Construction method of characteristic map of artemisia sacopiformis and preparation thereof

Abstract

The present application relates to the technical field of traditional Chinese medicine detection, and specifically provides a characteristic spectrum construction method of Herba Centipedae and its preparation. A specific elution procedure is obtained through a large number of experiments by using octadecylsilane bonded silica gel as a filler and a mobile phase including methanol and an acid-containing aqueous solution for gradient elution, 13 or more common characteristic peaks are obtained, and effective separation of characteristic peaks including neochlorogenic acid, caffeic acid, chlorogenic acid, cryptochlorogenic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, big pothos glycoside and thymotin lactone ketone is achieved, the peak shape is good, the baseline is smooth, the determined components can be completely separated, the detection time is short, and the method provides a basis for quality detection and control of Herba Centipedae and its preparation, can fully reflect the integrity and characteristics of Herba Centipedae and its preparation, and is simple to operate and applicable to quality control of different preparation types of Herba Centipedae.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine detection technology, specifically to a method for constructing characteristic spectra of *Gnaphalium affine* and its preparations. Background Technology

[0002] *Carpesium abrotanoides* L., a plant in the Asteraceae family, is the dried, mature fruit of the herb. It is harvested in autumn when the fruit is ripe, dried, and impurities removed. It is mainly used to treat ascariasis, pinworm infection, tapeworm infection, abdominal pain due to intestinal parasites, and infantile malnutrition. According to literature reports, the main chemical components of *Carpesium abrotanoides* are sesquiterpenoids (such as abrotanoid lactone and abrotanoid lactone alcohol), fatty acids, and organic acids (such as chlorogenic acid). The standard decoction of *Carpesium abrotanoides* is obtained through extraction, concentration, and drying of the herb.

[0003] The 2020 edition of the Chinese Pharmacopoeia includes quality control requirements for Artemisia annua (plant), such as the original plant variety, processing of the processed slices, characteristics of the slices, and physicochemical identification, but does not include content determination. Chinese patent CN116754688A discloses a detection method and quality control method for Artemisia annua, using octadecylsilane-bonded silica gel as a filler; methanol as mobile phase A; and 0.2% phosphoric acid aqueous solution as mobile phase B, eluting according to a specific gradient elution program, resulting in a chromatogram containing eight characteristic peaks. However, this patent has relatively few characteristic peaks, and the detection time is at least 95 minutes, which is time-consuming and cannot comprehensively and holistically detect and control the quality of Artemisia annua and its preparations. Summary of the Invention

[0004] The purpose of this invention is to provide a method for constructing characteristic spectra of grasshoppers and their preparations. The characteristic spectra constructed using the method of this invention have at least 13 characteristic peaks with good peak separation. Using the characteristic spectra for quality detection can improve the accuracy of the analytical results.

[0005] Therefore, this invention discloses a method for constructing a characteristic spectrum of the grasshopper and its preparations, comprising the following steps:

[0006] (1) Preparation of the test solution;

[0007] (2) The test solution was analyzed by ultra-high performance liquid chromatography. The packing material was octadecylsilane-bonded silica gel, and the mobile phase included methanol and acidic aqueous solution.

[0008] Gradient elution was performed, and the gradient elution program included: 0 → 12 minutes → 18 minutes → 20 minutes → 25 minutes → 28 minutes → 34 minutes → 42 minutes, with the volume percentage of methanol in the mobile phase being 7% → 18% → 30% → 35% → 40% → 45% → 47% → 60%.

[0009] Further, step (1) includes:

[0010] 1) Take the analyte, add solvent to extract, and obtain the extract;

[0011] 2) Separate the solid and liquid components of the extract, and take the liquid, which is the test solution;

[0012] Preferably, step (1) also satisfies any one or more of the following AEs:

[0013] A. In step 1), the solvent is selected from at least one of water, methanol, and ethanol;

[0014] B. In step 1), the mass ratio of the analyte to the volume of the solvent is 0.1-2 g: 10-250 mL;

[0015] C. In step 1), the extraction method is reflux extraction or ultrasonic extraction, and the extraction time is 15 min-120 min.

[0016] D. In step 2), the solid-liquid separation is performed by centrifugation or filtration;

[0017] E. In step 1), the test substance is selected from one or more of the following: *Hemiptera chinensis* medicinal materials, *Hemiptera chinensis* slices, *Hemiptera chinensis* granules, *Hemiptera chinensis* decoction, and *Hemiptera chinensis* powder.

[0018] Further, the acidic aqueous solution is selected from at least one of formic acid aqueous solution and phosphoric acid aqueous solution; preferably, the volume percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.1-0.4%; preferably, the volume percentage of formic acid in the formic acid aqueous solution is 0.05-0.1%.

[0019] Furthermore, the chromatographic conditions for ultra-high performance liquid chromatography in step (2) also include: a detection wavelength of 210-280 nm, a flow rate of 0.23-0.27 ml / min, a column temperature of 25-33 °C, a column length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.6-1.8 μm; preferably, the detection wavelength is 240 nm; preferably, the column temperature is 30 °C; preferably, the injection volume is 2 μl.

[0020] Furthermore, the gradient elution program also includes 42 minutes → 43 minutes, with the volume percentage of methanol in the mobile phase being 60% → 7%, or the gradient elution program also includes 42 minutes → 43 minutes → 48 minutes, with the volume percentage of methanol in the mobile phase being 60% → 7% → 7%.

