Isolation method, characteristic chromatogram and construction method of five substances in yibei mother

Abstract

The application discloses a separation method, characteristic spectrum and construction method of five substances in Ibeimu. The separation method comprises the following steps: subjecting a test sample solution to high performance liquid chromatography-evaporative light scattering detector detection to separate the components to be detected; the components to be detected comprise peimisine, sipeimine, ibeimine A, sipeimine and sushabeimine; and the preparation method of the test sample solution comprises the following steps: extracting alkaloids from Ibeimu standard decoction or pieces by using an ammonia solution to obtain a mixed solution; and heating and refluxing the mixed solution and a mixed solvent. The separation method can effectively separate the substances simultaneously, and the separation time is relatively short. The application establishes a quality evaluation method for qualitatively evaluating the Ibeimu standard decoction, and the method is relatively systematic, simple, easy to implement, specific and good in repeatability. The characteristic spectrum of the pieces and the standard decoction has high similarity. The method can be used for quality control of the Ibeimu standard decoction, and provides basic data support for subsequent formula granules and other researches.

Description

Technical Field

[0001] This invention specifically relates to a method for separating five substances from Fritillaria thunbergii, its characteristic chromatograms, and a method for constructing them. Background Technology

[0002] Fritillaria thunbergii, a precious and authentic medicinal herb of Xinjiang Uygur Autonomous Region, is mainly distributed in the Ili River Basin and belongs to the Fritillaria genus of the Liliaceae family. Fritillaria walujewii Regel or Ili Fritillaria Fritillaria pallidiflora The dried bulb of Schrenk. Fritillaria thunbergii is slightly cold in nature, bitter in taste, and enters the lung and heart meridians. Clinically, it is often used to treat hemoptysis, dry cough due to lung heat, and other symptoms. It is a commonly used medicinal material in traditional Chinese medicine production and prescriptions. Fritillaria thunbergii contains various components such as alkaloids, saponins, polysaccharides, coumarins, starches, and flavonoids. Among these, the medicinally active ingredient, isosteroidal alkaloids, has been studied in greater depth and possesses pharmacological effects such as clearing heat and moistening the lungs, resolving phlegm and relieving cough, relieving asthma, antispasmodic, antibacterial, and antioxidant properties.

[0003] Standardized decoctions of traditional Chinese medicine (TCM) serve as a crucial bridge to improving the quality of TCM granules, ensuring accurate clinical medication and consistent dosage. The quality standards for standardized TCM decoctions combine quantitative and qualitative aspects. Quantitative analysis primarily consists of parameters such as the content and transfer rate of indicator components, yield, pH, and relative density. Qualitative analysis focuses on HPLC-ELSD characteristic chromatograms and thin-layer chromatography identification, which best reflect the overall composition. Existing literature on *Fritillaria cirrhosa* mainly focuses on the raw materials and artificial cultivation, with no reports on its standardized decoctions. Therefore, establishing a quality evaluation system for standardized decoctions not only helps ensure medication safety and efficacy but also contributes to the inheritance and research of TCM decoctions, granules, and classic prescriptions.

[0004] Characteristic chromatograms, as a whole, are more effective than single-component or even multi-component qualitative and quantitative methods for quality control of a drug. However, no research reports on the characteristic chromatograms of Fritillaria thunbergii have been found in the current literature. Therefore, in order to better monitor the content of each component in Fritillaria thunbergii, there is an urgent need in this field to establish a simple and convenient characteristic chromatogram for Fritillaria thunbergii. This is also of great significance for more effectively controlling the quality of Fritillaria thunbergii and comprehensively and objectively evaluating it. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiency of the lack of research on standard decoction of fritillaria tablets in the prior art, and to provide a method for separating five substances in fritillaria, characteristic chromatograms and construction method.

[0006] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0007] This invention provides a method for separating five substances from Fritillaria thunbergii, which includes the following steps: performing high performance liquid chromatography-evaporative light scattering detector on the test solution to separate the analytes;

[0008] The components to be tested include: fritillary, fritillary glycoside, iberine A, fritillary alkaloid and fritillary alkaloid;

[0009] The preparation method of the test solution includes the following steps: S1 Extracting alkaloids from the standard decoction of Fritillaria thunbergii or Fritillaria thunbergii slices using an ammonia solution to obtain a mixed solution; S2 Heating the mixed solution and a mixed solvent under reflux to obtain the test solution.

[0010] Mobile phase A is a 0.1-0.5% aqueous solution of triethylamine, and mobile phase B is a mixed solution of acetonitrile and triethylamine; in mobile phase B, the volume ratio of acetonitrile to triethylamine is (99.50-99.90):(0.10-0.50).

[0011] The gradient elution conditions for mobile phase A and mobile phase B are as follows: 0-20 min, 35%→42% mobile phase B; 20-40 min, 42%→64% mobile phase B; 40-50 min, 64%→90% mobile phase B; 50-55 min, 90%→35% mobile phase B.

[0012] In this invention, the mobile phase A is preferably a 0.2-0.3% triethylamine aqueous solution, for example, a 0.25% triethylamine aqueous solution.

[0013] In this invention, the volume ratio of acetonitrile to triethylamine in the mobile phase B is preferably 99.75:(0.20-0.30), for example 99.75:0.25.

[0014] In this invention, "0-20 min, 35%→42% mobile phase B" generally refers to the volume ratio of the mobile phase gradually changing from 35% to 42% within the range of 0-20 min.

[0015] In this invention, the preferred chromatographic column is the Zafex Supfex AQ-C18 column.

[0016] The Zafex Supfex AQ-C18 column can have a size of 4.6 mm × 150 mm and a diameter of 5 μm.

[0017] In this invention, the column temperature is preferably 25-40℃, for example 30℃ or 35℃.

[0018] In this invention, the flow rate of the test solution is preferably 0.6-1.5 mL / min, more preferably 1 mL / min.

[0019] In this invention, the injection volume is preferably 15-25µL, and more preferably 20µL.

[0020] In this invention, the preferred parameters of the evaporative light scattering detector are: drift tube temperature 102℃, gas flow rate 3.0L / min, and gain value 2.

[0021] In a preferred embodiment of the present invention, the detection conditions of the high-performance liquid chromatography-evaporative light scattering detector are as follows: the chromatographic column is a Zafex Supfex AQ-C18 column (4.6 mm × 150 mm, 5 μm); mobile phase A is a 0.25% triethylamine aqueous solution, and mobile phase B is acetonitrile:triethylamine (volume ratio 99.75:0.25); gradient elution (0-20 min, 35%→42%B; 20-40 min, 42%→64%B; 40-50 min, 64%→90%B; 50-55 min, 90%→35%B); column temperature 30℃; flow rate 1 mL / min; ELSD detector: drift tube temperature 102℃, gas flow rate 3.0 L / min, gain value 2; injection volume 20 μL.

[0022] In this invention, a mixed reference solution is generally also prepared. The preparation method of the mixed reference solution generally includes the following steps: accurately weighing appropriate amounts of fritillaria syringa, fritillaria alkaloid, iberine alkaloid A, fritillaria alkaloid, and fritillaria alkaloid reference standards, and adding methanol to prepare a solution containing 200 mg / mL of each. g of mixed reference solution.

