Construction method and application of chelidonium majus sample specific chromatogram

Abstract

The invention provides a construction method and application of a chelidonium album sample specific chromatogram, the method adopts ultrahigh performance liquid chromatography to establish the specific chromatogram of chelidonium album formula granules or upstream raw materials thereof, and the method comprises the following specific steps: preparation of a test solution, preparation of a reference medicinal material solution, preparation of a reference substance solution and acquisition of the specific chromatogram. The specific chromatogram construction method provided by the invention makes up the vacancy of research on water-soluble components of the chelidonium majus, and the method is simple and convenient to operate, short in analysis period, good in common peak separation degree and stable in baseline, and provides a new analysis means for quality control of the chelidonium majus formula granules.

Description

Technical Field

[0001] The invention belongs to the field of quality control of traditional Chinese medicines, relates to a quality evaluation method for celandine formula granules, and specifically relates to a method for constructing a UPLC characteristic spectrum of celandine formula granules. Background Art

[0002] Greater celandine is the dried whole herb of Greater celandine, a plant of the genus Greater celandine of the Papaveraceae family. It is also known as Rehmannia glutinosa, Niujinhua, Tuhuanglian, Babujin, Duanchangcao, and Realgar Grass. It tastes bitter, is cool in nature, and is poisonous. The whole herb has the effects of analgesia, cough relief, diuresis, and detoxification. It is mainly used to treat stomachache, abdominal pain, enteritis, dysentery, chronic bronchitis, whooping cough, cough, jaundice, edema, ascites, scabies, and snake and insect bites. Studies on the chemical components of Greater celandine have shown that the main chemical components in Greater celandine are alkaloids, and it also contains flavonoids, triterpenoid saponins, volatile oils, vitamin C, and other components. The active ingredients it contains are mainly isoquinoline alkaloids, which have multiple effects such as anti-inflammatory, antibacterial, cough relief, and analgesia.

[0003] Greater celandine is included in the 2020 edition of the "Chinese Pharmacopoeia", which only includes identification items (physical and chemical identification with Greater celandine control materials and chelerythrine as reference) and content determination items (HPLC content determination with chelerythrine as reference) to control the quality of traditional Chinese medicine, and there are no relevant standards for fingerprint spectra or characteristic spectra.

[0004] The Greater Chelidonium formula granules are granules prepared by decocting the Greater Chelidonium medicinal material in water. They are consistent with the efficacy and quality of clinical decoctions. Under the guidance of traditional Chinese medicine theory, they can be flexibly prepared by clinical physicians and are easy to use.

[0005] In the prior art, although there are many studies on Greater celandine, most of them focus on the processing of medicinal materials, the preparation process of related ingredients, dosage forms, etc., while there is a gap in the research on Greater celandine formula granules or water-soluble extracts (especially quality control and quality evaluation research).

[0006] Reference 1 used UPLC-UV and QTOF-MS technology to establish characteristic spectra for 10 batches of chelidonine medicinal materials, and identified protopine, chelidonine, coptisine, berberine, chelerythrine, sanguinarine, and tetrahydrocoptisine through reference substances. However, this method requires a large amount of sample, a complex extraction method, and some common peaks have small peak areas, poor separation, and unstable baselines, which increases the difficulty of identification. In addition, this method is only applicable to the medicinal materials themselves and cannot take into account other derivative forms.

[0007] References:

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

[0009] Problem that the invention aims to solve

[0010] The 2020 edition of the "Chinese Pharmacopoeia" stipulates that the content index of chelandine is chelerythrine. However, preliminary studies have found that this ingredient cannot be detected in the standard decoction, extract and formula granules obtained by decocting chelandine slices by traditional water technology. Therefore, this analytical method cannot be used for quality control of chelandine formula granules.

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

[0012] Solutions for solving problems

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

[0014] [1] The present invention provides a method for constructing a characteristic spectrum of a celandine sample, comprising the following steps:

[0015] (1) Preparation of test solution: accurately weigh a sample of Greater celandine, accurately add an extraction solvent and weigh it, extract it, and pretreat the sample extract by column chromatography to obtain a test solution;

[0016] (2) Preparation of a control medicinal material solution: taking a control medicinal material of Greater celandine, adding an extraction solvent, extracting, and pretreating the control medicinal material extract by column chromatography to obtain a control medicinal material solution;

[0017] (3) Preparation of reference substance solution: using at least two of magnolamine, coptisine hydrochloride, tetrahydrocoptisine, hydroprotopine, chelidonine, dihydrochelerythrine and dihydrosanguinarine as reference substances, and adding reference substance solvents respectively to obtain reference substance solutions;

[0018] (4) Acquisition of characteristic spectrum: The test solution, the reference medicinal material solution and the reference substance solution are respectively detected by ultra-high performance liquid chromatography to obtain a characteristic spectrum of the celandine sample;

[0019] Wherein, the celandine sample is the celandine formula granules or the celandine medicinal material, decoction pieces, standard decoction or water extract used to prepare the formula granules.