[0021] Furthermore, the construction method further includes the step of preparing a reference solution using at least one of the following reference standards: neochlorogenic acid, caffeic acid, chlorogenic acid, cryptochlorogenic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, cosmososide, and gentamicin lactone; and the step of detecting the reference solution using ultra-high performance liquid chromatography according to any of the construction methods to obtain a reference standard reference spectrum.

[0022] Furthermore, the characteristic spectra of the *Gnaphalium affine* and its preparations obtained by the construction method are selected from any one of the following (1)-(2):

[0023] (1) The characteristic chromatogram of the grasshopper and its preparation has 13 characteristic peaks. With peak 9 as the reference peak, the relative retention times of characteristic peaks 1-8 and peaks 10-13 with the reference peak are within ±10% of the specified values. The specified values ​​are: 0.21 (peak 1), 0.25 (peak 2), 0.28 (peak 3), 0.48 (peak 4), 0.49 (peak 5), 0.56 (peak 6), 0.71 (peak 7), 0.93 (peak 8), 1.06 (peak 10), 1.09 (peak 11), 1.24 (peak 12), and 1.39 (peak 13).

[0024] (2) The characteristic chromatogram of the *Gnaphalium affine* and its preparation has 13 characteristic peaks. The peaks corresponding to the reference peaks of 1,5-dicaffeoylquinic acid are reference peaks. The relative retention times of characteristic peaks 1-8 and 10-13 with the reference peaks are within ±10% of the specified values. The specified values ​​are: 0.21 (peak 1), 0.25 (peak 2), 0.28 (peak 3), 0.48 (peak 4), 0.49 (peak 5), 0.56 (peak 6), 0.71 (peak 7), 0.93 (peak 8), 1.06 (peak 10), 1.09 (peak 11), 1.24 (peak 12), and 1.39 (peak 13).

[0025] The present invention also provides the application of the above-described construction method in the quality control of grasshoppers and their preparations.

[0026] The present invention also provides a quality control method for *Gnaphalium affine* and its preparations, comprising the step of comparing the characteristic spectrum of the *Gnaphalium affine* product to be tested with the characteristic spectrum of *Gnaphalium affine* and its preparations; wherein the characteristic spectrum of the *Gnaphalium affine* product to be tested is obtained by using the *Gnaphalium affine* product to be tested according to the above-described construction method, and the characteristic spectrum of *Gnaphalium affine* and its preparations is the aforementioned characteristic spectrum of *Gnaphalium affine* and its preparations.

[0027] This invention also provides a method for identifying the grasshopper and its preparations from counterfeit products, comprising:

[0028] Using the product to be identified as the test sample, the feature spectrum of the product to be identified was constructed according to the above construction method.

[0029] The feature map of the product to be identified is compared with the feature map constructed by the above construction method, and the identification is carried out based on the comparison results;

[0030] The counterfeit product is *Symplocos rubrum* and / or *Symplocos rubrum* preparations.

[0031] The technical solution of this invention has the following advantages:

[0032] 1. The method for constructing characteristic chromatograms of *Gnaphalium affine* and its preparations provided by this invention uses octadecylsilane-bonded silica gel as the packing material, and the mobile phase includes methanol and an acidic aqueous solution. Gradient elution is performed, and a specific elution program is obtained through extensive experimental screening. This yields 13 or more common characteristic peaks and achieves effective separation of characteristic peaks including neochlorogenic acid, caffeic acid, chlorogenic acid, cryptochlorogenic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, cosmososide, and gentianin lactone. The peaks are well-shaped, the baseline is stable, the components can be completely separated, and the detection time is short. This provides a basis for the quality detection and control of *Gnaphalium affine* and its preparations, and can fully reflect the integrity and characteristics of *Gnaphalium affine* and its preparations. The method is simple to operate and can be applied to the quality control of different types of *Gnaphalium affine* preparations.

[0033] 2. The method for constructing the characteristic chromatogram of *Gnaphalium affine* and its preparations provided by this invention, as identified by reference standards, shows that peaks 3, 4, 5, 6, 8, 9, 10, and 12 are, in order, neochlorogenic acid, caffeic acid, chlorogenic acid, cryptochlorogenic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, cosmososide, and gentamicin lactone. All peaks are well separated and have moderate areas. Using one or more of neochlorogenic acid, caffeic acid, chlorogenic acid, cryptochlorogenic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, cosmososide, and gentamicin lactone as references allows for precise qualitative quality control of *Gnaphalium affine*. The identification of 13 or more characteristic peaks in the characteristic chromatogram ensures the accuracy of this method for quality control of *Gnaphalium affine*.

[0034] 3. The quality control method for *Heliotropium indicum* and its preparations provided by this invention includes the step of comparing the characteristic chromatogram of the *Heliotropium indicum* product to be tested with the characteristic chromatogram of *Heliotropium indicum* and its preparations. This method can control the material transfer of indicator components in *Heliotropium indicum* medicinal materials, processed slices, granules, decoctions, and powders, and can also control the overall stability of the quality of characteristic components in *Heliotropium indicum* medicinal materials, processed slices, granules, decoctions, and powders. Moreover, the method is simple to operate, has high precision, good stability, good repeatability, high accuracy, and fast analysis rate.

[0035] 4. The method for identifying the grasshopper and its preparations and counterfeit products provided by this invention is highly specific and can quickly and accurately distinguish and identify the grasshopper and its drug preparations, the southern grasshopper and its drug preparations and their adulterants. Attached Figure Description

[0036] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0037] Figure 1 This is a chromatogram of the test solution and the reference solution in Example 1.

