A method for constructing HPLC characteristic chromatogram of ironwire tradescantia medicinal material, decoction piece, standard decoction and formula granules and application thereof

Abstract

The application belongs to the technical field of detection, and particularly relates to a construction method of HPLC characteristic spectrum of ironline throughbone medicinal materials, decoction pieces, standard decoction and formula granules and application thereof. The HPLC characteristic spectrum construction method provided by the application comprises the following steps: preparing a test sample, wherein the test sample is ironline throughbone medicinal materials, ironline throughbone decoction pieces, ironline throughbone standard decoction or ironline throughbone formula granules; dissolving the test sample to obtain a test sample solution; determining the test sample solution by using high performance liquid chromatography to obtain the HPLC characteristic spectrum corresponding to the test sample; and the chromatographic conditions of the high performance liquid chromatography are as follows: a C18 column is used as a chromatographic column; a mobile phase A is acetonitrile, a mobile phase B is a 0.5wt% phosphoric acid solution, and gradient elution is used. The characteristic spectrum construction method provided by the application can provide a more scientific basis for identification of ironline throughbone medicinal materials, decoction pieces, standard decoction and formula granules.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, and in particular relates to a method for constructing HPLC characteristic chromatograms of *Imperata cylindrica* herbal medicine, decoction pieces, standard decoctions, and formulation granules, and its application. Background Technology

[0002] Clematis intricate Bge, a plant in the Ranunculaceae family, is a dried aerial part of the herb. It has the effects of dispelling wind and dampness, clearing the meridians, and relieving pain. It is mainly used to treat rheumatoid arthritis, numbness and spasms in the limbs, psoriasis, and scabies. To ensure the uniformity and stability of the quality of Clematis intricate Bge medicinal materials, processed slices, standard decoctions, and formulated granules, it is essential to establish a new characteristic chromatographic method for quality control. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide a method for constructing HPLC characteristic chromatograms of *Imperata cylindrica* herbal materials, decoction pieces, standard decoctions, and formulation granules, and its application. The construction method provided by this invention can provide a more scientific basis for the identification of *Imperata cylindrica* herbal materials, decoction pieces, standard decoctions, and formulation granules.

[0004] This invention provides a method for constructing HPLC characteristic spectra of *Imperata cylindrica* herbal medicine, processed slices, standard decoctions, and formulated granules, comprising the following steps:

[0005] Prepare test samples, which are herbal medicine of *Imperata cylindrica*, sliced ​​*Imperata cylindrica*, standard decoction of *Imperata cylindrica*, or formula granules of *Imperata cylindrica*.

[0006] The test sample is dissolved to obtain a test sample solution;

[0007] The test solution was analyzed by high performance liquid chromatography to obtain the corresponding HPLC characteristic chromatogram of the test sample;

[0008] The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; mobile phase A is acetonitrile, mobile phase B is 0.5 wt% phosphoric acid solution, and gradient elution is used.

[0009] In the construction method provided by the present invention, the processed slices of *Imperata cylindrica* are processed products of *Imperata cylindrica* herbal material; the standard decoction of *Imperata cylindrica* is a freeze-dried powder prepared from *Imperata cylindrica* herbal material after processing; and the formulation granules of *Imperata cylindrica* are formulation granules prepared from *Imperata cylindrica* herbal material after processing and according to the main quality indicators of the standard decoction.

[0010] In the construction method provided by the present invention, the solvent used for dissolution is preferably a 50 vol% methanol solution. Under this solvent condition, the characteristic peaks of the chromatogram obtained have good peak shapes and moderate resolution. The ratio of the amount of the test sample to the solvent is preferably (0.2-0.5) g: 25 mL. Under this ratio adjustment, the peak shapes and resolution of the chromatogram obtained are good.

[0011] In the construction method provided by the present invention, the dissolution is preferably carried out under ultrasonic assistance to improve the dissolution efficiency; the ultrasonic assistance power is preferably 580-620W, more preferably 600W; the ultrasonic assistance frequency is preferably 35-45kHz, more preferably 40kHz; the ultrasonic assistance time is preferably 25-35min, more preferably 30min, and the chromatogram peak shape and resolution obtained under this ultrasonic time are better.

