream raw materials, and also provides a strong guarantee for quality control in the industrialization process. Attached Figure Description

[0044] Figure 1 UPLC plots based on wavelength: characteristic spectrum;

[0045] Figure 2 UPLC diagrams for pH testing - characteristic spectra;

[0046] Figure 3 UPLC plots for investigating buffer solution concentration - characteristic spectra;

[0047] Figure 4 UPLC diagrams and characteristic spectra of different amounts of neutral alumina;

[0048] Figure 5 UPLC chromatograms of different eluents - characteristic spectra;

[0049] Figure 6 UPLC plots with different elution volumes - characteristic spectra;

[0050] Figure 7 UPLC diagrams and characteristic spectra of different extraction solvents;

[0051] Figure 8Specificity test of the characteristics of Celandine granules (comparison between sample and blank);

[0052] Figure 9 Specificity test of the characteristics of Chelidonium majus formula granules (comparison of sample with control medicinal materials and control standards);

[0053] Figure 10 Chromatographic column analysis of the characteristic granules of Chelidonium majus formulation;

[0054] Figure 11 Column temperature study of the characteristic chromatogram of celandine formulation granules;

[0055] Figure 12 Flow rate study of the characteristic chromatogram of celandine formulation granules;

[0056] Figure 13 Overlay of characteristic images of multiple batches of Celandine (Hedychium erythrorhizon) medicinal materials;

[0057] Figure 14 Overlay of characteristic images of multiple batches of Celandine decoction pieces;

[0058] Figure 15 Overlay of characteristic spectra of multiple batches of freeze-dried powder of Celandine;

[0059] Figure 16 Superimposed image of particle characteristics from multiple batches of Celandine;

[0060] Figure 17 Overlay of characteristic maps of intermediates from multiple batches of Celandine;

[0061] Figure 18 The present invention provides a characteristic spectrum of Celandine sample. Detailed Implementation

[0062] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0063] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0064] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0065] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0066] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0067] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0068] In this instruction manual, when "room temperature" or "room temperature" is used, the temperature can be between 10-40℃.

[0069] First, this invention proposes a method for constructing a characteristic map of Chelidonium majus samples, comprising the following steps:

[0070] (1) Preparation of test solution: Accurately weigh the Chelidonium majus sample, accurately add the extraction solvent and weigh, extract, pre-treat the sample extract by column chromatography to obtain the test solution;

[0071] (2) Preparation of reference medicinal material solution: Take white celandine reference medicinal material, add extraction solvent, extract, and pretreat the reference medicinal material extract by column chromatography to obtain reference medicinal material solution;

[0072] (3) Preparation of reference solution: At least two of the following are reference standards: magnoflorine, berberine hydrochloride, tetrahydroberberine, protopine, chelidonine, dihydrochelidonine and dihydrosanthemin. Reference solvent is added to each to obtain reference solution.

[0073] (4) Acquisition of characteristic chromatograms: The test solution, the reference medicinal material solution and the reference solution were detected by ultra-high performance liquid chromatography to obtain the characteristic chromatograms of the Chelidonium majus sample;

[0074] The celandine sample is a celandine formulation granule or a celandine medicinal material, decoction piece, standard decoction or water extract used to prepare the formulation granule.

[0075] In some specific implementation schemes, the preparation method of the test solution may be as follows: take an appropriate amount of Chelidonium majus sample, grind it finely, weigh it accurately, add an extraction solvent accurately and weigh it, extract, cool, filter, add the filtrate to a chromatographic column pre-packed with a stationary phase, elute with an eluent, collect the eluent, evaporate to dryness, add a solvent to dissolve the residue, and obtain the test solution.

[0076] In some specific implementation schemes, the preparation method of the reference medicinal material solution can be as follows: weigh an appropriate amount of Chelidonium majus reference medicinal material, add an extraction solvent, extract, cool, filter, add the filtrate to a chromatographic column pre-packed with a stationary phase, elute with an eluent, collect the eluent, evaporate to dryness, and dissolve the residue with a solvent to obtain the reference medicinal material solution.

[0077] In some specific embodiments, the extraction solvent is water or a 10% to 100% v / v methanol aqueous solution, such as a 30% v / v methanol aqueous solution, a 50% v / v methanol aqueous solution, an 80% v / v methanol aqueous solution, or a 100% v / v methanol solution.

[0078] In some specific implementations, the extraction solvent is a 50% to 100% v / v methanol aqueous solution.

[0079] In some specific implementations, the extraction solvent is an 80% to 100% v / v methanol aqueous solution.