[0020] [2] The method according to [1], wherein the chromatographic conditions of the column chromatography include: using alumina as the stationary phase and alcohol or a mixture of alcohol and halogenated hydrocarbon as the eluent;

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

[0024] [3] The method according to [1] or [2], wherein the chromatographic conditions of the ultra-high performance liquid chromatography method include: using octadecyl embedded amide group bonded silica gel as the stationary phase, using a binary system consisting of nitrile and phosphate buffer as the mobile phase, using gradient elution as the elution mode, and the detection wavelength is 250-300nm;

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

[0026] and / or,

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

[0028] [4] The method according to any one of [1] to [3], wherein at least one of coptisine hydrochloride, dihydrosanguinarine, magnolialine, tetrahydrocoptisine, hydroprotopine, chelidonine, and dihydrochelerythrine is used as a reference substance;

[0029] Preferably, magnolia pine, coptisine hydrochloride, tetrahydrocoptisine, hydroprotopine, chelidonine, dihydrochelerythrine and dihydrosanguinarine are used as reference substances.

[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: from 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%; from 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%; from 18 to 22 min, the volume fraction of mobile phase A is 70%, and the volume fraction of mobile phase B is 30%; from 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%; from 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] The method according to [3], wherein the chromatographic conditions of the ultra-high performance liquid chromatography also 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 characteristic spectrum has at least ten characteristic peaks;

[0035] Preferably, peak 4 corresponding to the chromatographic peak of coptisine hydrochloride reference substance is taken as peak S1, and the relative retention times of peak 1, peak 2, peak 3, peak 5 and peak S1 are calculated; peak 10 corresponding to the chromatographic peak of dihydrosanguinarine reference substance is taken as peak S2, and the relative retention times of peaks 6 to 9 and peak S2 are calculated, and the relative retention times are all within the range of ±10% of the specified values, and the specified values ​​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 a use of a characteristic spectrum of a celandine sample constructed according to the method described in any one of [1] to [9] in quality detection of a celandine sample.

[0037]

[11] A quality inspection method for Greater Chelidonium formula granules, comprising the following steps: using a sample to be inspected as a test sample, constructing a characteristic spectrum according to the method described in any one of [1] to [9], and comparing the obtained characteristic spectrum with the characteristic spectrum constructed according to the method described in any one of [1] to [9] to obtain a quality inspection result.

[0038] Effects of the Invention

[0039] (1) The present invention adopts ultra-high performance liquid chromatography to establish a characteristic spectrum of the celandine granules or their upstream raw materials. The method is simple to operate and has low detection time and cost, thus providing a new analytical method for quality control of the celandine granules.

[0040] (2) The characteristic spectrum construction method proposed in the present invention is used to study the components in the water decoction of Greater celandine, filling the gap in the research on the water-soluble components of Greater celandine.

[0041] (3) The characteristic spectrum method proposed in the present invention uses neutral alumina to purify and enrich alkaloid components; a buffer salt is selected as the mobile phase, and phosphoric acid is selected to adjust to a suitable pH. This method abandons the traditional method of analyzing alkaloids (such as using ion pair reagents), and to a great extent ensures the reproducibility of the characteristic spectrum method and reduces the loss of the chromatographic column.

[0042] (4) The characteristic spectrum method of the present invention, using the control medicinal material of chelidonium as a reference, established a characteristic spectrum containing 10 characteristic peaks, and identified 7 common peaks, including magnolamine, hydroprotopine, chelidonine, coptisine hydrochloride, tetrahydrocoptisine, dihydrochelerythrine and dihydrosanguinarine, accounting for 70%.

[0043] (5) The present invention adopts UPLC to establish a characteristic spectrum method, which has a short analysis cycle, good common peak separation, and a stable baseline. At the same time, the characteristic spectrum research of medicinal materials, standard decoctions, extracts, and formula granules all have corresponding characteristic peaks, which can fully reflect the consistency of formula granules and pharmaceutical preparation intermediates, provide a basis for tracing the characteristic components in the Greater Chelidonium formula granules and upstream raw materials, and also provide a strong guarantee for the quality control of the industrialization process. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 : UPLC graph of wavelength investigation - characteristic spectrum;

[0045] Figure 2 : UPLC diagram of pH investigation - characteristic spectrum;

[0046] Figure 3 : UPLC diagram of buffer solution concentration investigation - characteristic spectrum;

[0047] Figure 4 : UPLC diagram of different neutral alumina dosages - characteristic spectrum;

[0048] Figure 5 : UPLC diagram of different eluents - characteristic spectrum;

[0049] Figure 6 : UPLC diagram of different elution volume dosage - characteristic spectrum;

[0050] Figure 7 : UPLC images of different extraction solvents - characteristic spectra;

[0051] Figure 8: Specificity test of celandine formula granules (comparison between sample and blank);

[0052] Fig. 9 : Specificity test of celandine formula granules (comparison between samples and control medicinal materials and reference substances);