[0023] In this invention, the high-performance liquid chromatography-evaporative light scattering detector detection method preferably includes the following steps:

[0024] S11 Prepare a mixed reference solution and a test solution respectively, and analyze the mixed reference solution and the test solution using the above detection method to obtain high performance liquid chromatograms of the mixed reference solution and the test solution respectively;

[0025] S12 Based on the high performance liquid chromatogram of the mixed reference solution, determine the compounds corresponding to each peak in the high performance liquid chromatogram of the test solution, and obtain the peak areas of the corresponding compounds in the mixed reference solution and the test solution.

[0026] In S11, the mixed reference solution can be prepared using a conventional solvent that can dissolve it, such as methanol.

[0027] In S11, the concentration of the mixed reference solution can be conventional in the art, and can be 30-500 μg / mL, for example 200 μg / mL.

[0028] In the preparation method of the test sample solution, in S1, the preparation method of the fritillaria cirrhosa standard decoction can be conventional in the art.

[0029] In a preferred embodiment, the preparation method of the standard decoction of Fritillaria thunbergii may include the following steps: Take 200 g of dried Fritillaria thunbergii slices and place them in a 3 L electrically controlled temperature ceramic automatic Chinese medicine pot. Add water and decoct twice. For the first decoction, add 7 times the amount of water, soak for 1 h, heat to a boil and decoct for 40 min. Filter through a 200-mesh filter cloth while hot, and squeeze the filter cloth until no obvious filtrate seeps out. For the second decoction, add 6 times the amount of water, heat to a boil and decoct for 30 min. Filter through a 200-mesh filter cloth while hot. Combine the two filtrates, transfer the filtrate to a round-bottom flask, and concentrate under low temperature and reduced pressure to 500 mL to obtain the decoction.

[0030] In the preparation method of the test sample solution, in S1, the yield of the standard decoction of Fritillaria cirrhosa is preferably 6.06%-10.12%, for example 6.06%, 6.43%, 6.44%, 6.75%, 7.41%, 7.56%, 7.58%, 7.63%, 7.76%, 8.00%, 8.48%, 8.96%, 9.02%, 9.23%, or 10.12%.

[0031] In the preparation method of the test solution, in step S1, the average yield of the standard decoction of Fritillaria cirrhosa is preferably 7.83%.

[0032] In the preparation method of the test sample solution, in S1, the pH value of the Fritillaria cirrhosa standard decoction is preferably 4.70-6.38, for example 4.70, 5.53, 5.55, 5.96, 6.13, 6.14, 6.19, 6.23, 6.24, 6.26, 6.27, 6.29, 6.35, 6.36 or 6.38.

[0033] In the preparation method of the test sample solution, in S1, the relative density value of the Fritillaria cirrhosa standard decoction is preferably 1.014-1.024, for example 1.014, 1.015, 1.016, 1.018, 1.019, 1.021, 1.024 or 1.022.

[0034] In the preparation method of the test solution, in S1, the volume-to-mass ratio of the ammonia solution and the fritillaria cirrhosa standard decoction can be (2-10) mL: (5.0-10.0) g, for example 2 mL: 7.5 g, 4 mL: 7.5 g, 6 mL: 7.5 g, 8 mL: 5.0 g, 8 mL: 7.5 g or 8 mL: 10 g.

[0035] In the preparation method of the test solution, in S1, the volume-to-mass ratio of the ammonia solution and the fritillaria cirrhosa slices can be (2-10) mL:3.0 g, for example, 2 mL:3.0 g, 4 mL:3.0 g, 6 mL:3.0 g or 8 mL:3.0 g.

[0036] In the preparation method of the test solution, in S1, the ammonia solution generally refers to undiluted, pure ammonia solution.

[0037] In the preparation method of the test solution, in S1, the extraction method can be conventional in the art, generally involving standing the standard decoction of Fritillaria thunbergii or the slices of Fritillaria thunbergii in the ammonia solution.

[0038] The settling time can be 0.3-2.5 hours, for example, 0.5 hours, 1 hour or 2 hours.

[0039] In the preparation method of the test solution, in step S2, the mixed solvent can be a mixed solution of haloalkanes and alcohol solvents.

[0040] The haloalkane is preferably dichloromethane or trichloromethane.

[0041] The preferred alcohol solvent is methanol.

[0042] The volume ratio of the haloalkane to the alcohol solvent is preferably (3.5-4.5):1, for example, 4:1.

[0043] In a preferred embodiment, the mixed solvent may be dichloromethane-methanol (volume ratio 4:1) or trichloromethane-methanol (volume ratio 4:1).

[0044] In the preparation method of the test solution, in S2, the mass-volume ratio of the fritillaria cirrhosa standard decoction and the mixed solvent can be 7.5g:(15-65)mL, for example 7.5g:20mL, 7.5g:40mL or 7.5g:60mL.

[0045] In the preparation method of the test solution, in S2, the mass-to-volume ratio of the fritillaria cirrhosa slices and the mixed solvent can be 3.0g:(15-65)mL, for example 3.0g:20mL, 3.0g:40mL or 3.0g:60mL.

[0046] In the preparation method of the test solution, in step S2, the temperature of the heating reflux is preferably 45-85℃, for example 50℃, 60℃, or 80℃.

[0047] In the preparation method of the test solution, in step S2, the heating reflux time is preferably 0.3-3.5 h, for example 0.5 h, 1 h, 2 h or 3 h.

[0048] In this invention, the product obtained in step S2 is generally cooled.

[0049] In the method for preparing the test solution, after step S2, preferably, the following steps are also performed:

[0050] S3 further dissolves the product obtained in S2 using the mixed solvent described in S2 (to make up for the weight loss due to heating and reflux) to obtain a second mixture;

[0051] S4 The second mixture is filtered once, the solvent is removed from the filtrate, and then dissolved in methanol solvent. After a second filtration, the sample to be tested is obtained.

[0052] In S4, the primary filtration method can be conventional in the art, generally using filter paper to remove impurities visible to the naked eye.

[0053] In S4, the secondary filtration method can be conventional in the art. For example, an organic microporous membrane can be used for filtration, preferably an organic microporous membrane with a pore size of 0.22µm or 0.45µm. According to common knowledge in the art, the filtration is microfiltration.

[0054] In S4, the alcohol solvent may be methanol.

[0055] In S4, the volume of the sample to be tested can be 0.25-0.75 times the volume of the filtrate taken before solvent removal, for example, 0.5 times.

[0056] In a preferred embodiment of the present invention, the weight loss is made up with a mixed solution of chloroform and methanol (volume ratio 4:1), shaken well, filtered through filter paper, 10 mL of the filtrate is accurately measured, concentrated to dryness under reduced pressure, the residue is dissolved in methanol and diluted to a volumetric flask of 5 mL, shaken well, filtered through a 0.45 µm organic microporous membrane, and the filtrate is obtained.

[0057] In a preferred embodiment of the present invention, the method for preparing the test solution includes the following steps:

[0058] Add 8 mL of undiluted ammonia water to 7.5 g of standard fritillaria decoction or 3.0 g of fritillaria slices, let stand for 0.5 h, add 20 mL of dichloromethane-methanol (volume ratio 4:1) mixed solution, mix well, heat under reflux in a 60℃ water bath for 2 h, cool and filter, concentrate the filtrate to dryness under reduced pressure, dissolve the residue in methanol, and make up to 5 mL in a volumetric flask, shake well, filter through a 0.45µm organic microporous membrane, and collect the filtrate.