[0012] In the construction method provided by the present invention, when the test sample is *Imperata cylindrica* herbal medicine or *Imperata cylindrica* decoction pieces, the specific process of dissolving and preparing the test sample solution preferably includes: sieving the test sample and mixing it with a solvent, dissolving it with ultrasonic assistance, cooling it, shaking it, filtering it, and the resulting filtrate is the test sample solution; wherein, the preferred ratio of the amount of test sample to solvent is 0.5g:25mL.

[0013] In the construction method provided by the present invention, when the test sample is a standard decoction of *Hedyotis diffusa*, the specific process of dissolving and preparing the test sample solution preferably includes: mixing the test sample with a solvent, dissolving with ultrasonic assistance, cooling, shaking, filtering, and the resulting filtrate is the test sample solution; wherein, the ratio of the amount of test sample to solvent is preferably 0.2g:25mL.

[0014] In the construction method provided by the present invention, when the test sample is *Imperata cylindrica* granules, the specific process of dissolving and preparing the test sample solution preferably includes: grinding the test sample into a fine powder and mixing it with a solvent, dissolving it with ultrasonic assistance, cooling it, shaking it, filtering it, and the resulting filtrate is the test sample solution; wherein, the ratio of the amount of the test sample to the amount of the solvent is preferably 0.2 g: 25 mL.

[0015] In the construction method provided by this invention, the gradient elution process is preferably performed as follows when conducting the high-performance liquid chromatography determination:

[0016] 0–9 min, Phase A: 4–8 vol%, Phase B: 96–92 vol%;

[0017] 9–16 min, Phase A: 8–13 vol%, Phase B: 92–87 vol%;

[0018] 16–20 min, Phase A: 13–15 vol%, Phase B: 87–85 vol%;

[0019] 20–25 min, Phase A: 15–13 vol%, Phase B: 85–87 vol%;

[0020] 25–30 min, Phase A: 13 vol%, Phase B: 87 vol%.

[0021] In the construction method provided by the present invention, when performing the high performance liquid chromatography determination, the flow rate of the mobile phase is preferably 0.3 mL / min. Under this flow rate condition, the chromatogram peak shape is better and the resolution is moderate.

[0022] In the construction method provided by the present invention, when performing the high performance liquid chromatography determination, the detection wavelength is preferably 354 nm. Under this detection wavelength condition, the chromatogram information is larger and the baseline is more stable.

[0023] In the construction method provided by the present invention, the injection volume is preferably 1 μL when performing the high performance liquid chromatography determination.

[0024] In the construction method provided by the present invention, when performing the high performance liquid chromatography determination, the packing material in the chromatographic column is octadecylsilane-bonded silica gel; the column length is preferably 80-120 mm, more preferably 100 mm; the inner diameter of the chromatographic column is preferably 1.5-2.5 mm, more preferably 2.1 mm; the particle size of the packing material in the chromatographic column is preferably 1-2 μm, more preferably 1.7 μm.

[0025] In the construction method provided by the present invention, when performing the high performance liquid chromatography determination, the column temperature of the chromatographic column is preferably 30°C. Under this column temperature condition, the peak shape of the chromatogram is more symmetrical, the resolution is better, and the peak elution is more complete.

[0026] In the construction method provided by the present invention, when performing the high performance liquid chromatography determination, the theoretical plate number calculated based on the rutin peak should not be less than 5000.

[0027] The construction method provided by the present invention preferably further includes the following steps:

[0028] Dissolve the reference herb *Clematis chinensis* to obtain a reference herb solution; dissolve rutin to obtain a reference standard solution.

[0029] The reference solutions of the reference medicinal materials and the reference standard were determined by high performance liquid chromatography to obtain the chromatograms of the reference materials; and the components of the HPLC characteristic chromatograms of the test sample were identified based on the chromatograms of the reference materials.

[0030] In the construction method provided by the present invention, the specific process of dissolving and preparing the reference solution of the control medicinal material preferably includes: mixing the *Clematis chinensis* reference medicinal material with a solvent, dissolving with ultrasonic assistance, cooling, shaking, filtering, and the resulting filtrate is the reference solution of the control medicinal material; wherein, the solvent is preferably a 50 vol% methanol solution; the ratio of the *Clematis chinensis* reference medicinal material to the solvent is preferably, more preferably, 0.5 g: 25 mL; the ultrasonic assistance power is preferably 580-620 W, more preferably 600 W; the ultrasonic assistance frequency is preferably 35-45 kHz, more preferably 40 kHz; and the ultrasonic assistance time is preferably 25-35 min, more preferably 30 min.