[0038] Figure 2 This is a chromatogram of the reference solution of the control medicinal material in Example 1.

[0039] Figure 3 This is the comparative feature map in Example 2.

[0040] Figure 4 It is a superimposed chromatogram of the test solution in Example 3 - Lyophilized powder of *Hemiptera chinensis* standard decoction and lyophilized powder of *Hemiptera chinensis* standard decoction.

[0041] Figure 5 This is the chromatogram of the test solution in Example 4.

[0042] Figure 6 This is the chromatogram of the test solution in Comparative Example 1.

[0043] Figure 7 This is the chromatogram of the test solution in Comparative Example 2.

[0044] Figure 8 This is the chromatogram of the test solution in Comparative Example 3.

[0045] Figure 9 This is an investigation of the chromatogram of the test solution in Experiment Example 1, focusing on the type and concentration of the mobile phase.

[0046] Figure 10 This refers to the selection of wavelength in the chromatogram overlay of the test sample solution in Experiment Example 1.

[0047] Figure 11 This refers to the chromatogram overlay of the test sample solution in Experiment Example 1, specifically the selection of column temperature.

[0048] Figure 12 This is the chromatogram of the test solution in Experiment Example 1. The selected chromatographic column is Waters CORTECS T3.

[0049] Figure 13 This is the chromatogram of the test solution in Experiment Example 1. The selected chromatographic column is YMC aq-C18.

[0050] Figure 14 This is the chromatogram of the negative control solution in Experiment Example 3 - specificity test.

[0051] Figure 15 This is the chromatogram of the test solution in Experiment Example 3 - specificity test.

[0052] Figure label: Peak 3: Neochlorogenic acid; Peak 4: Caffeic acid; Peak 5: Chlorogenic acid; Peak 6: Cryptochlorogenic acid; Peak 8: 1,4-Dicaffeoylquinic acid; Peak 9(S): 1,5-Dicaffeoylquinic acid; Peak 10: Cosmos glycoside; Peak 12: Tianmingling lactone. Detailed Implementation

[0053] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0054] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0055] The instruments and reagents used in this invention are as follows:

[0056] 1. Chromatograph: (1) Waters ACQUITY UPLC I-Class chromatography system, including a quaternary solvent manager (ACQ-QSM), an autosampler (ACQ-FTN), an original imported column oven (ACQ-CM), a diode array UV detector (ACQ-TUV), and an Empower chromatography management system.

[0057] 2. Electronic analytical balances: Mettler Toledo NewClassic MS 0.0001 g balance, Jing TianFA2044A 0.0001 g balance.

[0058] 3. Chromatographic columns: (1) Waters CORTECS T3, 100mm×2.1mm, 1.6μm; (2) Agilent EclipsePlus C18 RRHD, 100mm×2.1mm, 1.8μm; (3) YMC aq-C18 column, 100mm×2.1mm, 1.9μm.

[0059] 4. Reagents: Methanol (chromatographic grade), water (ultrapure water), formic acid (analytical grade).

[0060] 5. Test Drugs: Caffeic acid (110885-201703, 99.7%) and chlorogenic acid (110753-202119, 96.3%) were purchased from the China National Institutes for Food and Drug Control; neochlorogenic acid (DSTDX001503, 98%) and cryptochlorogenic acid (DST210427-035, 98%) were purchased from Lemeitian Pharmaceutical Co., Ltd.; 1,4-dicaffeoylquinic acid (050184-202309, 98%), cosmososide (170088-202106, 98%), and tianming ginsenoside lactone (200162-202311, 98%) were purchased from Shanghai Hongyong Biotechnology Co., Ltd.; 1,5-dicaffeoylquinic acid (000322-202309, 98%) was purchased from Jiangxi Baicaoyuan Biotechnology Co., Ltd.

[0061] The reference material for the herb *Heliotropium indicum* (121131-202102) was purchased from the China National Institutes for Food and Drug Control.

[0062] Preparation method of freeze-dried powder of standard decoction of Artemisia annua: Take Artemisia annua slices, heat and reflux twice for extraction. For the first extraction, add water with a weight of 8 times the weight of the slices and soak for 30 minutes, then heat and reflux for 30 minutes and filter. For the second extraction, add water with a weight of 6 times the weight of the slices and extract for 25 minutes and filter. Combine the filtrates from the two filtrations and concentrate the filtrate to a relative density of 1.05 g / mL (detected at 60℃). Freeze-dry the concentrate to obtain the freeze-dried powder of standard decoction.

[0063] Following the preparation method of the standard decoction freeze-dried powder of *Hemiptera chinensis*, *Hemiptera chinensis* was replaced with *Hemiptera australis* to prepare the standard decoction freeze-dried powder of *Hemiptera australis*.

[0064] Example 1

[0065] This embodiment provides a method for constructing the characteristic spectrum of the grasshopper and its preparations, including the following steps:

[0066] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the stationary phase (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); methanol as mobile phase A and 0.2% phosphoric acid aqueous solution (V:V) as mobile phase B, with gradient elution as specified in Table 1; the flow rate was 0.25 mL / min, the column temperature was 30 °C, and the detection wavelength was 240 nm. The theoretical plate number, calculated based on 1,5-dicaffeoylquinic acid, should not be less than 5000.