[0053] Fig.10 :Characteristic spectrum of celandine formula granules chromatographic column investigation;

[0054] Fig.11 :Investigation on column temperature of characteristic spectrum of celandine formula granules;

[0055] Fig.12 :Investigation on the flow rate of characteristic spectrum of celandine formula granules;

[0056] Fig.13 : Overlay of characteristic spectra of multiple batches of celandine;

[0057] Fig.14 : Overlay of characteristic spectra of multiple batches of celandine slices;

[0058] Fig.15 : Overlay of characteristic spectra of multiple batches of lyophilized powder of Greater Chelidonium;

[0059] Fig.16 : Overlay of characteristic spectra of multiple batches of celandine particles;

[0060] Fig.17 : Overlay of characteristic spectra of multiple batches of intermediates of Greater Chelidonium;

[0061] Fig.18 : Characteristic spectrum of the celandine sample constructed by the present invention. DETAILED DESCRIPTION

[0062] Various exemplary embodiments, features and aspects of the present invention will be described in detail below. The word "exemplary" used here means "used as an example, embodiment or illustrative". Any embodiment described here as "exemplary" is not necessarily interpreted as being superior or better than other embodiments.

[0063] In addition, in order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present invention.

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

[0065] In this specification, the word "may" means both performing a certain process and not performing a certain process.

[0066] In this specification, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0067] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints numerical values ​​A and B.

[0068] In this specification, when "normal temperature" or "room temperature" is used, the temperature may be 10-40°C.

[0069] First, the present invention proposes a method for constructing a characteristic spectrum of a celandine sample, comprising the following steps:

[0070] (1) Preparation of test solution: accurately weigh a sample of Greater celandine, accurately add an extraction solvent and weigh it, extract it, and pretreat the sample extract by column chromatography to obtain a test solution;

[0071] (2) Preparation of a control medicinal material solution: taking a control medicinal material of Greater celandine, adding an extraction solvent, extracting, and pretreating the control medicinal material extract by column chromatography to obtain a control medicinal material solution;

[0072] (3) Preparation of reference substance solution: using at least two of magnolamine, coptisine hydrochloride, tetrahydrocoptisine, hydroprotopine, chelidonine, dihydrochelerythrine and dihydrosanguinarine as reference substances, and adding reference substance solvents respectively to obtain reference substance solutions;

[0073] (4) Acquisition of characteristic spectrum: The test solution, the reference medicinal material solution and the reference substance solution are respectively detected by ultra-high performance liquid chromatography to obtain a characteristic spectrum of the celandine sample;

[0074] Wherein, the celandine sample is the celandine formula granules or the celandine medicinal material, decoction pieces, standard decoction or water extract used to prepare the formula granules.

[0075] In some specific embodiments, the method for preparing the test solution can be to take an appropriate amount of celandine sample, grind it, accurately weigh it, accurately add an extraction solvent and then weigh it, extract, cool, filter, add the filtrate to a chromatographic column pre-filled with a stationary phase, elute with an eluent, collect the eluate, evaporate to dryness, add a solvent to the residue to dissolve it, and obtain a test solution.

[0076] In some specific embodiments, the preparation method of the control medicinal material solution can be to weigh an appropriate amount of Greater celandine control medicinal material, add an extraction solvent, extract, cool, filter, add the filtrate to a chromatographic column pre-filled with a stationary phase, elute with an eluent, collect the eluate, evaporate to dryness, add a solvent to the residue to dissolve it, and obtain a control medicinal material solution.

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

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

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

[0080] In some specific embodiments, in the method for preparing the test solution, the usage ratio of the extraction solvent to the Greater celandine reference medicinal material 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.

[0081] In some specific embodiments, in the method for preparing the control medicinal material solution, the ratio of the extraction solvent to the celandine 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, heating reflux or shaking extraction.

[0082] In some specific embodiments, the extraction is carried out by ultrasonic treatment.

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

[0084] In some specific embodiments, the chromatographic conditions of the column chromatography include: using aluminum oxide as the stationary phase and using alcohol or a mixture of alcohol and halogenated hydrocarbon as the eluent.

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

[0086] In some specific embodiments, the amount of the stationary phase is 4-8 g / g celandine sample, such as 4 g / g celandine sample, 6 g / g celandine sample or 8 g / g celandine sample.

[0087] In some specific embodiments, 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 embodiments, the amount of the eluent is 20-80 mL / g celandine sample, for example, 20 mL / g celandine sample, 40 mL / g celandine sample, 60 mL / g celandine sample, 80 mL / g celandine sample, etc.

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

[0090] In some specific embodiments, the type of 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 is the same as or different from the solvent.