[0031] In the construction method provided by the present invention, the specific process of dissolving and preparing the reference solution preferably includes: mixing rutin with a solvent to obtain the reference solution; wherein, the solvent is preferably methanol; the ratio of rutin to solvent is preferably (10-40) μg:1mL, more preferably 25 μg:1mL.

[0032] The construction method provided by the present invention preferably further includes the following steps:

[0033] The similarity of the HPLC characteristic chromatograms of *Imperata cylindrica* was evaluated using a chromatographic fingerprint similarity evaluation system for traditional Chinese medicine, resulting in an HPLC standard characteristic chromatogram of *Imperata cylindrica* consisting of eight characteristic peaks. In the HPLC standard characteristic chromatogram, peak 7, corresponding to the rutin reference peak, is peak S. The relative retention times of each characteristic peak and peak S were calculated, and the relative retention times were within ±10% of the specified values, which were: 0.35 (peak 1), 0.44 (peak 2), 0.55 (peak 3), 0.61 (peak 4), 0.75 (peak 5), 0.80 (peak 6), 1.00 (peak 7), and 1.16 (peak 8).

[0034] The construction method provided by the present invention preferably further includes the following steps:

[0035] The similarity of the HPLC characteristic chromatograms of *Imperata cylindrica* slices was evaluated using a chromatographic fingerprint similarity evaluation system for traditional Chinese medicine. A standard HPLC characteristic chromatogram of *Imperata cylindrica* slices consisting of eight characteristic peaks was obtained. In the standard HPLC characteristic chromatogram, peak 7, corresponding to the rutin reference peak, was designated as peak S. The relative retention times of each characteristic peak and peak S were calculated, and the relative retention times were within ±10% of the specified values. The specified values ​​were: 0.35 (peak 1), 0.44 (peak 2), 0.55 (peak 3), 0.61 (peak 4), 0.75 (peak 5), 0.80 (peak 6), 1.00 (peak 7), and 1.16 (peak 8).

[0036] The construction method provided by the present invention preferably further includes the following steps:

[0037] The similarity of the HPLC characteristic chromatograms of the standard decoction of *Imperata cylindrica* was evaluated using a chromatographic fingerprint similarity evaluation system for traditional Chinese medicine. An HPLC standard characteristic chromatogram of the standard decoction of *Imperata cylindrica* consisting of eight characteristic peaks was obtained. In the HPLC standard characteristic chromatogram, peak 7, corresponding to the rutin reference peak, is peak S. The relative retention times of each characteristic peak and peak S were calculated, and the relative retention times were within ±10% of the specified values. The specified values ​​were: 0.35 (peak 1), 0.44 (peak 2), 0.55 (peak 3), 0.61 (peak 4), 0.75 (peak 5), 0.80 (peak 6), 1.00 (peak 7), and 1.16 (peak 8).

[0038] The construction method provided by the present invention preferably further includes the following steps:

[0039] The similarity of the HPLC characteristic chromatograms of *Imperata cylindrica* formula granules was evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, resulting in an HPLC standard characteristic chromatogram of *Imperata cylindrica* formula granules consisting of 8 characteristic peaks. In the HPLC standard characteristic chromatogram, peak 7, corresponding to the rutin reference peak, is peak S. The relative retention times of each characteristic peak and peak S were calculated, and the relative retention times were within ±10% of the specified values, which were: 0.35 (peak 1), 0.44 (peak 2), 0.55 (peak 3), 0.61 (peak 4), 0.75 (peak 5), 0.80 (peak 6), 1.00 (peak 7), and 1.16 (peak 8).

[0040] The present invention also provides a method for identifying *Imperata cylindrica* herbal materials, processed slices, standard decoctions, and formulation granules. This method uses the HPLC characteristic chromatograms obtained by the construction method described above as the basis for identifying *Imperata cylindrica* herbal materials, processed slices, standard decoctions, and formulation granules.