[0059] This invention also provides a characteristic spectrum of *Fritillaria thunbergii*, which includes five common characteristic peaks, with peak number 3 as the control peak. The relative retention times of the common characteristic peaks are as follows:

[0060] The characteristic peak for peak 2 is fritillary, with a relative retention time of 0.610 and an RSD of 0.12%.

[0061] The characteristic peak at point 3 is fritillary alkaloid, with a relative retention time of 1.000 and an RSD of 0.00%.

[0062] Peak 4 is a characteristic peak of ibeline A, with a relative retention time of 1.490 and an RSD of 0.12%.

[0063] The characteristic peak at point 6 is fritillary alkaloid, with a relative retention time of 1.850 and an RSD of 0.18%.

[0064] The characteristic peak of peak 7 is fritillary alkaloid, with a relative retention time of 2.350 and an RSD of 0.16%.

[0065] In this invention, the high performance liquid chromatogram of the fritillaria thunbergii generally also includes peak 1 and peak 5;

[0066] The common characteristic relative retention time of peak 1 is 0.200, and the RSD is 0.25%.

[0067] The common characteristic relative retention time of peak 5 is 1.650, and the RSD is 0.14%.

[0068] The present invention also provides a method for constructing the characteristic spectrum of the aforementioned Fritillaria thunbergii, which includes the following steps: detecting the test solution using high performance liquid chromatography-evaporative light scattering detector, and generating a characteristic spectrum from the detection results;

[0069] The test solution and the detection method using the high-performance liquid chromatography-evaporative light scattering detector are as described above.

[0070] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0071] The reagents and raw materials used in this invention are all commercially available.

[0072] The positive and progressive effects of this invention are as follows:

[0073] In the separation method of the present invention, the peak height is increased and the peak width is narrowed, which reduces the influence of baseline noise. It can not only effectively separate fritillary glycoside, fritillary glycoside A, fritillary glycoside and fritillary glycoside from fritillary bulb, but also has a short separation and detection time, with peaks appearing within 52 minutes.

[0074] The characteristic spectrum established in this invention can isolate 7 compounds, and 5 compounds can be identified by reference standards, namely fritillary, fritillary glycoside, iberine A, fritillary glycoside, and fritillary glycoside.

[0075] This invention establishes a qualitative evaluation method for the quality of a standard decoction of Fritillaria thunbergii. This method is relatively systematic, simple, easy to implement, and exhibits good specificity and repeatability. The characteristic chromatograms of the processed medicinal pieces and the standard decoction show high similarity. It can be used for the quality control of the standard decoction of Fritillaria thunbergii and provides basic data support for subsequent research on formulation granules and other applications.

[0076] The research results of 15 batches of standard decoction of Fritillaria thunbergii prepared in the preferred embodiment show that the components of Fritillaria thunbergii slices from different producing areas are not significantly different. The number of chromatographic peaks and retention times of the slices and the standard decoction are similar, with a similarity of >0.99. This indicates that the standard decoction of Fritillaria thunbergii slices conforms to the basic properties of Fritillaria thunbergii slices, retains the characteristics of the original medicinal slices to the maximum extent, has good overall uniformity, and its quality is stable and controllable.

[0077] Attached illustrations to be added

[0078] Figure 1 Microscopic images of the first batch of Fritillaria cirrhosa slices. Figure 1 A represents the vascular bundles contained in the powder of Fritillaria thunbergii. Figure 1 B represents the starch granules contained in the fritillaria powder.

[0079] Figure 2 The image shows the thin-layer chromatogram of the sample. Serial number 1 is the fritillary alkaloid reference standard; serial number 2 is the reference medicinal material of *Fritillaria yiliense*; serial numbers 3-17 are batches 1-15 of *Fritillaria yiliense* slices.

[0080] Figure 3 The following are thin-layer chromatograms of the samples, where No. 1 is a negative sample; No. 2 is a reference standard for fritillary alkaloids; No. 3 is a reference medicinal material of Fritillaria thunbergii (Ili Fritillaria); and Nos. 4-18 are the standard decoctions of Fritillaria thunbergii from batches 1 to 15.

[0081] Figure 4 This is the high-performance liquid chromatogram of the mixed reference standard. Peak 2 is fritillary glycoside; peak 3 is fritillary glycoside; peak 4 is fritillary glycoside A; peak 6 is fritillary glycoside; and peak 7 is fritillary glycoside.

[0082] Figure 5 Characteristic spectrum of Fritillaria cirrhosa slices (for comparison) Figure 5 D) HPLC overlay chromatograms of the test solution prepared from 15 batches (S1-S15) of Fritillaria cirrhosa slices ( Figure 5 E).

[0083] Figure 6 Characteristic spectrum of the standard decoction of Fritillaria cirrhosa ( Figure 6F) and the HPLC overlay chromatogram of 15 batches (S1-S15) of the standard decoction of Fritillaria cirrhosa (F) Figure 6 G). Detailed Implementation

[0084] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0085] 1. Instruments and reagents

[0086] 1.1 Instruments

[0087] 1260 High Performance Liquid Chromatograph (Agilent Technologies, USA); ELSD-6100 Evaporative Light Scattering Detector (Alltech); DT500B Electronic Balance (Changshu Jiaheng Balance Instrument Co., Ltd.); MS205DU Electronic Balance (Mettler Toledo Instruments (Beijing) Co., Ltd., sensitivity: 0.01 mg); ME204E / 02 Electronic Balance (Mettler Toledo Instruments (Beijing) Co., Ltd.); RE-5205 Rotary Evaporator (Shanghai Yarong Biochemical Instrument Factory); GoodLook-1000 Thin-Layer Chromatography Imaging System (Shanghai Kezhe Biochemical Technology Co., Ltd.); XSP-8CA Microscope (Shanghai Guangmi Instrument Co., Ltd.); Sartorius Laboratory pH Meter (Beijing Sartorius Balance Co., Ltd.); 2% NaOH Silica G Thin-Layer Plate (10 cm × 20 cm, Qingdao Haiyang Chemical Co., Ltd.); HSG254 Silica Pre-Prepared Plate (10 cm × 20 cm). cm, 20190430, Yantai Chemical Industry Research Institute); Electrically controlled temperature ceramic automatic Chinese medicine cooker (Guangdong Midea Household Appliances Manufacturing Co., Ltd.).

[0088] 1.2 Drug Testing

[0089] Acetonitrile (Merck, Inc., Batch No.: 175935, chromatographic grade); Diethylamine (Shanghai Maclean Biochemical Technology Co., Ltd., Batch No.: C11275764, chromatographic grade); Triethylamine (Tianjin Zhiyuan Chemical Reagent Co., Ltd., Batch No. 20220101416, analytical grade); Methanol (Tianjin Beilian Fine Chemicals Development Co., Ltd., Batch No. 20211120, analytical grade); Chloroform (Sinopharm Chemical Reagent Co., Ltd., Batch No. 20210906, analytical grade); Dichloroform (Tianjin Zhiyuan Chemical Reagent Co., Ltd., Batch No. 20220501404, analytical grade); Ammonia (Sichuan Xilong Science Co., Ltd., Batch No. 2109011, analytical grade); Wahaha purified drinking water.