[0080] In some specific implementation schemes, in the preparation method of the test solution, the ratio of the extraction solvent to the Chelidonium majus reference material is 1mL:0.01-0.05g, for example, 1mL:0.01g, 1mL:0.02g, 1mL:0.025g, 1mL:0.03g, 1mL:0.04g, 1mL:0.05g, etc.

[0081] In some specific embodiments, in the preparation method of the control herbal solution, the ratio of the extraction solvent to the Chelidonium majus sample is 1 mL:0.01–0.05 g, for example, 1 mL:0.01 g, 1 mL:0.02 g, 1 mL:0.025 g, 1 mL:0.03 g, 1 mL:0.04 g, 1 mL:0.05 g, etc. In some specific embodiments, the extraction method is one of ultrasonic treatment, reflux heating, or shaking extraction.

[0082] In some specific implementations, the extraction method is ultrasonic processing.

[0083] In some specific implementations, the ultrasonic treatment time is 15 to 60 minutes.

[0084] In some specific embodiments, the chromatographic conditions of the column chromatography include: using alumina as the stationary phase and an alcohol or a mixture of an alcohol and a haloalkanes as the eluent.

[0085] In some specific implementations, the stationary phase is neutral alumina.

[0086] In some specific implementations, the amount of the stationary phase is 4 to 8 g / g of Celandine sample, such as 4 g / g Celandine sample, 6 g / g Celandine sample, or 8 g / g Celandine sample.

[0087] In some specific implementations, the eluent is methanol or a mixture of methanol and dichloromethane, such as methanol-dichloromethane (1:4), methanol-dichloromethane (3:7), methanol-dichloromethane (2:3), methanol-dichloromethane (1:1), methanol-dichloromethane (3:2), methanol-dichloromethane (7:3), methanol-dichloromethane (4:1), etc.

[0088] In some specific implementations, the amount of the eluent used is 20-80 mL / g of Celandine sample, such as 20 mL / g, 40 mL / g, 60 mL / g, or 80 mL / g.

[0089] In some specific implementations, the amount of the eluent used is 60-80 mL / g of Chelidonium majus sample.

[0090] In some specific implementations, the solvent is methanol or a mixture of methanol and dichloromethane, such as methanol-dichloromethane (1:4), methanol-dichloromethane (3:7), methanol-dichloromethane (2:3), methanol-dichloromethane (1:1), methanol-dichloromethane (3:2), methanol-dichloromethane (7:3), methanol-dichloromethane (4:1), etc. It should be noted that the eluent may be the same as or different from the solvent.

[0091] In some specific embodiments, the preparation method of the reference solution may involve using at least two of the following as reference standards: magnoflorine, berberine hydrochloride, tetrahydroberberine, protopine, chelidonine, dihydrochelidonine, and dihydrosanguisorbine. Appropriate amounts of each are taken and dissolved to obtain the reference solution. The concentration of each reference standard may be 40–60 μg / mL for magnoflorine, 40–60 μg / mL for tetrahydroberberine, 80–120 μg / mL for dihydrosanguisorbine, 40–60 μg / mL for berberine hydrochloride, 40–60 μg / mL for protopine, 40–60 μg / mL for chelidonine, and 40–60 μg / mL for dihydrochelidonine.

[0092] In some specific implementation schemes, the method for preparing the reference solution uses at least one of berberine hydrochloride, dihydrosantheminine, magnoflorine, tetrahydroberberine, protopine, chelidonine, and dihydrochelidonine as reference standards.

[0093] In some specific implementation schemes, the method for preparing the reference solution uses magnoflorine, berberine hydrochloride, tetrahydroberberine, protopine, chelidonine, dihydrochelidonine, and dihydrosanthemin as reference standards.

[0094] In some specific implementations, the method for obtaining the characteristic chromatogram can be to precisely pipette 1 μl each of the test sample solution, the reference medicinal material solution, and the reference solution, inject them into a liquid chromatograph, and detect them using ultra-high performance liquid chromatography to obtain the characteristic chromatogram of the celandine sample.

[0095] In some specific implementations, the chromatographic conditions of the ultra-high performance liquid chromatography include: using octadecyl endolaminated amide-bonded silica gel as the stationary phase, using a binary system composed of nitriles and phosphate buffer as the mobile phase, using gradient elution as the elution method, and a detection wavelength of 250-300 nm.

[0096] In some specific implementations, the chromatographic column using the stationary phase is an Acclaim PA2 column.

[0097] In some specific implementations, the nitrile is acetonitrile.

[0098] In some specific implementations, the phosphate buffer solution is an aqueous solution of potassium dihydrogen phosphate with a pH of 2.5-4.5, such as an aqueous solution of potassium dihydrogen phosphate with a pH of 3.0, 3.3, 3.5, 4.0, 4.5, etc.