[0041] Compared with existing technologies, this invention provides a method for constructing HPLC characteristic chromatograms of *Imperata cylindrica* herbal materials, processed slices, standard decoctions, and formulated granules, and its application. The method provided by this invention can effectively separate the components of *Imperata cylindrica* chromatographic peaks in a short time, with multiple peaks, high separation between peaks, and good peak shapes. This method can be used to rapidly and efficiently establish HPLC characteristic chromatograms of *Imperata cylindrica* herbal materials, processed slices, standard decoctions, and formulated granules, with good repeatability, precision, and stability, providing a more scientific basis for the identification of *Imperata cylindrica* herbal materials, processed slices, standard decoctions, and formulated granules. Attached Figure Description

[0042] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0043] Figure 1 Chromatograms of different mobile phases for the herbal medicine *Imperata cylindrica* provided in Example 1 of this invention;

[0044] Figure 2 This is a chromatogram of different wavelengths of the herbal medicine *Imperata cylindrica* provided in Example 1 of this invention;

[0045] Figure 3 This is a column temperature chromatogram of the herbal medicine *Imperata cylindrica* provided in Example 1 of the present invention.

[0046] Figure 4 This is a chromatogram of the flow rate of the herbal medicine *Imperata cylindrica* provided in Example 1 of this invention.

[0047] Figure 5 This is a delayed chromatogram of the herbal medicine *Imperata cylindrica* provided in Example 1 of the present invention.

[0048] Figure 6 This is a graph showing the experimental results of investigating the dissolution solvent during the preparation of the test solution of the *Imperata cylindrica* herbal medicine provided in Example 1 of this invention;

[0049] Figure 7 This is a graph showing the experimental results of investigating the dissolution time during the preparation of the test solution of the *Imperata cylindrica* herbal medicine provided in Example 1 of this invention;

[0050] Figure 8 This is a graph showing the experimental results of the solvent addition during the preparation of the test solution of the herbal medicine *Imperata cylindrica* provided in Example 1 of this invention.

[0051] Figure 9 This is a graph showing the experimental results of investigating the dissolution method during the preparation of the test solution of the *Imperata cylindrica* herbal medicine provided in Example 1 of this invention;

[0052] Figure 10 This is the chromatographic peak identification diagram of the characteristic chromatogram of the herbal medicine *Imperata cylindrica* provided in Embodiment 1 of the present invention;

[0053] Figure 11 This is the ultraviolet absorption spectrum of the rutin reference standard provided in Example 1 of the present invention;

[0054] Figure 12 This is the ultraviolet absorption spectrum of rutin in the *Imperata cylindrica* herbal sample provided in Example 1 of this invention;

[0055] Figure 13 This is a graph showing the experimental results of the *Iron Wire Penetrating Herbal Material* examined with different instruments, as provided in Embodiment 1 of the present invention.

[0056] Figure 14 This is a graph showing the experimental results of different chromatographic columns for the *Imperata cylindrica* herb provided in Example 1 of this invention;

[0057] Figure 15 These are the characteristic chromatogram verification images of 21 batches of *Imperata cylindrica* herbal materials provided in Embodiment 1 of the present invention;

[0058] Figure 16 This is a comparative atlas of the characteristic atlas of the herbal medicine *Imperata cylindrica* provided in Embodiment 1 of the present invention;

[0059] Figure 17 These are the characteristic chromatogram verification images of 21 batches of *Imperata cylindrica* decoction pieces provided in Embodiment 1 of the present invention;

[0060] Figure 18 This is a comparative chromatogram of the characteristic chromatogram of *Imperata cylindrica* slices provided in Embodiment 1 of the present invention;

[0061] Figure 19 Chromatograms of different mobile phases for the standard decoction of *Imperata cylindrica* provided in Example 2 of this invention;

[0062] Figure 20 This is a chromatogram of the standard decoction of *Imperata cylindrica* at different wavelengths provided in Example 2 of the present invention;

[0063] Figure 21 This is a column temperature chromatogram of the standard decoction of *Imperata cylindrica* provided in Example 2 of this invention;

[0064] Figure 22 This is a chromatogram of the flow rate of the standard decoction of *Imperata cylindrica* provided in Example 2 of the present invention.

[0065] Figure 23 This is a delayed chromatogram of the standard decoction of *Imperata cylindrica* provided in Example 2 of the present invention.

[0066] Figure 24 This is a graph showing the experimental results of investigating the dissolution solvent during the preparation of the test solution of the standard decoction of *Imperata cylindrica* provided in Example 2 of the present invention;

[0067] Figure 25 This is a graph showing the experimental results of investigating the dissolution method during the preparation of the test solution of the standard decoction of *Imperata cylindrica* provided in Example 2 of the present invention;

[0068] Figure 26 This is a graph showing the experimental results of investigating the dissolution time during the preparation of the test solution of the standard decoction of *Imperata cylindrica* provided in Example 2 of this invention;

[0069] Figure 27 This is a graph showing the experimental results of the solvent addition during the preparation of the test solution of the standard decoction of *Imperata cylindrica* provided in Example 2 of the present invention.