[0067] Table 1 Elution gradient

[0068]

[0069]

[0070] Preparation of the reference solution: Take 1g of the reference herb *Heliotropium indicum*, add 50ml of water, heat under reflux for 60 minutes, filter, evaporate the filtrate to dryness, add 25ml of 50% methanol aqueous solution (V:V) to the residue, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution for the reference herb. Take appropriate amounts of neochlorogenic acid, caffeic acid, chlorogenic acid, cryptochlorogenic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, cosmososide, and gentamicin lactone reference standards, accurately weigh them, and prepare a solution containing 0.2 mg of neochlorogenic acid, 0.1 mg of caffeic acid, 0.1 mg of chlorogenic acid, 0.1 mg of cryptochlorogenic acid, 0.1 mg of 1,4-dicaffeoylquinic acid, 0.2 mg of 1,5-dicaffeoylquinic acid, 0.1 mg of cosmososide, and 0.1 mg of gentamicin lactone per ml using 50% methanol aqueous solution (V:V). This solution will serve as the reference standard mixture.

[0071] Take appropriate amounts of neochlorogenic acid, caffeic acid, chlorogenic acid, cryptochlorogenic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, cosmososide, and gentamicin lactone reference standards, accurately weigh them, and prepare individual reference solutions containing 0.2 mg of neochlorogenic acid, 0.1 mg of caffeic acid, 0.1 mg of chlorogenic acid, 0.1 mg of cryptochlorogenic acid, 0.1 mg of 1,4-dicaffeoylquinic acid, 0.2 mg of 1,5-dicaffeoylquinic acid, 0.1 mg of cosmososide, and 0.1 mg of gentamicin lactone per ml using 50% methanol aqueous solution (V:V).

[0072] Preparation of the test solution: Take an appropriate amount of freeze-dried powder of the standard decoction of *Heliotropium indicum*, grind it into a fine powder, take about 0.1 g, place it in a stoppered conical flask, add 25 ml of 50% methanol aqueous solution (V:V), sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0073] The determination method involves precisely pipetting 2 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0074] Test results are shown Figure 1-2Table 2 shows 13 characteristic peaks in the chromatogram of the test solution, which should correspond to the retention times of the 13 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peaks 3, 4, 5, 6, 8, 9, 10, and 12 should correspond to the retention times of the reference peaks of neochlorogenic acid, caffeic acid, chlorogenic acid, cryptochlorogenic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, cosmos glycoside, and gentianin lactone. The peak corresponding to the reference peak of 1,5-dicaffeoylquinic acid is designated as the S peak. The relative retention times of each characteristic peak and the S peak are calculated. The relative retention times are within ±10% of the specified values, which are: 0.21 (peak 1), 0.25 (peak 2), 0.28 (peak 3), 0.48 (peak 4), 0.49 (peak 5), 0.56 (peak 6), 0.71 (peak 7), 0.93 (peak 8), 1.06 (peak 10), 1.09 (peak 11), 1.24 (peak 12), and 1.39 (peak 13).

[0075] Table 2 System Adaptability Parameters

[0076]

[0077] Example 2

[0078] Characteristic chromatograms of 18 batches of freeze-dried powder of standard decoction of *Hemiptera chinensis* were constructed according to the method in Example 1. The results are shown in Tables 3-4.

[0079] Table 3. Relative retention time results of characteristic chromatogram determination of 18 batches of *Heliotropium indicum* decoction (lyophilized powder).

[0080]

[0081]

[0082]

[0083] Table 4. Results of relative peak area determination of characteristic chromatograms of 18 batches of *Heliotropium indicum* decoction (lyophilized powder).

[0084]

[0085]

[0086] Using the fingerprint chromatogram similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission, a reference characteristic chromatogram was generated, such as... Figure 3 As shown in Table 5-6. According to... Figure 3The characteristic chromatograms shown can be used to analyze and compare the detection results of the characteristic chromatograms of the lyophilized powder of the standard decoction, and are used for the quality control of the standard decoction. Characteristic peaks were identified and designated using UPLC and LC / MS / MS, confirming 13 chromatographic peaks with good resolution in the characteristic chromatogram. Based on peak identification and peak localization in the chromatogram of the reference solution, peak 3 was confirmed as neochlorogenic acid, peak 4 as caffeic acid, peak 5 as chlorogenic acid, peak 6 as cryptochlorogenic acid, peak 8 as 1,4-dicaffeoylquinic acid, peak 9 as 1,5-dicaffeoylquinic acid, peak 10 as cosmososide, and peak 12 as gentianin lactone. The chromatographic peak of 1,5-dicaffeoylquinic acid has a retention time in the middle of the chromatogram, a large peak response, and is readily available as a reference. Therefore, 1,5-dicaffeoylquinic acid was used as the S peak of this characteristic chromatogram, and the relative retention times of each characteristic peak were calculated. The relative retention times of each characteristic peak are within ±10% of the specified values, which are: 0.21 (peak 1), 0.25 (peak 2), 0.28 (peak 3), 0.48 (peak 4), 0.49 (peak 5), 0.56 (peak 6), 0.71 (peak 7), 0.93 (peak 8), 1.06 (peak 10), 1.09 (peak 11), 1.24 (peak 12), and 1.39 (peak 13).

[0087] The characteristic chromatograms of 18 batches of freeze-dried powder of standard decoction of *Heliotropium indicum* were compared with the control characteristic chromatograms. The similarity was between 0.969 and 1. The results are shown in Table 7.

[0088] Table 5. Relative retention time of common patterns in standard decoctions for *Heliotropium indicum*

[0089]

[0090] Table 6 Common Pattern Matching Data for Standard Decoction of *Heliotropium indicum*

[0091]

[0092] Table 7. Similarity results of characteristic chromatograms of 18 batches of standard decoction for *Heliotropium indicum*

[0093]

[0094]

[0095] Example 3

[0096] This embodiment provides a method for differentiating Artemisia annua and its preparations from counterfeit products, including the following steps:

[0097] The test solution for the grasshopper was prepared according to the preparation method of the test solution in Example 1.