[0099] In some specific implementations, the phosphate buffer solution is an aqueous solution of potassium dihydrogen phosphate with a pH of 3.0 to 4.0.

[0100] In some specific implementations, the phosphate buffer solution is an aqueous solution of potassium dihydrogen phosphate with a pH of 3.0 to 3.5.

[0101] In some specific implementations, the concentration of the potassium dihydrogen phosphate aqueous solution is 0.005-0.01 mol / L, such as 0.005 mol / L, 0.006 mol / L, 0.007 mol / L, 0.008 mol / L, 0.009 mol / L, 0.01 mol / L, etc.

[0102] In some specific embodiments, the gradient elution includes the following procedure: 0–10 min, the volume fraction of mobile phase A increases from 15% to 20%, and the volume fraction of mobile phase B decreases from 85% to 80%; 10–18 min, the volume fraction of mobile phase A increases from 20% to 70%, and the volume fraction of mobile phase B decreases from 80% to 30%; 18–22 min, the volume fraction of mobile phase A is 70%, and the volume fraction of mobile phase B is 30%; 22–22.5 min, the volume fraction of mobile phase A decreases from 70% to 15%, and the volume fraction of mobile phase B increases from 30% to 85%; 22.5–27 min, the volume fraction of mobile phase A is 15%, and the volume fraction of mobile phase B is 85%.

[0103] In some specific implementations, the detection wavelength is 280 nm.

[0104] In some specific implementations, the chromatographic conditions of the ultra-high performance liquid chromatography method further include a flow rate of 0.2 to 0.4 mL / min, such as 0.2 mL / min, 0.25 mL / min, 0.3 mL / min, 0.35 mL / min, 0.4 mL / min, etc.

[0105] In some specific implementations, the flow rate is 0.3–0.4 mL / min.

[0106] In some specific implementations, the chromatographic conditions of the ultra-high performance liquid chromatography method also include: a column temperature of 15 to 50°C, such as 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, etc.

[0107] In some specific implementations, the column temperature is 15–25°C.

[0108] In some specific implementations, the feature map has at least ten, for example, ten feature peaks.

[0109] In some specific implementation schemes, peak 4, corresponding to the chromatographic peak of berberine hydrochloride reference standard, is designated as peak S1. The relative retention times of peaks 1, 2, 3, and 5 with peak S1 are calculated. Peak 10, corresponding to the chromatographic peak of dihydrosanthemin reference standard, is designated as peak S2. The relative retention times of peaks 6 to 9 with peak S2 are calculated. The relative retention times should all be within ±10% of the specified values, which are: 0.28 (peak 1), 0.72 (peak 2), 0.90 (peak 3), 1.14 (peak 5), 0.69 (peak 6), 0.71 (peak 7), 0.85 (peak 8), and 0.97 (peak 9).

[0110] Secondly, the present invention also provides the application of the celandine sample characteristic spectrum constructed according to the method of the present invention in the quality detection of celandine samples.

[0111] Finally, the present invention also provides a quality detection method for Celandine Extract granules, comprising the following steps: using the sample to be tested as the test sample, constructing a feature spectrum according to the method of the present invention, comparing the obtained feature spectrum with the feature spectrum constructed according to the method of the present invention, and obtaining the quality detection result.

[0112] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0113] Sample sources used in the examples:

[0114] Magnolia alkaloid reference standard and berberine hydrochloride reference standard were purchased from the National Institutes for Food and Drug Control, with batch numbers 112090-202201 and 112026-201802, respectively.

[0115] Tetrahydroberberine reference standard, proopioline reference standard, chelidonine reference standard, dihydrochelidonine reference standard, and dihydrosanthemin reference standard were purchased from Shanghai Shidande Standards Technical Service Co., Ltd., with batch numbers 8227, 4086, 13035, 10761, and 8232, respectively.

[0116] Chelidonium majus reference material was purchased from the National Institutes for Food and Drug Control, batch number 121143-201604.

[0117] Chelidonium majus formula granules (prepared by Jiangyin Tianjiang Pharmaceutical Co., Ltd. using water decoction process, batch numbers: KL-1, KL-2, KL-3).

[0118] Example 1: Investigation of Detection Wavelength

[0119] Preparation of test sample: Take an appropriate amount of Chelidonium majus formula granules, grind them finely, take about 0.5g, place them in a stoppered conical flask, add 20ml of methanol, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, take 15ml of the filtrate, add it to a neutral alumina column (100-200 mesh, 4g, inner diameter 10mm, dry packing), elute with 30ml of methanol, collect the eluent, evaporate to dryness, add 5ml of methanol to dissolve the residue, and use it as the test sample solution.