[0070] Figure 28 This is the chromatographic peak identification diagram of the characteristic chromatogram of the standard decoction of *Imperata cylindrica* provided in Example 2 of the present invention;

[0071] Figure 29 This is the ultraviolet absorption spectrum of the rutin reference standard provided in Example 2 of the present invention;

[0072] Figure 30 This is the ultraviolet absorption spectrum of rutin in the standard decoction of *Imperata cylindrica* provided in Example 2 of the present invention;

[0073] Figure 31 This is a graph showing the experimental results of the standard decoction of *Imperata cylindrica* provided in Example 2 of this invention, examined using different instruments.

[0074] Figure 32 This is a graph showing the experimental results of different chromatographic columns for the standard decoction of *Imperata cylindrica* provided in Example 2 of this invention;

[0075] Figure 33 These are the characteristic chromatogram verification diagrams of 21 batches of *Imperata cylindrica* standard decoction provided in Example 2 of this invention;

[0076] Figure 34 This is a comparative chromatogram of the characteristic chromatogram of the standard decoction of *Imperata cylindrica* provided in Example 2 of the present invention;

[0077] Figure 35 Chromatograms of different mobile phases for the *Imperata cylindrica* formulation granules provided in Example 3 of this invention;

[0078] Figure 36 This is a chromatogram of different wavelengths of the *Imperata cylindrica* formula granules provided in Example 3 of the present invention;

[0079] Figure 37 This is a column temperature chromatogram of the *Imperata cylindrica* formula granules provided in Example 3 of the present invention.

[0080] Figure 38 This is a chromatogram of the flow rate of the *Imperata cylindrica* formulation granules provided in Example 3 of the present invention.

[0081] Figure 39 This is a chromatogram of the delayed effect of the Iron Wire Clematis Formula Granules provided in Example 3 of the present invention;

[0082] Figure 40 This is a graph showing the experimental results of investigating the dissolution solvent during the preparation of the test solution of the Iron Wire Clematis chinensis formula granules provided in Example 3 of the present invention;

[0083] Figure 41 This is a graph showing the experimental results of investigating the dissolution method during the preparation of the test solution of the Iron Wire Clematis Formula Granules provided in Example 3 of the present invention;

[0084] Figure 42 This is a graph showing the experimental results of investigating the dissolution time during the preparation of the test solution of the Iron Wire Clematis Formula Granules provided in Example 3 of the present invention;

[0085] Figure 43 This is a graph showing the experimental results of the solvent addition during the preparation of the test sample solution of the *Imperata cylindrica* formula granules provided in Example 3 of the present invention.

[0086] Figure 44 This is the chromatographic peak identification diagram of the characteristic granules of *Imperata cylindrica* provided in Embodiment 3 of the present invention;

[0087] Figure 45 This is the ultraviolet absorption spectrum of the rutin reference standard provided in Example 3 of the present invention;

[0088] Figure 46 This is the ultraviolet absorption spectrum of rutin in the *Imperata cylindrica* formula granules provided in Example 3 of the present invention;

[0089] Figure 47 This is a graph showing the experimental results of the Iron Wire Clematis Formula Granules provided in Embodiment 3 of the present invention under different instrument examinations;

[0090] Figure 48 This is a graph showing the experimental results of different chromatographic columns for the Iron Thread Herb Formula Granules provided in Example 3 of the present invention;

[0091] Figure 49 These are the characteristic spectral verification diagrams of three batches of *Imperata cylindrica* formula granules provided in Embodiment 3 of the present invention;

[0092] Figure 50 This is a comparative chromatogram of the characteristic chromatogram of the *Imperata cylindrica* formula granules provided in Example 3 of the present invention. Detailed Implementation

[0093] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0094] Example 1

[0095] Construction of HPLC characteristic chromatograms of *Imperata cylindrica* herbal medicine and processed medicinal slices:

[0096] 1) Materials, reagents and instruments

[0097] 1.1) Instruments and Materials

[0098] Ultra-high performance liquid chromatographs: Instrument 1 (Agilent 1290 ultra-high performance liquid chromatograph), Instrument 2 (Waters H-class ultra-high performance liquid chromatograph), Instrument 3 (Thermo Fisher Vanquish Flex ultra-high performance liquid chromatograph). Unless otherwise specified, Instrument 1 will be used by default.