[0098] The freeze-dried powder of the standard decoction for *Hemiptera chinensis* was replaced with the freeze-dried powder of the standard decoction for *Hemiptera chinensis*, and three batches of *Hemiptera chinensis* test solutions were prepared according to the preparation method of the test solution in Example 1.

[0099] Take the test solutions of *Gnaphalium affine* and *Gnaphalium affine* and perform the determination according to the liquid chromatography method in Example 1.

[0100] Test results are shown Figure 4 The characteristic chromatogram results showed that none of the three batches of *Symplocos edulis* test solutions had the 13 characteristic peaks of *Symplocos edulis*. The identification method of this embodiment can distinguish between the two, which is beneficial for accurate medication.

[0101] Example 4

[0102] This embodiment provides a method for constructing characteristic chromatograms of *Gnaphalium affine* and its preparations. The construction method is basically the same as that in Example 1, except that the gradient elution program in the chromatographic conditions is different. The gradient elution program in this embodiment is shown in Table 8. Detection results are shown in... Figure 5 Table 9. The results in the table show that the 13 characteristic peaks in the chromatogram have good separation effect, good peak shape, and stable baseline.

[0103] Table 8 Gradient elution program

[0104]

[0105] Table 9 Test Results

[0106]

[0107]

[0108] Comparative Example 1

[0109] This comparative example provides a method for constructing characteristic chromatograms of *Gnaphalium affine* and its preparations. The test solution prepared in Example 1 was analyzed under the following chromatographic conditions: Waters CORTECS T3 column (100 mm × 2.1 mm, 1.6 μm), column temperature 30 °C, flow rate 0.3 mL / min, injection volume 1 μL, and gradient elution program as shown in Table 10. Detection results are shown in... Figure 6 The results from the chart show that the chromatographic peaks are concentrated between 22 and 26 minutes, indicating that some peaks were not effectively separated. Therefore, the elution gradient of the mobile phase needs further optimization.

[0110] Table 10 Gradient elution program

[0111]

[0112] Comparative Example 2

[0113] This comparative example provides a method for constructing characteristic chromatograms of *Gnaphalium affine* and its preparations. The test solution prepared in Example 1 was analyzed under the following chromatographic conditions: Waters CORTECS T3 column (100 mm × 2.1 mm, 1.6 μm), column temperature 30 °C, flow rate 0.3 mL / min, injection volume 1 μL, and gradient elution program as shown in Table 11. Detection results are shown in... Figure 7 The results in the chart show that although the method used in this comparative example advanced the chromatographic peak at 22-26 minutes and further improved the separation effect of the chromatographic peak, there are still some peaks with poor separation effect in the chromatogram. Therefore, the elution gradient of the mobile phase needs to be further optimized.

[0114] Table 11 Gradient elution program

[0115]

[0116] Comparative Example 3

[0117] This comparative example provides a method for constructing characteristic chromatograms of *Gnaphalium affine* and its preparations. The test solution prepared in Example 1 was analyzed under the following chromatographic conditions: Waters CORTECS T3 column (100 mm × 2.1 mm, 1.6 μm), column temperature 30 °C, flow rate 0.3 mL / min, injection volume 1 μL, and gradient elution program as shown in Table 12. Detection results are shown in... Figure 8 The results in the chart show that although the method used in this comparative example further improved the separation of the middle chromatographic peak, the separation of the chromatographic peaks in the first 2-4 minutes was still poor.

[0118] Table 12 Gradient elution program

[0119]

[0120] Example 1: Establishment of Liquid Chromatography Conditions

[0121] 1. Investigation of the type and concentration of the mobile phase

[0122] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); methanol was used as mobile phase A, and 0.05% formic acid aqueous solution, 0.1% formic acid aqueous solution, 0.2% phosphoric acid aqueous solution, and 0.4% phosphoric acid aqueous solution (V:V) were used as mobile phase B, respectively, according to the specifications in Table 1; the flow rate was 0.25 mL per minute, the column temperature was 30 °C, and the detection wavelength was 240 nm.

[0123] The test solution was prepared according to the preparation method of the test solution in Example 1, and the test was performed according to the chromatographic conditions under the section "Investigation of Mobile Phase Type and Concentration" and the determination method in Example 1.

[0124] Test results are shown Figure 9 The results show that, compared with formic acid, phosphoric acid, when used as the aqueous elution phase, provides a more stable baseline, symmetrical peak shapes, and better separation. When comparing different concentrations of phosphoric acid, a 0.2% phosphoric acid aqueous solution as the mobile phase exhibits better separation of characteristic peaks; therefore, a methanol-0.2% phosphoric acid aqueous solution is the preferred mobile phase.

[0125] 2. Selection of detection wavelength

[0126] Based on the determination of the mobile phase type and concentration, the effects of detection wavelengths of 210 nm, 240 nm, 260 nm, and 280 nm on the chromatographic peak response of the test sample solution were observed using a full-wavelength detection method.

[0127] Test results are shown Figure 10 As shown in the figure, compared with 210nm, 260nm and 280nm, the baseline in the chromatogram corresponding to the detection wavelength of 240nm is stable and the response is high. Therefore, 240nm is the preferred detection wavelength for the characteristic spectrum.