[0120] Measurement: Collect chromatograms of the sample at 210 nm, 280 nm and 360 nm.

[0121] The test results are attached. Figure 1As shown, the results indicate that at a wavelength of 280 nm, there are more chromatographic peaks, the response is moderate, and the baseline is relatively stable.

[0122] Example 2: Determination of Chromatographic Conditions – Investigation of pH

[0123] Preparation of test sample: Take an appropriate amount of Chelidonium majus formula granules, grind them finely, take about 0.5g, place them in a stoppered conical flask, add 20ml of methanol, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, take 15ml of the filtrate, add it to a neutral alumina column (100-200 mesh, 4g, inner diameter 10mm, dry packing), elute with 30ml of methanol, collect the eluent, evaporate to dryness, add 5ml of methanol to dissolve the residue, and use it as the test sample solution.

[0124] Determination: High-purity silica gel bonded with octadecyl endomemide groups was used as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 2.2 μm); acetonitrile was used as mobile phase A, and 0.005 mol / L potassium dihydrogen phosphate solution, 0.005 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.0 with phosphoric acid), 0.005 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.3 with phosphoric acid), and 0.005 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.5 with phosphoric acid) were used as mobile phase B, respectively, according to the specifications in the table below; the flow rate was 0.35 mL / min; the column temperature was 20℃; and the detection wavelength was 280 nm. The theoretical plate number, calculated based on the berberine hydrochloride peak, should not be less than 3000.

[0125] Table 1 Gradient elution program

[0126]

[0127] The characteristic spectra obtained from the measurements are attached. Figure 2 As shown, by appendix Figure 2 It can be seen that the peak shape and resolution of the chromatographic peaks obtained at different pH values ​​are different. Among them, the resolution and peak shape of each chromatographic peak are the best when pH = 3.3.

[0128] Example 3: Determination of Chromatographic Conditions – Investigation of Buffer Salt Concentration

[0129] Preparation of test sample: Take an appropriate amount of Chelidonium majus formula granules, grind them finely, take about 0.5g, place them in a stoppered conical flask, add 20ml of methanol, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, take 15ml of the filtrate, add it to a neutral alumina column (100-200 mesh, 4g, inner diameter 10mm, dry packing), elute with 30ml of methanol, collect the eluent, evaporate to dryness, add 5ml of methanol to dissolve the residue, and use it as the test sample solution.

[0130] The column was packed with octadecyl endomemide-bonded high-purity silica gel (100 mm column length, 2.1 mm inner diameter, 2.2 μm particle size); acetonitrile was used as mobile phase A, and 0.005 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.3 with phosphoric acid) and 0.01 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.3 with phosphoric acid) were used as mobile phase B, respectively, according to the specifications in Table 1; the flow rate was 0.35 mL / min; the column temperature was 20 °C; and the detection wavelength was 280 nm. The theoretical plate number, calculated based on the berberine hydrochloride peak, should be no less than 3000.

[0131] The characteristic spectra obtained from the measurements are attached. Figure 3 As shown, by appendix Figure 3 It can be seen that the peak shape and resolution of the chromatographic peaks obtained by different concentrations of buffer salts are different. It was found that the resolution and peak shape of each chromatographic peak are the best when the buffer concentration is 0.01 mol / L.

[0132] Example 4: Determination of Sample Pretreatment Method – Investigation of Neutral Alumina Dosage

[0133] Preparation of test samples: Take an appropriate amount of Chelidonium majus formula granules (batch number: KL-1), grind them finely, take about 0.5g, a total of 3 groups, place them in stoppered conical flasks, add 20ml of methanol to each, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, take 15ml of the filtrate respectively, add it to neutral alumina columns (100-200 mesh, inner diameter 10mm, dry packing) of different dosages (2g, 3g, 4g), elute with 30ml of methanol, collect the eluent, evaporate to dryness, add 5ml of methanol to dissolve the residue, and use it as the test sample solution.

[0134] Determination: Accurately pipette 1 μl of each test solution and inject it into the liquid chromatograph. Calculate the peak area of ​​the characteristic peak / sample weight. Chromatographic conditions: Use octadecyl endomemide-bonded high-purity silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 2.2 μm); use acetonitrile as mobile phase A and 0.01 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.3 with phosphoric acid) as mobile phase B, and perform gradient elution according to the specifications in Table 1; flow rate 0.35 mL / min; column temperature 20℃; detection wavelength 280 nm. The theoretical plate number, calculated based on the berberine hydrochloride peak, should be no less than 3000.