[0099] Electronic balances: ME204E / 02, MS205DM, XP26 (Mettler-Toledo Instruments Ltd.);

[0100] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0101] Ultrasonic cleaner: KQ-600DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0102] Chromatographic column: Column 1 (Manufacturer: Waters; Model: ACQUITY) HSS T3), column 2 (manufacturer: YMC; model: YMC-Triart C18), and column 3 (manufacturer: DIKMA; model: Endeavorsil C18) are all packed with octadecylsilane-bonded silica gel, with a column length of 100 mm, an inner diameter of 2.1 mm, and a packing particle size of 1.7 μm. Unless otherwise specified, column 1 is used by default.

[0103] 1.2) Reagents and reagents

[0104] Acetonitrile and phosphoric acid were of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0105] Rutin (China National Institutes for Food and Drug Control, batch number: 100080-202012, purity: 91.6%);

[0106] Reference material of *Imperata cylindrica* (Chengdu Desite Biotechnology Co., Ltd., batch number: DSTYT007801);

[0107] Iron Wire Penetrating Bone Herbal Material (Prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., Batch Nos.: YC-01, YC-02, YC-03, YC-04, YC-05, YC-06, YC-07, YC-08, YC-09, YC-10, YC-11, YC-12, YC-13, YC-14, YC-15, YC-16, YC-17, YC-18, YC-19, YC-20, YC-21);

[0108] Iron-wire-penetrating herb slices (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: YP-01, YP-02, YP-03, YP-04, YP-05, YP-06, YP-07, YP-08, YP-09, YP-10, YP-11, YP-12, YP-13, YP-14, YP-15, YP-16, YP-17, YP-18, YP-19, YP-20, YP-21).

[0109] 2) Feature map detection method

[0110] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material (column length 100 mm, inner diameter 2.1 mm, packing material particle size 1.7 μm); acetonitrile was used as mobile phase A, and 0.5 wt% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 1; the flow rate was 0.3 mL per minute; the column temperature was 30 °C; the detection wavelength was 354 nm; the theoretical plate number calculated based on the rutin peak should not be less than 5000.

[0111] Table 1 Gradient elution program

[0112]

[0113] Preparation of reference solution: Take 0.5g of *Clematis chinensis* reference material, place it in a stoppered conical flask, add 25mL of 50vol% methanol solution, stopper tightly, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference material; separately take an appropriate amount of rutin reference standard, accurately weigh it, add methanol to prepare a solution containing 25μg of rutin per 1mL, as the reference solution of the reference standard.

[0114] Preparation of the test solution: Take 1g of the powder (passed through a No. 3 sieve), place it in a stoppered conical flask, add 50mL of 50vol% methanol solution, stopper tightly, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0115] Determination method: Accurately pipette 1 μL of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0116] 3) Investigation of chromatographic conditions

[0117] 3.1) Selection of mobile phase

[0118] Based on the experimental conditions outlined above, the separation effects of two different mobile phases were investigated: methanol (phase A) - 0.5 vol% phosphoric acid (phase B) and acetonitrile (phase A) - 0.5 vol% phosphoric acid (phase B). The results are shown in [Figure 1]. Figure 1 , Figure 1Chromatograms of different mobile phases for the herbal medicine *Imperata cylindrica* provided in Example 1 of this invention. (Through...) Figure 1 It can be seen that the chromatogram baseline is relatively stable and there are many chromatographic peaks under the gradient elution condition of acetonitrile-0.5 vol% phosphoric acid solution. Therefore, the gradient elution of acetonitrile-0.5 vol% phosphoric acid solution is used as the mobile phase for the determination of the characteristic chromatogram of *Imperata cylindrica*.

[0119] 3.2) Wavelength Selection

[0120] Based on the above-specified experimental conditions, a diode array detector was used to perform a full-band scan of the test solution, and chromatograms of the test solution at wavelengths of 330 nm, 354 nm, and 370 nm were extracted. The results are shown in the figure. Figure 2 , Figure 2 This is a chromatogram of different wavelengths of the *Imperata cylindrica* herb provided in Embodiment 1 of the present invention. (Through...) Figure 2 It can be seen that the chromatographic peak information is greater and the chromatographic baseline is more stable when the detection wavelength is 354nm, so the detection wavelength is determined to be 354nm.