[0090] 1.3 Reference Standards and Samples

[0091] Fritillaria alkaloid reference standard (National Institutes for Food and Drug Control, batch number 111917-201202, purity: 96.4%); Fritillaria alkaloid reference standard (National Institutes for Food and Drug Control, batch number 110767-202111, purity: 99.4%); Fritillaria alkaloid reference standard (National Institutes for Food and Drug Control, batch number 111892-201402, purity: 100%); Fritillaria alkaloid reference standard (Shanghai Hongyong Biotechnology Co., Ltd., batch number 080101-202106, purity: 100%); Iberine A reference standard (Lemeitian Pharmaceutical / Dest Biotechnology, batch number DST-201010-5) 14. Purity: 95%); Fritillaria thunbergii (Ili Fritillaria) reference material (China National Institutes for Food and Drug Control, batch number 121739-201701); For Fritillaria thunbergii with multiple origins, its origin was fixed as Fritillaria thunbergii. 15 Fritillaria thunbergii samples were collected from Ili, Xinjiang Uygur Autonomous Region, the main producing area of ​​Fritillaria thunbergii. The samples were identified by Researcher Feng Ying of the Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, as the bulbs of Fritillaria thunbergii (Ili Fritillaria). Fritillaria thunbergii slices were prepared according to the processing regulations for Fritillaria thunbergii under Part I of the 2020 edition of the Pharmacopoeia of the People's Republic of China (hereinafter referred to as the "Chinese Pharmacopoeia"): removing mud and sand, sun-drying, and then removing the fibrous roots and outer skin. Sample source information is shown in Table 1.

[0092] Table 1. Information on the Source of Fritillaria thunbergii Medicinal Material

[0093]

[0094] 2. Methods and Results

[0095] 2.1 Quality Assessment of Fritillaria cirrhosa Slices

[0096] According to the inspection methods for Fritillaria thunbergii (Ili Fritillaria) in the 2020 edition of the Chinese Pharmacopoeia, 15 batches of Fritillaria thunbergii slices were tested for moisture, ash, extractives, microscopic identification, thin-layer chromatography identification, and content determination. The results showed that all 15 batches of Fritillaria thunbergii slices met the requirements of the Pharmacopoeia. The results are shown in Table 2 below. Figure 1 and Figure 2 .

[0097] Table 2. Quality Inspection Results of 15 Batches of Fritillaria cirrhosa Slices

[0098]

[0099] The test methods for each item in Table 2 are as follows:

[0100] (1) Moisture content determination: Accurately weigh 2 g of crushed Fritillaria cirrhosa slices and place them in a weighing bottle that has been dried to constant weight. Open the bottle cap and dry at 100-105℃ for 5 hours. Close the bottle cap, transfer it to a desiccator, cool for 30 minutes, and accurately weigh it. Dry at the above temperature for another hour, cool, and weigh again until the difference between two consecutive weighings does not exceed 5 mg. Calculate the water content (%) in the sample based on the weight loss. The moisture content should not exceed 15.0%.

[0101] Moisture=(W1-W2 / W1-W0) 100% (W0 weighing bottle constant weight, W1 weighing bottle and sample before drying, W2 weighing bottle and sample after drying)

[0102] (2) Ash content determination: Take 2g of fritillaria powder that can pass through a No. 2 sieve, place it in a crucible heated to constant weight, weigh it, and slowly raise the temperature to 500-600℃ to completely ashed and reach constant weight. Calculate the total ash content of the sample based on the weight of the residue. The ash content shall not exceed 4.5%.

[0103] Ash content = (W2 - W0 / W1 - W0) 100% (W0: mass of crucible at constant weight, W1: weight of crucible and sample before ignition, W2: weight of weighing bottle and sample after ignition)

[0104] (3) Determination of extract: Accurately weigh approximately 4g of Fritillaria cirrhosa slices and place them in a 250-300ml Erlenmeyer flask. Accurately add 100ml of 70% ethanol, seal tightly, and allow to soak under cold conditions. Shake frequently for the first 6 hours, then let stand for 18 hours. Filter quickly using a desiccator. Accurately measure 20ml of the filtrate and place it in an evaporating dish that has been dried to constant weight. Evaporate to dryness on a water bath, dry at 105℃ for 3 hours, cool in a desiccator for 30 minutes, and quickly and accurately weigh. The extract should not be less than 9.0%.

[0105] Leachate = (W1 - W0) V / W (1-moisture%) V1 100% (W0 is the constant weight of the evaporating dish, W1 is the total mass of the evaporating dish and the alcohol-soluble extract, W is the sample weight, V is the amount of 70% ethanol added, and V1 is the volume of the subsequent filtrate taken.)

[0106] (4) Content determination: The content determination method is as specified in Part I of the Chinese Pharmacopoeia 2020, under the section on Fritillaria. The total amount of fritillaria alkaloids and fritillaria alkaloids shall not be less than 0.070%.

[0107] Figure 1 Microscopic images of the first batch of Fritillaria cirrhosa slices. Figure 1 A represents the vascular bundles contained in the powder of Fritillaria thunbergii. Figure 1B represents the starch granules contained in the *Fritillaria thunbergii* powder. According to the 2020 edition of the Chinese Pharmacopoeia under the *Fritillaria thunbergii* entry, a microscopic image of *Fritillaria thunbergii* must show the structures of vessels and starch granules. Therefore, all 15 batches of collected *Fritillaria thunbergii* slices contained the structures (starch granules and vessels) specified in the pharmacopoeia, and thus passed the microscopic identification test.

[0108] The experiment was conducted according to the thin-layer chromatography identification method for Fritillaria thunbergii in the 2020 edition of the Chinese Pharmacopoeia, and the results were obtained. Figure 2 . Figure 2 This is a thin-layer chromatogram of the sample. Serial number 1 is the fritillary alkaloid reference standard; serial number 2 is the reference medicinal material of *Fritillaria yiliense*; serial numbers 3-17 are batches 1-15 of *Fritillaria yiliense* slices. Figure 2 It can be seen that the 15 batches of collected fritillaria cirrhosa slices showed the same color spots in the same positions as the reference standard and reference medicinal materials, which meets the requirements of the pharmacopoeia and is qualified for thin-layer identification.

[0109] get Figure 2 The specific experimental methods are as follows: Take 5 g of each of the 1st to 15th batches of Fritillaria thunbergii powder, add 2 mL of concentrated ammonia solution and 20 mL of chloroform, shake, let stand overnight, filter, evaporate the filtrate to dryness, dissolve the filtrate in 0.5 mL of chloroform, and use this as the test solution for each of the 1st to 15th batches. Separately, take 5 g of Fritillaria thunbergii reference material and prepare a reference material solution using the same method. Separately, take fritillary alkaloid reference standard and prepare a 0.5 mg / mL reference solution with chloroform. According to the thin-layer chromatography method (General Rule 0502), take 2-4 μL of the three solutions and spot them separately on the same silica gel G thin-layer plate prepared with 2% sodium hydroxide solution. Use the lower layer solution of chloroform-ethyl acetate-methanol-water (8:8:3:2) placed below 10℃ as the developing solvent. After development, remove and dry the plate, and spray it with dilute bismuth potassium iodide test solution and sodium nitrite test solution as color developing agents. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material; and spots of the same color appear at the corresponding positions as in the chromatogram of the reference substance.