[0135] Table 2 Comparison of extraction efficiency with different amounts of neutral alumina (total peak area / sample weight)

[0136]

[0137] The characteristic spectra of the measurement results are attached. Figure 4 As shown in Table 2, the extraction efficiency is as follows: Figure 4As shown in Table 2, the number of chromatographic peaks obtained with different amounts of neutral alumina is consistent. Further comparison of the total peak area / sample weight of each component obtained with different amounts of alumina reveals that the extraction efficiency of each component is higher when the amount of alumina is 3g.

[0138] Example 5: Determination of Sample Pretreatment Method – Investigation of Eluents

[0139] (1) Investigation of eluent types

[0140] Preparation of test samples: Take an appropriate amount of Chelidonium majus formula granules (batch number: KL-1), grind them finely, take about 0.5g, a total of 4 groups, place them in stoppered conical flasks, add 20ml of methanol to each, and sonicate them separately (power 250W, frequency 40kHz) for 30 minutes, cool them, take 15ml of the filtrate, add it to a neutral alumina column (100-200 mesh, 4g, inner diameter 10mm, dry packing), and elute with 30ml of methanol, methanol-dichloromethane (1:1), methanol-dichloromethane (3:7), and methanol-dichloromethane (7:3), respectively. Collect the eluent, evaporate it to dryness, add 5ml of methanol to dissolve the residue, and use it as the test sample solution.

[0141] Determination: Accurately pipette 1 μl of each test solution and inject it into the liquid chromatograph. Determine the peak area of ​​the characteristic peak under the specified chromatographic conditions and calculate the ratio of peak area to sample weight. Chromatographic conditions: Use octadecyl endomemide-bonded high-purity silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 2.2 μm); use acetonitrile as mobile phase A and 0.01 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.3 with phosphoric acid) as mobile phase B, and perform gradient elution according to the specifications in Table 1; flow rate 0.35 mL / min; column temperature 20℃; detection wavelength 280 nm. The theoretical plate number, calculated based on the berberine hydrochloride peak, should be no less than 3000.

[0142] Table 3 Comparison of extraction efficiency for different elution volumes (peak area / sample weight)

[0143]

[0144] The characteristic spectra of the measurement results are attached. Figure 5 As shown in Table 3, the extraction efficiency is as follows. The results show that, based on the analysis of the total peak area / sample weight of each component, there is no significant difference in extraction efficiency among different elution solvents; however, when methanol is used as the elution solvent, the peak area of ​​peak 6 increases significantly. Therefore, in order to ensure complete elution, methanol is the preferred elution solvent.

[0145] (2) Investigation of eluent dosage

[0146] Preparation of test sample: Take an appropriate amount of Chelidonium majus formula granules (batch number: KL-1), grind them into a fine powder, take about 0.5g, make 3 groups, place them in stoppered conical flasks, add 20ml of methanol to each, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, take 15ml of the filtrate from each, add it to a neutral alumina column (100-200 mesh, 4g, inner diameter 10mm, dry packing), elute with 10ml, 20ml, 30ml and 40ml of methanol respectively, collect the eluent, evaporate to dryness, add 5ml of methanol to dissolve the residue, and use it as the test sample solution.

[0147] Determination: Accurately pipette 1 μl of each test solution and inject it into the liquid chromatograph. Determine the chromatographic conditions described above and calculate the peak area of ​​the characteristic peak / sample weight.

[0148] Table 4 Comparison of extraction efficiency with different elution volumes (peak area / sample weight)

[0149]

[0150] The characteristic spectra of the measurement results are attached. Figure 6 As shown in Table 4, the extraction efficiency is as follows. The results show that, based on the analysis of the total peak area / sample weight of each component, there is no significant difference in extraction efficiency with different amounts of elution solvent. However, when the amount of elution solvent is increased to 30 ml, the growth trend of peak area of ​​peak 6 tends to be slow. Therefore, considering the complete elution and the need to save solvent, the amount of elution solvent was determined to be 30 ml.

[0151] Example 6: Investigation of the extraction solvent

[0152] Preparation of test samples: Take an appropriate amount of Chelidonium majus formula granules (batch number: KL-1), grind them finely, take about 0.5g, a total of 5 groups, place them in stoppered conical flasks, add 20ml each of water, 30% methanol, 50% methanol, 80% methanol, and methanol, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, take 15ml of the filtrate, add it to neutral alumina columns (100-200 mesh, inner diameter 10mm, dry packing) of different dosages (2g, 3g, 4g), elute with 30ml of methanol, collect the eluent, evaporate to dryness, add 5ml of methanol to dissolve the residue, and use it as the test sample solution.