[0091] In some specific embodiments, the preparation method of the reference solution can be to use at least two of magnolia pine, coptisine hydrochloride, tetrahydrocoptisine, hydroprotopine, chelidonine, dihydrochelerythrine and dihydrosanguinarine as reference substances, take an appropriate amount of each, add a dissolving agent, and obtain a reference solution; wherein the concentration of each reference substance can be magnolia pine 40-60 μg / mL, tetrahydrocoptisine 40-60 μg / mL, dihydrosanguinarine 80-120 μg / mL, coptisine hydrochloride 40-60 μg / mL, hydroprotopine 40-60 μg / mL, chelidonine 40-60 μg / mL, and dihydrochelerythrine 40-60 μg / mL.

[0092] In some specific embodiments, the method for preparing the reference solution uses at least one of coptisine hydrochloride and dihydrosanguinarine and magnolialine, tetrahydrocoptisine, hydroprotopine, chelidonine, and dihydrochelerythrine as reference substances.

[0093] In some specific embodiments, the method for preparing the reference solution uses magnolia pine, coptisine hydrochloride, tetrahydrocoptisine, hydroprotopine, chelidonine, dihydrochelerythrine and dihydrosanguinarine as reference substances.

[0094] In some specific embodiments, the method for obtaining the characteristic spectrum can be to accurately pipette 1 μl of the test solution, control medicinal material solution and reference substance solution respectively, inject them into a liquid chromatograph, and detect them by ultra-high performance liquid chromatography to obtain the characteristic spectrum of the celandine sample.

[0095] In some specific embodiments, the chromatographic conditions of the ultra-high performance liquid chromatography include: using octadecyl embedded amide group bonded silica gel as the stationary phase, using a binary system consisting of nitrile and phosphate buffer as the mobile phase, using gradient elution as the elution mode, and a detection wavelength of 250-300nm.

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

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

[0098] In some specific embodiments, the phosphate buffer is an aqueous solution of potassium dihydrogen phosphate with a pH of 2.5-4.5, for example, 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 embodiments, the phosphate buffer is an aqueous solution of potassium dihydrogen phosphate with a pH of 3.0 to 4.0.

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

[0101] In some specific embodiments, the concentration of the potassium dihydrogen phosphate aqueous solution is 0.005-0.01 mol / L, for example, 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 procedures: from 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%; from 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%; from 18 to 22 min, the volume fraction of mobile phase A is 70%, and the volume fraction of mobile phase B is 30%; from 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%; from 22.5 to 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 embodiments, the detection wavelength is 280 nm.

[0104] In some specific embodiments, the chromatographic conditions of the ultra performance liquid chromatography also include: a flow rate of 0.2 to 0.4 mL / min, for example, 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 embodiments, the flow rate is 0.3-0.4 mL / min.

[0106] In some specific embodiments, the chromatographic conditions of the ultra performance liquid chromatography also include: a column temperature of 15 to 50°C, for example, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, etc.

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

[0108] In some specific embodiments, the characteristic spectrum has at least ten, for example ten characteristic peaks.

[0109] In some specific implementation schemes, peak 4 corresponding to the chromatographic peak of coptisine hydrochloride reference substance is taken as peak S1, and the relative retention times of peak 1, peak 2, peak 3, peak 5 and peak S1 are calculated; peak 10 corresponding to the chromatographic peak of dihydrosanguinarine reference substance is taken as peak S2, and the relative retention times of peaks 6 to 9 and peak S2 are calculated, and the relative retention times should be within the range of ±10% of the specified values, and the specified values ​​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 a use of a characteristic spectrum of a celandine sample constructed according to the method of the present invention in quality detection of the celandine sample.

[0111] Finally, the present invention also provides a quality detection method for Greater celandine formula granules, comprising the following steps: using the product to be detected as a test product, constructing a characteristic spectrum according to the method of the present invention, comparing the obtained characteristic spectrum with the characteristic spectrum constructed according to the method of the present invention, and obtaining a quality detection result.

[0112] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0113] Sources of samples used in the examples:

[0114] The magnolia pine reference substance and coptisine hydrochloride reference substance were purchased from China Food and Drug Inspection Institute with batch numbers 112090-202201 and 112026-201802, respectively.

[0115] Tetrahydrocoptisine reference substance, hydroorthoopine reference substance, chelidonine reference substance, dihydrochelerythrine reference substance, and dihydrosanguinarine reference substance were purchased from Shanghai Standard Standard Technical Service Co., Ltd. with batch numbers 8227, 4086, 13035, 10761, and 8232, respectively.

[0116] The control drug of Greater celandine was purchased from China Food and Drug Inspection Institute with batch number 121143-201604.

[0117] Greater celandine formula granules (prepared by water decoction process by Jiangyin Tianjiang Pharmaceutical Co., Ltd., 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 celandine granules, grind them into powder, take about 0.5g, put it in a stoppered conical flask, add 20ml of methanol, ultrasonically treat (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 column), elute with 30ml of methanol, collect the eluate, evaporate to dryness, add 5ml of methanol to the residue to dissolve it, and use it as the test sample solution.