[0110] 2.2 Preparation of Standard Decoction of Fritillaria thunbergii

[0111] The preparation method of the standard decoction of Fritillaria cirrhosa includes the following steps:

[0112] Take 200 g of dried fritillaria cirrhosa slices and place them in a 3 L electrically controlled temperature ceramic automatic Chinese medicine pot. Add water and decoct twice. For the first decoction, add 7 times the amount of water, soak for 1 hour, heat to a boil and decoct for 40 minutes. Filter through a 200-mesh filter cloth while hot, and squeeze the filter cloth until no obvious filtrate seeps out. For the second decoction, add 6 times the amount of water, heat to a boil and decoct for 30 minutes. Filter through a 200-mesh filter cloth while hot. Combine the two filtrates and transfer them to a round-bottom flask. Concentrate under low temperature and reduced pressure to 500 mL to obtain the final product.

[0113] 2.3 Thin-layer chromatographic identification of the standard decoction of Fritillaria cirrhosa

[0114] Preparation of the test solution: Take 15 g of the standard decoction of Fritillaria cirrhosa, add 2 mL of undiluted ammonia solution and 20 mL of chloroform solution, reflux in a water bath at 80℃ for 1 h, filter, evaporate the filtrate to dryness, add 1 mL of methanol to dissolve, and use as the test solution.

[0115] Preparation of reference solution: Take fritillary alkaloid reference standard and add methanol to prepare a reference solution of 0.5 mg / mL.

[0116] Preparation of negative solution: Take 15 g of water and prepare a negative solution according to the preparation method of the test solution.

[0117] Reference solution of Fritillaria thunbergii (Ili Fritillaria): Take 5g of Fritillaria thunbergii reference material and prepare the reference solution using the same method as the test solution.

[0118] Take 5 μL of the above reference solution, and 10 μL each of the test solution, negative solution, and reference medicinal material solution, and spot them separately onto the same HSGF254 silica gel pre-prepared plate. The developing solvent is ethyl acetate-methanol-ammonia-water (volume ratio 18:2:1:0.1). After development, remove and air dry, spray with dilute bismuth potassium iodide reagent and sodium nitrite reagent, and examine under sunlight. The results are shown in the figure. Figure 3 .

[0119] Figure 3 The images show thin-layer chromatograms of the samples, where number 1 is the negative sample; number 2 is the cisperidin reference standard; number 3 is the *Fritillaria yiliense* (Ili fritillaria) reference medicinal material; and numbers 4-18 are the standard decoctions of *Fritillaria yiliense* from batches 1-15. Figure 3 It can be seen that the spots of the standard decoction of Fritillaria cirrhosa showed consistent color at the corresponding positions of the fritillaria cirrhosa alkaloid reference standard and Fritillaria cirrhosa (Ili Fritillaria cirrhosa) reference material, while the negative sample showed no spots at this position.

[0120] Example 1: Selection of the preparation method for the test solution in the characteristic chromatogram of the standard decoction of Fritillaria cirrhosa.

[0121] Chromatographic conditions: The column was a Zafex Supfex AQ-C18 column (4.6 mm × 150 mm, 5 μm). Mobile phase A was 0.25% triethylamine, and mobile phase B was acetonitrile:triethylamine (99.75:0.25). Gradient elution was used (0–20 min, 35% → 42% B; 20–40 min, 42% → 64% B; 40–50 min, 64% → 90% B; 50–55 min, 90% → 35% B). Column temperature was 30 °C; flow rate was 1 mL / min; ELSD detector: drift tube temperature 102 °C, gas flow rate 3.0 L / min, gain 2; injection volume was 20 μL.

[0122] 1. Investigation of different sample sizes

[0123] Accurately weigh 5 g, 7.5 g, and 10 g of the first batch of *Fritillaria thunbergii* standard decoction prepared from medicinal slices and place them in a round-bottom flask. Add 8 mL of 25% ammonia water (v / v), let stand for 2 h, add 40 mL of chloroform-methanol (4:1) mixed solution, mix well, and heat under reflux in a 60℃ water bath for 2 h. After cooling, filter, concentrate the filtrate to dryness under reduced pressure, dissolve the residue in methanol, transfer and dilute to a 5 mL volumetric flask, shake well, filter, and take the filtrate to obtain the test solution. Perform the determination according to the aforementioned chromatographic conditions. The sample weight, retention time, peak area, and resolution are shown in Table 3 below.

[0124] Table 3. Characteristic Spectrum of Standard Decoction of Fritillaria thunbergii, Sample Size Assessment Table

[0125]

[0126] The test results are shown in the table. When the sample weight of the standard decoction of Fritillaria cirrhosa is 7.5 g, the peak area of ​​the characteristic peak is suitable and the resolution is greater than 1.6. Therefore, the optimal sample weight is 7.5 g.

[0127] 2. Investigation of different ammonia concentrations

[0128] Accurately weigh 7.5 g of the first batch of *Fritillaria cirrhosa* standard decoction prepared from the medicinal slices and place it in a round-bottom flask. Add 8 mL of 25% ammonia water (v / v), 60% ammonia water (v / v), and undiluted ammonia water, respectively. Let stand for 2 h. Add 40 mL of chloroform-methanol (4:1) mixed solution, mix well, and heat under reflux in a 60℃ water bath for 2 h. After cooling, filter, concentrate the filtrate to dryness under reduced pressure, dissolve the residue in methanol, transfer and dilute to a 5 mL volumetric flask, shake well, filter, and collect the filtrate to obtain the test solution. Perform the determination according to the aforementioned chromatographic conditions. The ammonia concentration, retention time, peak area, and resolution are shown in Table 4 below.

[0129] Table 4. Characteristic spectrum of Fritillaria thunbergii standard decoction and ammonia concentration investigation table

[0130]

[0131] The results are shown in the table. When the ammonia water in the standard decoction of Fritillaria thunbergii was undiluted, the characteristic peak separation was more suitable and the peak area was larger. Therefore, the optimal ammonia water concentration was 100%.

[0132] 3. Investigation of different ammonia water volumes

[0133] Accurately weigh 7.5 g of the first batch of *Fritillaria cirrhosa* standard decoction prepared from the medicinal slices and place it in a round-bottom flask. Add 4 ml, 6 ml, and 8 mL of ammonia water respectively, let stand for 2 h, add 40 mL of chloroform-methanol (4:1) mixed solution, mix well, and heat under reflux in a 60℃ water bath for 2 h. After cooling, filter, concentrate the filtrate to dryness under reduced pressure, dissolve the residue in methanol, transfer and dilute to a 5 mL volumetric flask, shake well and filter. Take the filtrate to obtain the test solution. Determine the chromatographic properties according to the aforementioned chromatographic conditions. The ammonia water volume, retention time, peak area, and resolution are shown in Table 5 below.

[0134] Table 5. Characteristic spectrum of Fritillaria cirrhosa standard decoction, ammonia volume analysis table.

[0135]

[0136] The results are shown in the table. When the volume of ammonia water in the extraction of the standard decoction of Fritillaria thunbergii is 8 mL, the characteristic peak separation is more suitable and the peak area is larger. Therefore, the optimal volume of ammonia water is 8 mL.

[0137] 4. Investigation of different ammonia water standing times

[0138] Accurately weigh 7.5 g of the first batch of *Fritillaria cirrhosa* standard decoction prepared from the medicinal slices and place it in a round-bottom flask. Add 8 mL of ammonia water, and let it stand for 0.5 h, 1 h, and 2 h respectively. Add 40 mL of chloroform-methanol (4:1) mixed solution, mix well, and heat under reflux in a 60℃ water bath for 2 h. After cooling, filter, concentrate the filtrate to dryness under reduced pressure, dissolve the residue in methanol, transfer and dilute to a 5 mL volumetric flask, shake well, filter, and collect the filtrate to obtain the test solution. Perform the determination according to the aforementioned chromatographic conditions. The ammonia water standing time, retention time, peak area, and resolution are shown in Table 6 below.