[0153] Determination: Accurately pipette 1 μl of each test solution and inject it into the liquid chromatograph. Calculate the peak area of ​​the characteristic peak / sample weight. Chromatographic conditions: Use octadecyl endomemide-bonded high-purity silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 2.2 μm); use acetonitrile as mobile phase A and 0.01 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.3 with phosphoric acid) as mobile phase B, and perform gradient elution according to the specifications in Table 1; flow rate 0.35 mL / min; column temperature 20℃; detection wavelength 280 nm. The theoretical plate number, calculated based on the berberine hydrochloride peak, should be no less than 3000.

[0154] Table 5 Comparison of extraction efficiency of different extraction solvents

[0155]

[0156] The characteristic spectra of the measurement results are attached. Figure 7 As shown in Table 5, the extraction efficiency is as follows. Figure 7 As shown in Table 5, the number of chromatographic peaks obtained after extraction with each extraction solvent was consistent. Further comparison of the total peak area / sample weight values ​​of each component obtained with each extraction solvent revealed that the extraction efficiency of each component was higher when the methanol volume was 80%.

[0157] In addition, different extraction methods and extraction times were investigated. For extraction methods, ultrasonic treatment, reflux extraction, and shaking extraction were used. The results showed that the number of chromatographic peaks obtained by the three methods was consistent, and ultrasonic treatment and reflux extraction were equally efficient and superior to shaking extraction. For extraction time, extractions were performed for 15 min, 30 min, and 45 min. The results showed that the number of chromatographic peaks obtained by different extraction times was consistent, and there was no significant difference in the total peak area / sample weight ratio for each component.

[0158] Example 7: Specificity Examination

[0159] Preparation of test sample: Take an appropriate amount of test sample, grind it into a fine powder, take 0.5 g, place it in a stoppered conical flask, add 20 ml of 80% methanol, sonicate (power 250 W, frequency 40 kHz) for 30 minutes, cool, filter, take 15 ml of the filtrate, add it to a neutral alumina column (100-200 mesh, 4 g, inner diameter 10 mm, dry packing), elute with 30 ml of methanol, collect the eluent, evaporate to dryness, add 5 ml of methanol to dissolve the residue, and use it as the test sample solution.

[0160] The following preparation methods were used to prepare the following solutions: *Cephalotaxus fortunei* granule test solution (sample), *Cephalotaxus fortunei*-deficient negative solution (blank), reference herb solution, and reference standard solution. These solutions were injected into a liquid chromatograph under the following chromatographic conditions: octadecyl endomemide-bonded high-purity silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 2.2 μm); acetonitrile as mobile phase A; and 0.01 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.3 with phosphoric acid) as mobile phase B, with gradient elution as specified in Table 1; flow rate 0.35 mL / min; column temperature 20 °C; and detection wavelength 280 nm. The theoretical plate number, calculated based on the berberine hydrochloride peak, should be no less than 3000.

[0161] The test results are attached. Figure 8 and attached Figure 9 As shown, the results indicate that the negative solution did not have any chromatographic peaks at the locations corresponding to the retention times of the characteristic peaks in the chromatogram of the test sample solution, indicating that the solvent did not interfere with the determination of Chelidonium majus formulation granules, and that this method is specific for determining the characteristic chromatogram of Chelidonium majus formulation granules.

[0162] Example 8: Precision Examination

[0163] Samples from the same batch were used. The preparation method for the test sample is described in Example 7. The test sample solution was injected six times consecutively, 1 μl each time, under the chromatographic conditions described in Example 7. The retention times of the characteristic peaks were recorded, and the relative retention times were calculated. The results are shown in Tables 6 and 7.

[0164] Table 6. Precision Experiment Results (Relative Retention Time)

[0165]

[0166] Table 7. Precision Experiment Results (Relative Peak Area)

[0167]

[0168] The results show that the relative retention time and relative peak area of ​​each characteristic peak have an RSD of less than 1%, indicating good precision.

[0169] Example 9: Intermediate Precision Examination

[0170] Take the same batch of Celandine granules formula, and the test sample preparation method is as described in Example 7. Two experimenters, A and B, each processed 3 samples, and injected 1 μl into each sample on an Agilent and a Thermo instrument. The chromatographic conditions are as described in Example 6. Record the retention time of the characteristic peak, calculate the relative retention time, and the results are shown in Table 8.

[0171] Table 8 Intermediate Precision Assessment (Relative Retention Time)

[0172]

[0173] The results showed that the RSD of the characteristic peak relative retention time of the sample was less than 3%, indicating good intermediate precision.