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

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

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

[0123] Preparation of test sample: Take an appropriate amount of celandine granules, grind them into powder, take about 0.5g, put it in a stoppered conical flask, add 20ml of methanol, ultrasonically treat (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 column), elute with 30ml of methanol, collect the eluate, evaporate to dryness, add 5ml of methanol to the residue to dissolve it, and use it as the test sample solution.

[0124] Determination: Use octadecyl embedded amide group bonded high purity silica gel as filler (column length 100mm, inner diameter 2.1mm, particle size 2.2μm); use acetonitrile as mobile phase A, 0.005mol / L potassium dihydrogen phosphate solution, 0.005mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.0 with phosphoric acid), 0.005mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.3 with phosphoric acid), 0.005mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.5 with phosphoric acid) as mobile phase B, and perform gradient elution according to the provisions in the following table; flow rate is 0.35ml per minute; column temperature is 20℃; detection wavelength is 280nm. The number of theoretical plates calculated based on the coptisine hydrochloride peak should not be less than 3000.

[0125] Table 1 Gradient elution program

[0126]

[0127] The characteristic spectrum obtained by the determination is shown in the attached Figure 2 As shown in the attached Figure 2 It can be seen that the peak shapes and separations of the chromatographic peaks obtained at different pH values ​​are different. When pH = 3.3, the separation and peak shape of each chromatographic peak are the best.

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

[0129] Preparation of test sample: Take an appropriate amount of celandine granules, grind them into powder, take about 0.5g, put it in a stoppered conical flask, add 20ml of methanol, ultrasonically treat (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 column), elute with 30ml of methanol, collect the eluate, evaporate to dryness, add 5ml of methanol to the residue to dissolve it, and use it as the test sample solution.

[0130] High-purity silica gel bonded with octadecyl embedded amide groups was used as filler (column length: 100 mm, inner diameter: 2.1 mm, particle size: 2.2 μm); acetonitrile was used as mobile phase A, 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, and gradient elution was performed according to the provisions in Table 1; the flow rate was 0.35 ml per minute; the column temperature was 20°C; the detection wavelength was 280 nm. The theoretical plate number calculated based on the coptisine hydrochloride peak should be no less than 3000.

[0131] The characteristic spectrum obtained by the determination is shown in the attached Figure 3 As shown in the attached Figure 3 It can be seen that the peak shapes and separations of the chromatographic peaks obtained with different concentrations of buffer salts are different. It is found that when the buffer concentration is 0.01 mol / L, the separation and peak shape of each chromatographic peak are the best.

[0132] Example 4: Determination of sample pretreatment method - Investigation of the amount of neutral alumina used

[0133] Preparation of test sample: Take an appropriate amount of Greater Chelidonium formula granules (batch number: KL-1), grind it into powder, take about 0.5g, put it in 3 groups, put it in a stoppered conical flask, add 20ml of methanol each, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, cool it, take 15ml of the filtrate respectively, add it to a neutral alumina column (100-200 mesh, inner diameter 10mm, dry column) with different amounts (2g, 3g, 4g), elute with 30ml of methanol, collect the eluate, evaporate it 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, inject into the liquid chromatograph, and calculate the characteristic peak area / sample weight. Chromatographic conditions: Use octadecyl embedded amide group bonded high-purity silica gel as filler (column length is 100 mm, inner diameter is 2.1 mm, particle size is 2.2 μm); acetonitrile is mobile phase A, 0.01 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.3 with phosphoric acid) is mobile phase B, and gradient elution is performed according to the provisions in Table 1; the flow rate is 0.35 ml per minute; the column temperature is 20°C; the detection wavelength is 280 nm. The number of theoretical plates calculated based on the coptisine hydrochloride peak should not be less than 3000.

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

[0136]

[0137] The characteristic spectrum of the measurement results is shown in the attached Figure 4 The extraction efficiency is shown in Table 2. Figure 4As can be seen from 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 values ​​of each component obtained with different amounts of alumina shows that when the amount of alumina is 3 g, the extraction efficiency of each component is higher.

[0138] Example 5: Determination of sample pretreatment method - Investigation of eluent

[0139] (1) Investigation of eluent types

[0140] Preparation of test sample: Take an appropriate amount of Greater celandine granules (batch number: KL-1), grind it into powder, take about 0.5g, and place it in 4 groups. Put it in a stoppered conical flask, add 20ml of methanol each, and ultrasonically treat it (power 250W, frequency 40kHz) for 30 minutes respectively. Cool it, take 15ml of the filtrate, add it to a neutral alumina column (100-200 mesh, 4g, inner diameter 10mm, dry column loading), and elute it with 30ml of methanol, methanol-dichloromethane (1:1), methanol-dichloromethane (3:7), and methanol-dichloromethane (7:3), respectively. Collect the eluate, evaporate it to dryness, add 5ml of methanol to dissolve the residue, and use it as the test sample solution.