[0139] Table 6. Characteristic spectrum of the standard decoction of Fritillaria thunbergii, and the effect of ammonia water standing time.

[0140]

[0141] The results are shown in the table. When the ammonia water standing time in the extract of Fritillaria cirrhosa standard decoction is 0.5 h, the characteristic peak separation is more suitable, the peak area is larger and time is saved. Therefore, the optimal ammonia water standing time is 0.5 h.

[0142] 5. Investigation of different solvents

[0143] Accurately weigh the first batch of *Fritillaria cirrhosa* slices into seven round-bottom flasks, add 8 mL of ammonia water, let stand for 0.5 h, then add 40 mL of chloroform-methanol (4:1) and chloroform-methanol (4:1) mixed solutions respectively. After mixing thoroughly, heat under reflux in a 60℃ water bath for 2 h, cool, filter, concentrate the filtrate to dryness under reduced pressure, dissolve the residue in methanol, transfer and dilute to a 5 mL volumetric flask, shake well, filter, and collect the filtrate to obtain the test solution. Perform the determination under the aforementioned chromatographic conditions. The different solvents, retention times, peak areas, and resolutions are shown in Table 7 below.

[0144] Table 7. Characteristic chromatograms of Fritillaria cirrhosa slices and their effects on different solvents.

[0145]

[0146] The test results are shown above. When the extraction solvent of Fritillaria cirrhosa slices is dichloromethane-methanol (4:1), the extraction effect is similar to that of chloroform-methanol (4:1). However, chloroform is a precursor chemical and is more harmful to the human body. Therefore, the optimal extraction solvent is dichloromethane-methanol (4:1).

[0147] 6. Different solvent volumes

[0148] Accurately weigh 7.5g of the first batch of *Fritillaria cirrhosa* standard decoction prepared from the medicinal slices and place it in a round-bottom flask. Add 8mL of ammonia water and let stand for 0.5h. Then add 20mL, 40mL, and 60mL of dichloromethane-methanol (4:1) respectively, mix well, and heat under reflux in a 60℃ water bath for 2h. After cooling, filter, concentrate the filtrate to dryness under reduced pressure, dissolve the residue in methanol, transfer and dilute to a 5mL volumetric flask, shake well, filter, and collect the filtrate to obtain the test solution. Perform the determination under the aforementioned chromatographic conditions. The solvent volume, retention time, peak area, and resolution are shown in Table 8 below.

[0149] Table 8. Characteristic Spectrum of Fritillaria cirrhosa Standard Decoction: Solvent Volume Analysis

[0150]

[0151] The results are shown above. When the extraction solvent volume of the *Fritillaria cirrhosa* standard decoction was 20 mL, the characteristic peak separation was good. Considering all factors, the optimal extraction solvent volume was 20 mL.

[0152] 7. Different reflux times

[0153] Accurately weigh 7.5 g of the first batch of *Fritillaria cirrhosa* standard decoction prepared from the medicinal slices and place it in a round-bottom flask. Add 8 mL of ammonia water and let stand for 0.5 h. Then add 40 mL of dichloromethane-methanol (4:1) and mix well. Heat under reflux in a 60℃ water bath for 0.5 h, 1 h, and 2 h, respectively. After cooling, filter and concentrate the filtrate to dryness under reduced pressure. Dissolve the residue in methanol, transfer and dilute to a 5 mL volumetric flask, shake well and filter. Take the filtrate to obtain the test solution. Perform the determination under the aforementioned chromatographic conditions. The reflux time, retention time, peak area, and resolution are shown in Table 9 below.

[0154] Table 9. Characteristic Spectrum of Fritillaria cirrhosa Standard Decoction: Reflux Time Study

[0155]

[0156] The results are shown above. When the reflux time of the Fritillaria cirrhosa standard decoction is 2 h, the characteristic peak area is larger and the separation is better. Taking all factors into consideration, the optimal reflux time is 2 h.

[0157] In summary, the optimal preparation method for the test solution includes the following steps: accurately weigh 7.5 g of the standard decoction of Fritillaria cirrhosa and place it in a round-bottom flask. Add 8 mL of ammonia water and let it stand for 0.5 h. Add 20 mL of dichloromethane-methanol (4:1) and mix well. Heat the mixture under reflux for 2 h in a 60℃ water bath. After cooling, filter the solution. Concentrate the filtrate under reduced pressure to dryness. Dissolve the residue in methanol, transfer the solution to a 5 mL volumetric flask, shake well, filter, and collect the filtrate.

[0158] Example 2: Method for establishing HPLC characteristic chromatograms of fritillaria bulb slices and standard decoction

[0159] 1. Chromatographic conditions

[0160] The chromatographic column was a Zafex Supfex AQ-C18 column (4.6 mm × 150 mm, 5 μm). Mobile phase A was 0.25% triethylamine aqueous solution, and mobile phase B was a mixture of acetonitrile and triethylamine (volume ratio 99.75:0.25). Gradient elution was shown in Table 10. Column temperature was 30℃; flow rate was 1 mL / min; ELSD detector: drift tube temperature 102℃, gas flow rate 3.0 L / min, gain 2; injection volume was 20 μL.

[0161] Table 10 Mobile Phase Gradient Table

[0162]

[0163] 2. Preparation of mixed reference solution: Accurately weigh appropriate amounts of fritillaria syringa, fritillaria alkaloid, iberine alkaloid A, fritillaria alkaloid, and fritillaria alkaloid reference standards, and add methanol to prepare a solution containing 200 mg / mL of each. The mixed standard solution of g is obtained.

[0164] 3. Preparation of the test solution:

[0165] Test solution 1 (Fritillaria thunbergii slices): Accurately weigh about 3 g of Fritillaria thunbergii slices, place them in a round-bottom flask, add 8 mL of undiluted ammonia water, let stand for 0.5 h, add 20 mL of dichloromethane-methanol (4:1) mixed solution to the slices and the standard decoction respectively, mix well, heat under reflux in a 60℃ water bath for 2 h, cool and filter, concentrate the filtrate to dryness under reduced pressure, dissolve the residue in methanol, transfer and make up to volume in a 5 mL volumetric flask, shake well, filter through a 0.45µm organic microporous membrane, and collect the filtrate.

[0166] Test solution 2 (Fritillaria thunbergii standard decoction): Replace about 3 g of raw Fritillaria thunbergii slices in test solution 1 with about 7.5 g of standard decoction. All other operations and conditions are the same as in test solution 1 to obtain test solution 2.

[0167] 4. Accurately pipette 10 μL of test solution 1, 10 μL of test solution 2, and 10 μL of mixed reference solution, and inject them into the high performance liquid chromatograph, measure and record the chromatograms within 60 min.

[0168] The chromatograms were processed using characteristic chromatogram software, resulting in a characteristic chromatogram of Fritillaria cirrhosa slices or standard decoctions. Seven common peaks were observed, from which seven compounds could be separated. Five compounds were identified using reference standards: Fritillaria cirrhosa, Fritillaria cirrhosa glycoside, Fritillaria cirrhosa glycoside A, Fritillaria cirrhosa glycoside, and Fritillaria cirrhosa glycoside. These five common peaks constitute the fingerprint characteristics of Fritillaria cirrhosa slices or standard decoctions, serving as the standard characteristic chromatogram for Fritillaria cirrhosa slices or standard decoctions.