[0174] Example 10: Stability Study

[0175] Take the same batch of Celandine granules formula, and the test sample preparation method is as described in Example 7. Inject 1 μl of the test sample solution at 0, 2, 4, 6, 8, 12, 18 and 24 hours respectively, record the retention time of the characteristic peak, calculate the relative retention time, and the results are shown in Tables 9 and 10.

[0176] Table 9. Stability Study (Relative Retention Time)

[0177]

[0178] Table 10. Stability test results (relative peak area)

[0179]

[0180] The results show that the relative retention time and relative peak area RSD of each characteristic peak are both less than 2%, indicating good stability.

[0181] Example 11: Repeatability Test

[0182] Six parallel groups of celandine granules from the same batch were prepared. The preparation method of the test sample is described in Example 7. 1 μl of the test sample solution was injected into each group, and the retention time of the characteristic peak was recorded. The relative retention time was calculated, and the results are shown in Tables 11 and 12.

[0183] Table 11 Repeatability Tests (Relative Retention Times)

[0184]

[0185] Table 12 Repeatability Tests (Relative Peak Areas)

[0186]

[0187] The results show that the method has good repeatability.

[0188] Example 12: Chromatographic Column Investigation

[0189] The same batch of Celandine granules was used. The sample preparation method is described in Example 7. The separation performance of three different chromatographic columns (1-Acclaim RSLC 120 C18 (Thermo, 2.1×100mm, 2.2μm); 2-ZORBAX Eclipse Plus C18 RRHD (Agilent, 2.1×100mm, 1.8μm); 3-Acclaim PA2 (Thermo, 2.1×100mm, 2.2μm)) was investigated. 1μl of each column was injected, and the corresponding chromatograms were recorded. The results are shown in Appendix 1. Figure 10 .

[0190] The results showed that the Acclaim PA2 column (100 mm in length, 2.1 mm in inner diameter, and 2.2 μm in particle size) had the best separation performance.

[0191] Example 13: Column Temperature Investigation

[0192] The same batch of Celandine granules was used. The preparation method for the test sample is described in Example 7. Samples were injected at three temperatures: 15℃, 20℃, and 25℃. The retention times of the characteristic peaks were recorded, and the relative retention times were calculated. The results are shown in Table 13 and Appendix. Figure 11 .

[0193] Table 13 Column Temperature Investigation (Relative Retention Time)

[0194]

[0195] The results showed that the measurement results of the samples at column temperatures ranging from 15℃ to 25℃ all met the retention time requirements specified in the text, indicating good robustness.

[0196] Example 14: Flow Rate Investigation

[0197] The same batch of Celandine granules was used. The preparation method for the test sample is described in Example 7. Three flow rates of 0.30 ml, 0.35 ml, and 0.40 ml per minute were investigated. 1 μl of each sample was injected, and the retention time of the characteristic peak was recorded. The relative retention time was calculated according to the requirements in the main text. The results are shown in Table 14 and Appendix. Figure 12 .

[0198] Table 14 Flow velocity analysis (relative retention time)

[0199]

[0200] The results showed that the sample determination results within the flow rate range of 0.30 ml / min to 0.40 ml / min all met the retention time requirements specified in the text, indicating good robustness.

[0201] Example 15: Sample Determination

[0202] Sixteen batches of Chelidonium majus medicinal materials, Chelidonium majus slices, standard decoction (lyophilized powder form), three batches of Chelidonium majus formula granules, and intermediates were collected. The preparation method for the test samples is described in Example 7. The samples were tested separately, and the results are shown below. Figure 13-17 Table 15-19.

[0203] Table 15. Test results (relative retention time) of 16 batches of Chelidonium majus medicinal material samples.

[0204]

[0205] Table 16. Results of the determination of relative retention time for 16 batches of Chelidonium majus decoction pieces.

[0206]

[0207]

[0208] Table 17 Results of freeze-dried powder analysis of multiple batches of Celandine (relative retention time)

[0209]

[0210] Table 18. Results of particle determination of multiple batches of Celandine officinalis (relative retention time)

[0211]

[0212] Table 19 Results of Chelidonium majus intermediate determination (relative retention time)

[0213]