[0128] 3. Column temperature selection

[0129] Based on the determination of the type and concentration of the mobile phase and the detection wavelength, the test solution was tested at column temperatures of 25℃ and 30℃, respectively. The other liquid phase conditions were the same as those in the section on "Investigation of Mobile Phase Type and Concentration".

[0130] Test results are shown Figure 11 As can be seen from the figure, the separation of each chromatographic peak is better at a column temperature of 30℃. Therefore, 30℃ is the preferred column temperature for the characteristic chromatogram of the grasshopper.

[0131] 4. Selection of chromatographic column

[0132] Based on the determination of the mobile phase type and concentration, detection wavelength, and column temperature, the test solution was detected on Waters CORTECS T3 and YMC aq-C18 columns, respectively. The other liquid phase conditions were consistent with the method under "Investigation of Mobile Phase Type and Concentration".

[0133] Test results are shown Figure 12-13 As can be seen from the figure, the chromatographic peaks under the Waters CORTECS T3 column have good resolution. Therefore, the Waters CORTECS T3 is the preferred chromatographic column for the characteristic chromatogram of the grasshopper.

[0134] 5. Determination of chromatographic conditions

[0135] Based on the above optimization results, the chromatographic conditions for the characteristic chromatograms are determined as follows:

[0136] Octadecylsilane-bonded silica gel was used as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); methanol was used as mobile phase A and 0.2% phosphoric acid aqueous solution was used as mobile phase B, and gradient elution was performed according to the specifications in Table 13; the flow rate was 0.25 ml per minute, the column temperature was 30 °C, and the detection wavelength was 240 nm.

[0137] Table 13 Gradient elution program

[0138]

[0139] Example 2: Establishment of a method for preparing the test solution

[0140] Based on the chromatographic conditions determined in Experiment 1, “Establishment of Liquid Chromatography Conditions”, the preparation method of the test sample solution was further investigated.

[0141] The prepared test solution was tested according to the chromatographic conditions determined under "Establishment of Liquid Chromatography Conditions" in Experiment Example 1.

[0142] 1. Selection of processing method

[0143] The effects of different extraction methods on the extraction efficiency of freeze-dried powder of standard decoction of Artemisia annua were investigated, with the information content of chromatographic peaks and system suitability as the main evaluation indicators.

[0144] Extraction method: Take about 0.1g of freeze-dried powder of standard decoction of *Heliotropium indicum*, add 25ml of 70% methanol aqueous solution (V:V), and perform ultrasonic treatment (300W power, 40kHz frequency) and reflux extraction for 30 minutes each. Remove, cool, weigh again, make up the weight loss with 70% methanol aqueous solution, shake well, filter, and collect the filtrate to obtain the final product.

[0145] The test results are shown in Table 14. The results indicate that the chromatographic peaks obtained by both extraction methods exhibited superior system adaptability parameters, good resolution, and symmetry. There was no significant difference in extraction between the different extraction methods. Considering ease of operation, ultrasonic treatment was the preferred extraction method for the test sample.

[0146] Table 14 Comparison of Extraction Methods and Chromatographic Peak System Adaptability Parameters

[0147]

[0148] 2. Selection of extraction solvent

[0149] Based on the above-determined treatment method, water, ethanol, 30% ethanol aqueous solution, 50% ethanol aqueous solution, 70% ethanol aqueous solution, methanol, 30% methanol aqueous solution, 50% methanol aqueous solution, and 70% methanol aqueous solution (V:V) were selected as solvents to prepare the test solution.

[0150] The test results are shown in Table 15. The results show that, compared with ethanol, 30% ethanol aqueous solution, 50% ethanol aqueous solution, and 70% ethanol aqueous solution, pure water, 30% methanol aqueous solution, 50% methanol aqueous solution, 70% methanol aqueous solution, and methanol can extract the chromatographic peaks better. Among these, 50% methanol aqueous solution yields better peak shapes, relatively better system adaptability parameters, and a larger characteristic peak response; therefore, 50% methanol aqueous solution is the preferred extraction solvent.

[0151] Table 15 Comparison of Extraction Solvent-Chromatographic Peak System Adaptability Parameters

[0152]

[0153]

[0154]

[0155]

[0156] 3. Examination of extraction time

[0157] Based on the treatment method and extraction solvent determined above, 15, 30, 45, and 60 minutes were selected as extraction times to prepare the test solution.

[0158] The test results are shown in Table 16. The results show that when the ultrasound time is 30-60 minutes, the peak area is higher than that when the ultrasound time is 15 minutes, and the difference between the two is not significant. Therefore, 30 minutes is the preferred ultrasound time.

[0159] Table 16 Comparison of Extraction Time-Chromatographic Peak System Adaptability Parameters

[0160]

[0161]

[0162] 4. Investigation of solvent usage

[0163] Based on the treatment method, extraction solvent, and extraction time determined above, 10 ml, 25 ml, and 50 ml were selected as the dosages of 50% methanol aqueous solution to prepare the test sample solution.

[0164] The test results are shown in Table 17. The results show that when the solvent volume is 10-50 ml, the peak area decreases proportionally, indicating that complete extraction can be achieved with solvent volumes of 10-50 ml. Considering the chromatographic peak response, 25 ml is the preferred solvent volume for extraction.

[0165] Table 17 Comparison of Solvent Usage-Chromatographic Peak System Adaptability Parameters

[0166]

[0167]

[0168] 5. Selection of injection volume

[0169] Based on the determined treatment method, extraction solvent, extraction time, and solvent volume, 1 μl, 2 μl, and 3 μl were selected as injection volumes for the test sample solution.