[0141] Determination: Accurately pipette 1μl of each test solution, inject it into the liquid chromatograph, determine according to the chromatographic conditions, and calculate the characteristic peak area / sample weight. Chromatographic conditions: Use octadecyl embedded amide group bonded high-purity silica gel as filler (column length is 100mm, inner diameter is 2.1mm, particle size is 2.2μm); acetonitrile is mobile phase A, 0.01mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.3 with phosphoric acid) is mobile phase B, and gradient elution is performed according to the provisions in Table 1; the flow rate is 0.35ml per minute; the column temperature is 20℃; the detection wavelength is 280nm. The number of theoretical plates calculated based on the coptisine hydrochloride peak should not be less than 3000.

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

[0143]

[0144] The characteristic spectrum of the measurement results is shown in the attached Figure 5 The extraction efficiency is shown in Table 3. The results show that from the numerical analysis of the total peak area / sample weight of each component, there is no significant difference in the extraction efficiency of different elution solvents; but when methanol is 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 Greater Chelidonium formula granules (batch number: KL-1), grind it into powder, take about 0.5g, and place it in 3 groups. Put it in a stoppered conical flask, add 20ml of methanol each, and ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes. Cool it, take 15ml of the filtrate respectively, add it to a neutral alumina column (100-200 mesh, 4g, inner diameter 10mm, dry column), and elute it with 10ml, 20ml, 30ml, and 40ml of methanol respectively. Collect the eluate, evaporate it 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, inject into liquid chromatograph, determine according to the above chromatographic conditions, and calculate the peak area / sample weight of the characteristic peak.

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

[0149]

[0150] The characteristic spectrum of the measurement results is shown in the attached Figure 6 The extraction efficiency is shown in Table 4. The results show that from the numerical analysis of the total peak area / sample weight of each component, there is no significant difference in the extraction efficiency of different elution solvent dosages; but when the elution solvent dosage is increased to 30 ml, the growth trend of Peak 6 peak area tends to be flat, so from the perspective of complete elution and solvent saving, the elution solvent dosage is determined to be 30 ml.

[0151] Example 6: Investigation of extraction solvent

[0152] Preparation of test sample: Take an appropriate amount of Greater Chelidonium formula granules (batch number: KL-1), grind it into powder, take about 0.5g, and place it in a stoppered conical flask. Add 20ml of water, 30% methanol, 50% methanol, 80% methanol, and methanol respectively. Ultrasonic treatment (power 250W, frequency 40kHz) for 30 minutes, cool it, take 15ml of the filtrate respectively, and add it to a neutral alumina column (100-200 mesh, inner diameter 10mm, dry column) with different amounts (2g, 3g, 4g), elute with 30ml of methanol, collect the eluate, evaporate it 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, inject into the liquid chromatograph, and calculate the characteristic peak area / sample weight. Chromatographic conditions: Use octadecyl embedded amide group bonded high-purity silica gel as filler (column length is 100 mm, inner diameter is 2.1 mm, particle size is 2.2 μm); acetonitrile is mobile phase A, 0.01 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.3 with phosphoric acid) is mobile phase B, and gradient elution is performed according to the provisions in Table 1; the flow rate is 0.35 ml per minute; the column temperature is 20°C; the detection wavelength is 280 nm. The number of theoretical plates calculated based on the coptisine hydrochloride peak should not be less than 3000.

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

[0155]

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

[0157] In addition, different extraction methods and extraction times were investigated. In terms of extraction methods, ultrasonic treatment, heating reflux, and shaking extraction were used for extraction. The results showed that the number of chromatographic peaks obtained by the three treatment methods was consistent, and the extraction efficiency of ultrasonic treatment and heating reflux was equivalent and better than shaking extraction. In terms of extraction time, 15min, 30min, and 45min were extracted respectively. 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 of each component.

[0158] Example 7: Specificity Investigation

[0159] Preparation of test sample: Take an appropriate amount of the test sample, grind it into powder, take 0.5 g, put it in a stoppered conical flask, add 20 ml of 80% methanol, ultrasonically treat (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 column), elute with 30 ml of methanol, collect the eluate, evaporate to dryness, add 5 ml of methanol to dissolve the residue, and use it as the test sample solution.

[0160] According to the above preparation method, the test solution (sample) of celandine formula granules, the negative solution lacking celandine (blank), the control medicinal material solution, and the reference substance solution were prepared and injected into the liquid chromatograph. The chromatographic conditions were as follows: octadecyl embedded amide group bonded high-purity silica gel was used as the filler (column length was 100 mm, inner diameter was 2.1 mm, and particle size was 2.2 μm); acetonitrile was used as the mobile phase A, and 0.01 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.3 with phosphoric acid) was used as the mobile phase B, and gradient elution was performed according to the provisions in Table 1; the flow rate was 0.35 ml per minute; the column temperature was 20°C; and the detection wavelength was 280 nm. The number of theoretical plates calculated based on the coptisine hydrochloride peak should not be less than 3000.

[0161] The test results are as attached Figure 8 and attached Fig. 9 As shown, the results showed that the negative solution had no chromatographic peaks at the retention times corresponding to the characteristic peaks in the chromatogram of the test solution, indicating that the solvent had no interference with the determination of the Greater Chelidonium Formula Granules, and that this method was specific for determining the characteristic spectrum of the Greater Chelidonium Formula Granules.