[0214] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0215] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for constructing a characteristic spectrum of a Chelidonium majus sample, comprising the following steps: (1) Preparation of test solution: Accurately weigh the celandine sample, accurately add the extraction solvent and weigh, extract, pre-treat the sample extract by column chromatography to obtain the test solution; (2) Preparation of reference medicinal material solution: Take white celandine reference medicinal material, add extraction solvent, extract, and pretreat the reference medicinal material extract by column chromatography to obtain reference medicinal material solution; (3) Preparation of reference solution: Magnolia alkaloid, berberine hydrochloride, tetrahydroberberine, protopine, chelidonine, dihydrochelidonine and dihydrosanthemin were used as references. Reference solvents were added to each to obtain reference solution. (4) Acquisition of characteristic chromatograms: The test solution, the reference medicinal material solution and the reference solution were detected by ultra-high performance liquid chromatography to obtain the characteristic chromatograms of the Chelidonium majus sample; The chromatographic conditions for the ultra-high performance liquid chromatography (UHPLC) include: using octadecyl endomemide-bonded silica gel as the stationary phase, a binary system consisting of acetonitrile and phosphate buffer as the mobile phase, gradient elution as the elution mode, and a detection wavelength of 250-300 nm; the phosphate buffer is an aqueous solution of potassium dihydrogen phosphate with a pH of 3.0-3.5; and the concentration of the potassium dihydrogen phosphate aqueous solution is 0.005-0.01 mol / L. The gradient elution procedure is as follows: 0-10 min, the volume fraction of mobile phase A increases from 15% to 20%, and the volume fraction of mobile phase B decreases from 85% to 80%; 10-18 min, the volume fraction of mobile phase A increases from 20% to 70%, and the volume fraction of mobile phase B decreases from 80% to 30%; 18-22 min, the volume fraction of mobile phase A is 70%, and the volume fraction of mobile phase B is 30%; 22-22.5 min, the volume fraction of mobile phase A decreases from 70% to 15%, and the volume fraction of mobile phase B increases from 30% to 85%; 22.5-27 min, the volume fraction of mobile phase A is 15%, and the volume fraction of mobile phase B is 85%. The chromatographic conditions for the column chromatography method include: using neutral alumina as the stationary phase and methanol or a mixture of methanol and dichloromethane as the eluent. The extraction solvent is a 50%~100% v / v methanol aqueous solution; The celandine sample is celandine formula granules, celandine medicinal material, celandine decoction pieces, or celandine standard decoction.

2. The method according to claim 1, characterized in that, The chromatographic conditions for the column chromatography method include: the amount of the stationary phase is 4~8 g / g of Chelidonium majus sample; The amount of eluent used is 20~80mL / g of Chelidonium majus sample.

3. The method according to claim 2, characterized in that, The amount of eluent used is 60-80 mL / g of Chelidonium majus sample.

4. The method according to any one of claims 1 to 3, characterized in that, The chromatographic conditions for the ultra-high performance liquid chromatography method include: a detection wavelength of 280 nm; The chromatographic column used for the stationary phase is an Acclaim PA2 column.

5. The method according to any one of claims 1 to 3, characterized in that, The extraction solvent is an 80%~100% v / v methanol aqueous solution.

6. The method according to any one of claims 1 to 3, characterized in that, The extraction is performed using one of the following methods: ultrasonic treatment, heating and reflux, or shaking extraction.

7. The method according to claim 6, characterized in that, The extraction was performed using ultrasonic treatment.

8. The method according to claim 4, characterized in that, The chromatographic conditions for the ultra-high performance liquid chromatography method also include: a flow rate of 0.2~0.4 mL / min and a column temperature of 15~50℃.

9. The method according to claim 8, characterized in that, The flow rate is 0.3~0.4 mL / min.

10. The method according to claim 8, characterized in that, The column temperature is 15~25℃.

11. The method according to any one of claims 1 to 3, characterized in that, The feature spectrum has ten characteristic peaks.

12. The method according to claim 11, characterized in that, The characteristic chromatograms are defined with peak 4, corresponding to the chromatographic peak of berberine hydrochloride reference standard, as peak S1. The relative retention times of peaks 1, 2, 3, and 5 with peak S1 are calculated. Peak 10, corresponding to the chromatographic peak of dihydrosandricaline reference standard, is defined as peak S2. The relative retention times of peaks 6 to 9 with peak S2 are calculated. The relative retention times are all within ±10% of the specified values. The specified values ​​for peak 1 are 0.28, peak 2 are 0.72, peak 3 are 0.90, peak 5 are 1.14, peak 6 are 0.69, peak 7 are 0.71, peak 8 are 0.85, and peak 9 are 0.

97.

13. The use of a celandine sample characteristic spectrum constructed by the method according to any one of claims 1 to 12 in the quality detection of celandine samples.

14. A method for quality testing of Celandine Extract granules, comprising the following steps: The sample to be tested is used as the test sample, and a feature spectrum is constructed according to the method described in any one of claims 1 to 12. The obtained feature spectrum is compared with the feature spectrum constructed according to the method described in any one of claims 1 to 12 to obtain the quality test result.

Images