[0170] The test results are shown in Table 18. The results show that compared with the injection volumes of 1 μl and 3 μl, the chromatographic peaks corresponding to the injection volume of 2 μl have better resolution, and the peak height and peak width are relatively moderate. Therefore, the preferred injection volume is 2 μl.

[0171] Table 18 Comparison of Injection Volume-Chromatographic Peak System Adaptability Parameters

[0172]

[0173]

[0174] 6. Determination of the preparation method of the test solution

[0175] Based on the above optimization results, the preparation method of the test solution was determined as follows: Take about 0.1g of freeze-dried powder of the standard decoction of *Heliotropium indicum*, place it in a stoppered conical flask, add 25ml of 50% methanol aqueous solution, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution; the injection volume was determined to be 2μl.

[0176] Experiment Example 3: Methodological Validation

[0177] 1. Precision test

[0178] The test solution was prepared according to the preparation method of the test solution in Example 1. The test solution was injected repeatedly 6 times under the chromatographic conditions in Example 1. The retention time was recorded and the relative retention time (t / ts) of the common peak was calculated.

[0179] Using peak 9 (1,5-dicaffeoylquinic acid) as the S peak, the relative retention times of peaks 1-8 and 10-13 were calculated.

[0180] The results are shown in Table 19 below. The results show that the RSD of the relative retention time of each characteristic peak and the reference S peak is less than 2%, indicating that the instrument has good precision.

[0181] Table 19 Results of Instrument Precision and Relative Retention Time Tests

[0182]

[0183]

[0184] 2. Method repeatability test

[0185] Six test solutions were prepared using the same batch of freeze-dried licorice standard decoction powder according to the preparation method of the test solution in Example 1. The solutions were measured under the liquid phase conditions in Example 1, the retention times were recorded, and the relative retention times (t / ts) of the common peaks were calculated.

[0186] The results are shown in Table 20 below. The results show that the RSD of the relative retention times of each characteristic peak and the reference S peak is less than 2%, indicating that the method has good repeatability.

[0187] Table 20 Results of the method repeatability relative retention time test

[0188]

[0189]

[0190] 3. Intermediate precision test (by different operators)

[0191] Three inspectors at different times prepared test solutions using the same batch of freeze-dried *Heliotropium indicum* standard decoction powder according to the preparation method of the test solution in Example 1. The retention times of the prepared test solutions were measured using the same equipment, and the relative retention times (t / ts) of the common peaks were calculated.

[0192] The results are shown in Table 21 below. The results show that the RSD of the relative retention times of each characteristic peak and the reference peak S is less than 2%, indicating good intermediate precision of this method.

[0193] Table 21 Results of Intermediate Precision Relative Retention Time Tests (Different Operators)

[0194]

[0195] 4. Specificity test

[0196] A blank solvent (50% methanol aqueous solution) was selected as the negative control solution. The test solution was prepared according to the preparation method of the test solution in Example 1. The negative control solution and the test solution were detected under the chromatographic conditions described in Example 1.

[0197] Test results are shown Figure 14-15 As can be seen from the figure, there is no interference from negative results, indicating that the method has good specificity.

[0198] 5. Stability test

[0199] Following the preparation method of the test solution in Example 1, the same batch of lyophilized powder of the standard decoction for licorice was used to prepare the test solution, and the test solution was tested under the liquid phase conditions in Example 1. The test solution was injected at 0, 2, 4, 8, 12 and 24 hours after preparation, and the retention time of the common peak was recorded and the relative retention time was calculated.

[0200] The results are shown in Table 22 below. The results show that the relative retention times of each characteristic peak and the reference peak S are less than 2%, indicating that the test solution is stable within 24 hours and meets the determination requirements.

[0201] Table 22 Results of the relative retention time test for stability

[0202]

[0203] 6. Durability test

[0204] A. Investigation of different flow velocities

[0205] The test solution was prepared according to the preparation method of the test solution in Example 1. The test solution was injected and detected under the chromatographic conditions in Example 1, except that the determination was carried out at flow rates of 0.23 ml / min, 0.25 ml / min and 0.27 ml / min to examine the separation effect of each characteristic peak when the flow rate changed.

[0206] The results are shown in Table 23 below. The results indicate that all chromatographic peaks were detected at different flow rates, demonstrating good separation performance.

[0207] Table 23 Results of relative retention time for different flow velocities

[0208]

[0209]

[0210] B. Investigation at different column temperatures

[0211] The test solution was prepared according to the preparation method of the test solution in Example 1. The test solution was injected and detected under the chromatographic conditions in Example 1. The only difference was that the determination was carried out at column temperatures of 27℃, 30℃ and 33℃ to examine the separation effect of each characteristic peak when the column temperature changed.

[0212] The results are shown in Table 24 below. The results indicate that all chromatographic peaks were detected at different column temperatures, demonstrating good separation performance.

[0213] Table 24 Comparison of relative retention time results at different column temperatures

[0214]

[0215] C. Investigation using different instruments

[0216] The test solution was prepared according to the preparation method of the test solution in Example 1. The test solution was injected and detected under the chromatographic conditions in Example 1. The only difference was that the detection was carried out on Waters instrument and Thermo ultra-high performance liquid chromatography to examine the separation effect of each characteristic peak of different instruments.

[0217] The results are shown in Table 25 below. The results indicate that the chromatographic peaks were detected on all instruments, demonstrating good peak separation.