[0169] Example 3 Similarity Evaluation

[0170] The test solutions of 15 batches of Fritillaria cirrhosa slices and corresponding standard decoctions were prepared according to the preparation method of the test solution in Example 2. The obtained test solutions of 15 batches of Fritillaria cirrhosa slices and corresponding standard decoctions were detected under the HPLC chromatographic conditions of Example 2 to generate reference characteristic chromatograms. The characteristic chromatograms of the 15 batches of Fritillaria cirrhosa slices and corresponding standard decoctions were measured, and the similarity was evaluated and calculated using the "Similarity Evaluation System for Chromatographic Characteristic Chromatography of Traditional Chinese Medicine (2012 Edition)".

[0171] Figure 4 This is a high-performance liquid chromatogram of a mixed reference standard. Figure 5 Characteristic spectrum of Fritillaria cirrhosa slices (for comparison) Figure 5 D) HPLC overlay chromatograms of the test solution prepared from 15 batches (S1-S15) of Fritillaria cirrhosa slices ( Figure 5 E). Figure 6 Characteristic spectrum of the standard decoction of Fritillaria cirrhosa ( Figure 6 F) and the HPLC overlay chromatogram of 15 batches (S1-S15) of the standard decoction of Fritillaria cirrhosa (F) Figure 6 G).

[0172] Depend on Figure 5-6 It can be seen that there are a total of 7 peaks in the fritillaria cirrhosa slices or standard decoction. By comparing with the reference standard, 5 characteristic peaks were identified, namely peak 2 fritillaria cirrhosa, peak 3 fritillaria cirrhosa glycoside, peak 4 fritillaria cirrhosa glycoside A, peak 6 fritillaria cirrhosa glycoside, and peak 7 fritillaria cirrhosa glycoside. All 5 characteristic peaks are present in both fritillaria cirrhosa slices and standard decoction.

[0173] The characteristic chromatograms of 15 batches of Fritillaria cirrhosa slices show the relative retention time, relative peak area, and similarity of the peaks as shown in Tables 11-13 below.

[0174] Table 11. Characteristic chromatograms of 15 batches of Fritillaria cirrhosa slices: relative retention times of common peaks.

[0175]

[0176] Table 12. Characteristic Spectra of 15 Batches of Fritillaria thunbergii Slices: Total Peaks and Relative Peak Areas

[0177]

[0178] Table 13 Similarity of Feature Maps of 15 Batches of Medicinal Herbs

[0179]

[0180] Conclusion: Tables 11-13 show that the relative retention times of the characteristic chromatograms of different batches of Fritillaria cirrhosa slices were less than 1%, while the relative peak areas varied significantly. The similarity of the characteristic chromatograms of all 15 batches of Fritillaria cirrhosa slices and the control was greater than 0.90. This indicates that the sample quality is uniform and the homogeneity among batches of slices is good.

[0181] In summary, the similarity of the characteristic chromatograms of the 15 batches of Fritillaria thunbergii slices all exceeded 0.9, with small differences between batches. Five characteristic peaks were identified: fritillarin (peak 2), fritillarin glycoside (peak 3), fritillarin glycoside A (peak 4), fritillarin (peak 6), and fritillarin glycoside (peak 7). The established characteristic chromatogram method for Fritillaria thunbergii slices is stable and reliable, providing a certain scientific method and reference for the value transfer, quality analysis, and evaluation of standard decoctions and formulation granules of Fritillaria thunbergii.

[0182] The characteristic chromatograms of 15 batches of Yi Bei Mu decoction have the following peak relative retention time, relative peak area, and similarity as shown in Tables 14-16 below.

[0183] Table 14. Characteristic chromatograms of 15 batches of Iberia rubra standard decoction: relative retention times of common peaks.

[0184]

[0185] Table 15. Characteristic chromatograms of 15 batches of Iberian Fritillaria standard decoction: common peaks and relative peak areas.

[0186]

[0187] Table 16. Similarity of characteristic chromatograms of 15 batches of Iberia rubra standard decoction

[0188]

[0189] Conclusion: Tables 14-16 show that the relative retention time of the characteristic chromatograms of the 15 batches of Fritillaria cirrhosa standard decoction was less than 1%, and the similarity was greater than 0.90. This indicates that the transfer from medicinal slices to standard decoction is relatively stable, and the consistency of the standard decoction among batches is good.

[0190] In summary, the similarity of the characteristic chromatograms of all 15 batches of Fritillaria cirrhosa slices, standard decoction, and control was >0.99. This indicates that the samples are relatively stable, and the consistency between the slices and standard decoction is good among different batches.

[0191] Example 4: Investigation of Feature Mapping Methodology

[0192] (1) Precision test:

[0193] The experiment was conducted using the 15th batch of Fritillaria cirrhosa slices. Test solution 1 and test solution 2 from Example 2 were injected six times each under the chromatographic conditions described in Example 2. Using fritillaria cirrhosa glycoside (peak 3) as the reference peak S, the RSD of the relative peak area between each characteristic peak and peak S was calculated to be 0.70%–2.15%, and the RSD of the relative retention time was 0.06%–0.25% (n=6), indicating good instrument precision.

[0194] Table 17 Relative Retention Times in Precision Tests

[0195]

[0196] Table 18 Relative Peak Area of ​​Precision Test

[0197]

[0198] (2) Stability test.

[0199] The 15th batch of Fritillaria cirrhosa slices was used for the experiment. Test solutions 1 and 2 from Example 2 were injected at 0, 3, 7, 11, 16, 24, 32, and 40 h according to the chromatographic conditions in Example 2. Using fritillaria cirrhosa glycoside (peak 3) as the reference peak S, the RSD of the relative peak area between the characteristic peak and peak S was calculated to be 1.82%-4.57%, and the RSD of the relative retention time was 0.07%-0.84%, indicating that the test solutions were stable at room temperature for 40 h.

[0200] Table 19 Relative Retention Times in Stability Tests

[0201]

[0202] Table 20 Relative peak areas in stability tests

[0203]

[0204] (3) Repeatability test.

[0205] The experiment was conducted using the 15th batch of Fritillaria cirrhosa slices. Six parallel samples of test solution 1 and six parallel samples of test solution 2 from Example 2 were taken and analyzed under the chromatographic conditions described in Example 2. Using fritillaria cirrhosa glycoside (peak 3) as the reference peak S, the RSD of the relative peak area between the characteristic peak and peak S was calculated to be 0.66%–3.21%, and the RSD of the relative retention time was 0.03%–0.58%, indicating good repeatability of the method.