[0162] Example 8: Precision Investigation

[0163] Take the same batch number of samples, the test sample preparation method is shown in Example 7, the test sample solution is injected 6 times continuously, 1 μl each time, and the chromatographic conditions are shown in Example 7. Record the retention time of its characteristic peak, calculate the relative retention time, and the results are shown in Table 6 and Table 7.

[0164] Table 6 Precision test results (relative retention time)

[0165]

[0166] Table 7 Precision test results (relative peak area)

[0167]

[0168] The results showed that the RSDs of the relative retention time and relative peak area of ​​each characteristic peak were less than 1%, indicating good precision.

[0169] Example 9: Intermediate precision study

[0170] Take the same batch of Greater celandine granules, the test sample preparation method is shown in Example 7, and two experimenters A and B process 3 portions respectively, inject 1 μl on Agilent and Thermo instruments respectively, the chromatographic conditions are shown in Example 6, record the characteristic peak retention time, calculate the relative retention time, and the results are shown in Table 8.

[0171] Table 8 Intermediate precision study (relative retention time)

[0172]

[0173] The results showed that the RSD of the relative retention time of the characteristic peaks of the samples was less than 3%, and the intermediate precision test was good.

[0174] Example 10: Stability Study

[0175] Take the same batch of Greater celandine granules, the test sample preparation method is shown in Example 7, and inject 1 μl of the test sample solution at 0, 2, 4, 6, 8, 12, 18, and 24 hours, record the retention time of its characteristic peak, and calculate the relative retention time. 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 showed that the RSD of the relative retention time and relative peak area of ​​each characteristic peak was less than 2%, indicating good stability.

[0181] Example 11: Repeatability Study

[0182] Take the same batch of Greater celandine granules, and prepare 6 groups in parallel. The test sample preparation method is shown in Example 7. 1 μl of the test sample solution is injected respectively, and the retention time of its characteristic peak is recorded. The relative retention time is calculated. The results are shown in Tables 11 and 12.

[0183] Table 11 Repeatability test (relative retention time)

[0184]

[0185] Table 12 Repeatability test (relative peak area)

[0186]

[0187] The results showed that the repeatability of the method was good.

[0188] Example 12: Chromatographic column inspection

[0189] The same batch of celandine granules was taken. The preparation method of the test sample was as shown in Example 7. The separation effects of three different types of chromatographic columns (1-Acclaim RSLC 120 C18 (Thermo, 2.1×100mm, 2.2μm); 2-ZORBAX Eclipse PlusC18 RRHD (Agilent, 2.1×100mm, 1.8μm); 3-Acclaim PA2 (Thermo, 2.1×100mm, 2.2μm)) were investigated. 1μl was injected respectively, and the corresponding chromatograms were recorded. The results are shown in the attached Fig.10 .

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

[0191] Example 13: Column Temperature Investigation

[0192] Take the same batch of celandine granules, the test sample preparation method is shown in Example 7, and examine the three temperatures of 15°C, 20°C, and 25°C, inject 1 μl respectively, record the retention time of its characteristic peak, and calculate its relative retention time. The results are shown in Table 13 and Attached Fig.11 .

[0193] Table 13 Column temperature investigation (relative retention time)

[0194]

[0195] The results show that the column temperature in the range of 15℃~25℃ meets the retention time requirements specified in the text and has good durability.

[0196] Example 14: Flow rate investigation

[0197] Take the same batch of celandine granules, the test sample preparation method is shown in Example 7, examine the three flow rates of 0.30ml, 0.35ml, and 0.40ml per minute, inject 1μl respectively, record the retention time of the characteristic peak, and calculate the relative retention time according to the requirements of the text. The results are shown in Table 14 and Appendix Fig.12 .

[0198] Table 14 Flow rate investigation (relative retention time)

[0199]

[0200] The results show that the measurement results of samples in the flow rate range of 0.30ml / min to 0.40ml / min all meet the retention time requirements specified in the text and have good durability.

[0201] Example 15: Sample determination

[0202] Take 16 batches of celandine medicinal materials, celandine slices, standard decoction (freeze-dried powder form), 3 batches of celandine formula granule samples and intermediates, the preparation method of the test sample is shown in Example 7, and the test results are shown in Figure 13-17 , Table 15-19.