[0218] Table 25 Comparison of relative retention time measurement results from different instruments

[0219]

[0220]

[0221] D. Investigation of different chromatographic columns

[0222] The test solution was prepared according to the preparation method of the test solution in Example 1. The test solution was injected and detected under basically the chromatographic conditions in Example 1. The only difference was that Waters CORTECS T3, 100mm×2.1mm, 1.6μm and Agilent EclipsePlus C18 RRHD, 100mm×2.1mm, 1.8μm columns were used for injection analysis.

[0223] The results are shown in Table 26 below. The results indicate that the characteristic peaks in the spectra corresponding to different chromatographic columns were well separated.

[0224] Table 26 Comparison of relative retention time determination results for different chromatographic columns

[0225]

[0226] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing a characteristic spectrum of the grasshopper and its preparations, characterized in that, Includes the following steps: (1) Preparation of test solution: including, 1) taking the analyte, adding solvent and extracting by ultrasonication to obtain an extract; 2) separating the solid and liquid of the extract and taking the liquid, which is the test solution; the solvent is methanol; (2) The test solution and the reference solution were detected by ultra-high performance liquid chromatography. The packing material was octadecylsilane-bonded silica gel, and the mobile phase included methanol and an acidic aqueous solution. The acidic aqueous solution was an aqueous solution of phosphoric acid. Gradient elution was performed, with the following elution program: 0 → 12 min → 18 min → 20 min → 25 min → 28 min → 34 min → 42 min. The volume percentage of methanol in the mobile phase was 7% → 18% → 30% → 35% → 40% → 45% → 47% → 60%. The method for preparing the reference solution includes the steps of preparing the reference solution using neochlorogenic acid, caffeic acid, chlorogenic acid, cryptochlorogenic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, cosmos glycoside, and gentamicin lactone as reference standards.

2. The construction method according to claim 1, characterized in that, Step (1) also satisfies any one or more of the following AD: A. In step 1), the mass ratio of the analyte to the volume of the solvent is 0.1-2 g: 10-250 mL; B. In step 1), the extraction time is 15 min-120 min; C. In step 2), the solid-liquid separation is performed by centrifugation or filtration; D. In step 1), the test substance is selected from one or more of the following: *Hemiptera chinensis* medicinal materials, *Hemiptera chinensis* slices, *Hemiptera chinensis* granules, *Hemiptera chinensis* decoction, and *Hemiptera chinensis* powder.

3. The construction method according to claim 1, characterized in that, The volume percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.1-0.4%.

4. The construction method according to claim 1, characterized in that, Step (2) The chromatographic conditions for ultra-high performance liquid chromatography also include: detection wavelength of 210-280 nm, flow rate of 0.23-0.27 ml / min, column temperature of 25-33 ℃, column length of 100 mm, inner diameter of 2.1 mm, and particle size of 1.6-1.8 μm.

5. The construction method according to claim 4, characterized in that, The detection wavelength is 240nm.

6. The construction method according to claim 4, characterized in that, The column temperature is 30℃.

7. The construction method according to claim 4, characterized in that, The injection volume was 2 μl.

8. The construction method according to claim 1, characterized in that, The gradient elution program also includes 42 minutes → 43 minutes, with the volume percentage of methanol in the mobile phase being 60% → 7%, or the gradient elution program also includes 42 minutes → 43 minutes → 48 minutes, with the volume percentage of methanol in the mobile phase being 60% → 7% → 7%.

9. The construction method according to any one of claims 1-8, characterized in that, The characteristic spectra of the *Gnaphalium affine* and its preparations obtained by the construction method are selected from any one of the following (1)-(2): (1) The characteristic spectrum of the grasshopper and its preparation has 13 characteristic peaks, with peak 9 as the reference peak. The relative retention times of characteristic peaks 1-8 and 10-13 with the reference peak are within ±10% of the specified values. The specified values ​​of characteristic peaks 1-8 and 10-13 are as follows: 0.21, 0.25, 0.28, 0.48, 0.49, 0.56, 0.71, 0.93, 1.06, 1.09, 1.24, 1.

39. (2) The characteristic chromatogram of the grasshopper and its preparation has 13 characteristic peaks. The peaks corresponding to the reference peaks of 1,5-dicaffeoylquinic acid are reference peaks. The relative retention times of characteristic peaks 1-8 and 10-13 with the reference peaks are within ±10% of the specified values. The specified values ​​of characteristic peaks 1-8 and 10-13 are as follows: 0.21, 0.25, 0.28, 0.48, 0.49, 0.56, 0.71, 0.93, 1.06, 1.09, 1.24, and 1.39, respectively.

10. The application of the construction method according to any one of claims 1-9 in the quality control of Artemisia annua and its preparations.

11. A quality control method for *Gnaphalium affine* and its preparations, characterized in that, The method includes the step of comparing the characteristic spectrum of the test product with the characteristic spectrum of the grasshopper and its preparation; the characteristic spectrum of the test product is obtained by using the test product according to any one of the construction methods described in claims 1-8, and the characteristic spectrum of the grasshopper and its preparation is the characteristic spectrum of the grasshopper and its preparation as described in claim 9.

12. A method for identifying the grasshopper and its preparations from counterfeit products, characterized in that, include: The characteristic spectrum of the product to be identified is constructed using the test sample according to any one of the construction methods described in claims 1-9; The obtained feature map of the product to be identified is compared with the feature map constructed by any of the construction methods described in claims 1-9, and the identification is performed based on the comparison results. The counterfeit product is *Symplocos rubrum* and / or *Symplocos rubrum* preparations.

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