[0206] Table 21 Relative Retention Time in Repeatability Tests

[0207]

[0208] Table 22 Relative peak area in repeatability tests

[0209]

Claims

1. A method for separating five substances from Fritillaria thunbergii, characterized in that, It includes the following steps: detecting the test solution using high performance liquid chromatography-evaporative light scattering detector to separate the analyte; The components to be tested include: fritillary, fritillary glycoside, iberine A, fritillary alkaloid and fritillary alkaloid; The preparation method of the test sample solution includes the following steps: S1 Extracting alkaloids from the standard decoction of Fritillaria thunbergii or Fritillaria thunbergii slices using an ammonia solution to obtain a mixed solution; S2 Heating the mixed solution and the mixed solvent under reflux to obtain the test sample solution; In the preparation method of the test sample solution, in S1, the volume-to-mass ratio of the ammonia solution to the standard decoction of Fritillaria thunbergii is (6-8) mL: (7.5-10.0) g; In S2, the mixed solvent is a mixture of a haloalkane and an alcohol solvent; the haloalkane is dichloromethane or trichloromethane; the alcohol solvent is methanol; and the volume ratio of the haloalkane to the alcohol solvent is (3.5-4.5):

1. Mobile phase A is a 0.1-0.5% aqueous solution of triethylamine, and mobile phase B is a mixed solution of acetonitrile and triethylamine; in mobile phase B, the volume ratio of acetonitrile to triethylamine is (99.50-99.90):(0.10-0.50). The gradient elution conditions for mobile phase A and mobile phase B are as follows: 0-20 min, 35%→42% mobile phase B; 20-40 min, 42%→64% mobile phase B; 40-50 min, 64%→90% mobile phase B; 50-55 min, 90%→35% mobile phase B; the chromatographic column is a Zafex Supfex AQ-C18 column.

2. The method for separating five substances from Fritillaria thunbergii as described in claim 1, characterized in that, The separation method satisfies one or more of the following conditions (1)-(6): (1) The mobile phase A is a 0.2-0.3% triethylamine aqueous solution; (2) In the mobile phase B, the volume ratio of acetonitrile to triethylamine is 99.75:(0.20-0.30); (3) The column temperature is 25-40℃; (4) The flow rate of the test solution is 0.6-1.5 mL / min; (5) The injection volume is 15-25 µL; (6) The parameters of the evaporative light scattering detector are: drift tube temperature 102℃, gas flow rate 3.0 L / min, and gain value 2.

3. The method for separating five substances from Fritillaria thunbergii as described in claim 1, characterized in that, The separation method satisfies one or more of the following conditions (1)-(5): (1) The mobile phase A is a 0.25% triethylamine aqueous solution; (2) In the mobile phase B, the volume ratio of acetonitrile to triethylamine is 99.75:0.25; (3) The column temperature is 30℃ or 35℃; (4) The flow rate of the test solution is 1 mL / min; (5) The injection volume is 20µL.

4. The method for separating five substances from Fritillaria thunbergii as described in claim 1, characterized in that, The high-performance liquid chromatography-evaporative light scattering detector (ELSD) conditions were as follows: The column was a Zafex Supfex AQ-C18 column, 4.6 mm × 150 mm, 5 μm; mobile phase A was a 0.25% triethylamine aqueous solution, and mobile phase B was a mixture of acetonitrile and triethylamine at a volume ratio of 99.75:0.25; gradient elution conditions were: 0–20 min, 35% → 42% mobile phase B; 20–40 min, 42% → 64% mobile phase B; 40–50 min, 64% → 90% mobile phase B; 50–55 min, 90% → 35% mobile phase B; column temperature 30℃; flow rate 1 mL / min; ELSD detector: drift tube temperature 102℃, gas flow rate 3.0 L / min, gain 2; injection volume 20 μL.

5. The method for separating five substances from Fritillaria thunbergii as described in claim 1, characterized in that, The high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) detection method includes the following steps: S11 Prepare a mixed reference solution and the test solution respectively, and use the detection method to analyze and detect the mixed reference solution and the test solution to obtain high performance liquid chromatograms of the mixed reference solution and the test solution respectively; S12 Based on the high performance liquid chromatogram of the mixed reference solution, determine the compounds corresponding to each peak in the high performance liquid chromatogram of the test solution, and obtain the peak areas of the corresponding compounds in the mixed reference solution and the test solution.

6. The method for separating five substances from Fritillaria thunbergii as described in claim 5, characterized in that, In S11, the concentration of the mixed reference solution is 30-500 μg / mL.

7. The method for separating five substances from Fritillaria thunbergii as described in claim 5, characterized in that, In S11, the concentration of the mixed reference solution is 200 μg / mL.

8. The method for separating five substances from Fritillaria thunbergii as described in claim 1, characterized in that, The preparation method of the test solution satisfies one or more of the following conditions (1)-(7): (1) In S1, the volume-to-mass ratio of the ammonia solution and the fritillaria cirrhosa standard decoction is 6 mL: 7.5 g, 8 mL: 7.5 g or 8 mL: 10 g; (2) In S1, the volume-to-mass ratio of the ammonia solution to the fritillaria cirrhosa slices is (2-10) mL: 3.0 g; (3) In S1, the extraction method includes the following steps: letting the standard decoction of Fritillaria thunbergii or the slices of Fritillaria thunbergii stand in the ammonia solution; (4) In S2, the mass-to-volume ratio of the standard decoction of Fritillaria thunbergii and the mixed solvent is 7.5 g: (15-65) mL; (5) In S2, the mass-to-volume ratio of the fritillaria bulb slices to the mixed solvent is 3.0 g: (15-65) mL; (6) In S2, the temperature of the heating reflux is 45-85℃; (7) In S2, the heating reflux time is 0.3-3.5h.

9. The method for separating five substances from Fritillaria thunbergii as described in claim 8, characterized in that, The preparation method of the test solution satisfies one or more of the following conditions (1)-(3): (1) In S1, the volume-to-mass ratio of the ammonia solution to the fritillaria cirrhosa slices is 2 mL: 3.0 g, 4 mL: 3.0 g, 6 mL: 3.0 g, or 8 mL: 3.0 g; (2) The settling time is 0.3-2.5 hours; (3) The volume ratio of the haloalkane and the alcohol solvent is 4:

1.

10. The method for separating five substances from Fritillaria thunbergii as described in claim 8, characterized in that, The settling time is 0.5h, 1h or 2h.

11. The method for separating five substances from Fritillaria thunbergii as described in claim 8, characterized in that, The preparation method of the test solution satisfies one or more of the following conditions (1)-(4): (1) In S2, the mass-volume ratio of the fritillaria cirrhosa standard decoction and the mixed solvent is 7.5g:20 mL, 7.5g:40 mL or 7.5g:60 mL; (2) In S2, the mass-to-volume ratio of the fritillaria cirrhosa slices and the mixed solvent is 3.0g:20 mL, 3.0g:40 mL, or 3.0g:60 mL; (3) In S2, the temperature of the heating reflux is 50°C, 60°C or 80°C; (4) In S2, the heating reflux time is 0.5 h, 1 h, 2 h or 3 h.

12. The method for separating five substances from Fritillaria thunbergii as described in claim 1, characterized in that, The method for preparing the test solution includes the following steps: Add 8 mL of ammonia water to 7.5 g of standard fritillaria decoction or 3.0 g of fritillaria slices, let stand for 0.5 h, add 20 mL of a mixture of dichloromethane and methanol in a volume ratio of 4:1, mix well, heat under reflux in a 60℃ water bath for 2 h, cool and filter, concentrate the filtrate to dryness under reduced pressure, dissolve the residue in methanol, and make up to 5 mL in a volumetric flask, shake well, filter through a 0.45µm organic microporous membrane, and collect the filtrate.

13. A method for constructing a characteristic spectrum of Fritillaria thunbergii, comprising the following steps: detecting a test solution using high performance liquid chromatography-evaporative light scattering detector, and generating a characteristic spectrum from the detection results; The test solution and the detection method using high-performance liquid chromatography-evaporative light scattering detector are both as described in any one of claims 1-12.

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