[0203] Table 15 Determination results of 16 batches of celandine medicinal material samples (relative retention time)

[0204]

[0205] Table 16 Determination results of 16 batches of celandine slices samples (relative retention time)

[0206]

[0207]

[0208] Table 17 Celandine multi-batch freeze-dried powder measurement results (relative retention time)

[0209]

[0210] Table 18 Determination results of multiple batches of celandine particles (relative retention time)

[0211]

[0212] Table 19 Celandine intermediate determination results (relative retention time)

[0213]

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

[0215] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or technical improvements in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A method for constructing a characteristic spectrum of a celandine sample, comprising the following steps: (1) Preparation of test solution: accurately weigh a sample of Greater celandine, accurately add an extraction solvent and weigh it, extract it, and pretreat the sample extract by column chromatography to obtain a test solution; (2) Preparation of a control medicinal material solution: taking a control medicinal material of Greater celandine, adding an extraction solvent, extracting, and pretreating the control medicinal material extract by column chromatography to obtain a control medicinal material solution; (3) Preparation of reference substance solution: using at least two of magnolamine, coptisine hydrochloride, tetrahydrocoptisine, hydroprotopine, chelidonine, dihydrochelerythrine and dihydrosanguinarine as reference substances, and adding reference substance solvents respectively to obtain reference substance solutions; (4) Acquisition of characteristic spectrum: The test solution, the reference medicinal material solution and the reference substance solution are respectively detected by ultra-high performance liquid chromatography to obtain a characteristic spectrum of the celandine sample; Wherein, the celandine sample is the celandine formula granules or the celandine medicinal material, decoction pieces, standard decoction or water extract used to prepare the formula granules.

2. The method according to claim 1, characterized in that The chromatographic conditions of the column chromatography include: using aluminum oxide as the stationary phase and alcohol or a mixture of alcohol and halogenated hydrocarbon as the eluent; Preferably, the stationary phase is neutral alumina; and / or, the amount of the stationary phase is 4 to 8 g / g celandine sample; and / or, Preferably, the eluent is methanol or a mixture of methanol and dichloromethane; and / or, the amount of the eluent is 20 to 80 mL / g celandine sample, preferably 60 to 80 mL / g celandine sample.

3. The method according to claim 1 or 2, characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatography method include: using octadecyl embedded amide group bonded silica gel as the stationary phase, using a binary system consisting of nitrile and phosphate buffer as the mobile phase, using gradient elution as the elution mode, and the detection wavelength is 250-300nm, preferably 280nm; Preferably, the chromatographic column using the stationary phase is an Acclaim PA2 chromatographic column; and / or, Preferably, the nitrile is acetonitrile; and / or the phosphate buffer is a potassium dihydrogen phosphate aqueous solution with a pH of 2.5 to 4.5, preferably 3.0 to 4.0, and more preferably 3.0 to 3.5, and preferably the concentration of the potassium dihydrogen phosphate aqueous solution is 0.005-0.01 mol / L.

4. The method according to any one of claims 1 to 3, characterized in that: Using at least one of coptisine hydrochloride and dihydrosanguinarine and magnolia alkaloids, tetrahydrocoptisine, hydroprotopine, chelidonine, and dihydrochelerythrine as reference substances; Preferably, magnolia pine, coptisine hydrochloride, tetrahydrocoptisine, hydroprotopine, chelidonine, dihydrochelerythrine and dihydrosanguinarine are used as reference substances.

5. The method according to any one of claims 1 to 4, characterized in that: The extraction solvent is water or 10% to 100% v / v, preferably 50% to 100% v / v, and more preferably 80% to 100% v / v methanol aqueous solution.

6. The method according to any one of claims 1 to 5, characterized in that: The extraction is one of ultrasonic treatment, heating reflux or shaking extraction, preferably ultrasonic treatment.

7. The method according to claim 3, characterized in that The procedure of the gradient elution is as follows: from 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%; from 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%; from 18 to 22 min, the volume fraction of mobile phase A is 70%, and the volume fraction of mobile phase B is 30%; from 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%; 22.5-27 min, the volume fraction of mobile phase A is 15%, and the volume fraction of mobile phase B is 85%.

8. The method according to claim 3, characterized in that The chromatographic conditions of the ultra-high performance liquid chromatography method also include: a flow rate of 0.2 to 0.4 mL / min, preferably 0.3 to 0.4 mL / min; and a column temperature of 15 to 50° C., preferably 15 to 25° C.

9. The method according to any one of claims 1 to 8, characterized in that: The characteristic spectrum has at least ten characteristic peaks; Preferably, peak 4 corresponding to the chromatographic peak of coptisine hydrochloride reference substance is taken as peak S1, and the relative retention times of peak 1, peak 2, peak 3, peak 5 and peak S1 are calculated; peak 10 corresponding to the chromatographic peak of dihydrosanguinarine reference substance is taken as peak S2, and the relative retention times of peaks 6 to 9 and peak S2 are calculated, and the relative retention times are all within the range of ±10% of the specified values, and the specified values ​​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).

10. Use of the characteristic spectrum of celandine samples constructed according to the method according to any one of claims 1 to 9 in quality detection of celandine samples.

11. A quality detection method for celandine granules, comprising the following steps: The product to be tested is used as a test product, a characteristic spectrum is constructed according to the method described in any one of claims 1 to 9, and the obtained characteristic spectrum is compared with the characteristic spectrum constructed by the method described in any one of claims 1 to 9 to obtain a quality test result.

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