[0121] 3.3) Column Temperature Investigation

[0122] Based on the above-specified experimental conditions, the test solution was subjected to chromatographic detection at column temperatures of 25℃, 30℃, and 35℃, respectively. The results are shown in the figure. Figure 3 , Figure 3 This is a column temperature chromatogram of the *Imperata cylindrica* herb provided in Example 1 of this invention. (The text repeats itself here.) Figure 3 It can be seen that when the column temperature is 30℃, the chromatogram peaks are more symmetrical, the separation is better, and the peaks are more complete. Therefore, the column temperature is determined to be 30℃.

[0123] 3.4) Flow velocity investigation

[0124] Based on the above-specified experimental conditions, the test solution was subjected to chromatographic detection at flow rates of 0.2 mL / min, 0.3 mL / min, and 0.4 mL / min, respectively. The results are shown in the figure. Figure 4 , Figure 4 This is a chromatogram for the flow rate determination of the *Imperata cylindrica* herb provided in Example 1 of the present invention. (Through...) Figure 4 It can be seen that when the flow rate is 0.3 mL / min, the chromatogram peak shape is good and the resolution is moderate, so the flow rate is determined to be 0.3 mL / min.

[0125] 3.5) Delayed Examination

[0126] Based on the above-specified experimental conditions, a delay test was conducted, and the results are shown below. Figure 5 , Figure 5 This is a delayed-action chromatogram of the *Imperata cylindrica* herb provided in Example 1 of the present invention. (Through...) Figure 5 It can be seen that the sample has basically no useful chromatographic peaks after 30 minutes, so the sample detection time is set at 30 minutes.

[0127] 4) Preparation of the test solution

[0128] 4.1) Investigation of the dissolving solvent

[0129] Take 1g of the powder (passed through a No. 3 sieve, batch number: YC-21) and place it in a stoppered conical flask. Dissolve it in 50mL each of methanol, 30vol% methanol, 50vol% methanol, 80vol% methanol, ethanol, 30vol% ethanol, 50vol% ethanol, 80vol% ethanol, and water. Seal the flask tightly and sonicate (600W power, 40kHz frequency) for 30 minutes. Cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0130] Under the experimental conditions outlined above, chromatographic determinations were performed on the test solutions prepared using different solvents. The results are shown below. Figure 6 , Figure 6 This is a graph showing the experimental results of investigating the dissolution solvent during the preparation of the *Imperata cylindrica* herbal sample solution provided in Example 1 of this invention. Figure 6 It can be seen that when the solvent is 50 vol% methanol, the peak shapes of each characteristic peak are good and the separation is moderate. Therefore, the extraction solvent is determined to be 50 vol% methanol.

[0131] 4.2) Investigation of dissolution time

[0132] Take 1g of the powder (passed through a No. 3 sieve, batch number: YC-21), place it in a stoppered conical flask, add 50mL of 50vol% methanol solution, seal tightly, and sonicate (power 600W, frequency 40kHz) for 20 minutes, 30 minutes, and 40 minutes respectively. Cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0133] Under the experimental conditions outlined above, chromatographic determinations were performed on the test solutions subjected to ultrasonic treatment for different times. The results are shown in the table below. Figure 7 , Figure 7 This is a graph showing the experimental results of investigating the dissolution time during the preparation of the *Imperata cylindrica* herbal sample solution provided in Example 1 of this invention. Figure 7 It can be seen that the chromatogram peak shape and resolution are better when the dissolution time is 30 min, so the dissolution time is determined to be 30 min.

[0134] 4.3) Investigation of the amount of solvent added

[0135] Take 1g of the powder (passed through a No. 3 sieve, batch number: YC-21) and place it in a stoppered conical flask. Add 25mL, 50mL and 100mL of 50vol% methanol solution respectively, seal tightly, and sonicate (power 600W, frequency 40kHz) for 30 minutes. Cool, shake well, filter, and take the filtrate to obtain the test solution.

[0136] Under the experimental conditions specified above, chromatographic determinations were performed on the test solutions with different solvent addition amounts. The results are shown in the figure. Figure 8 , Figure 8 This is a graph showing the experimental results of investigating the amount of solvent added during the preparation of the *Imperata cylindrica* herbal sample solution according to Example 1 of the present invention. Figure 8 It can be seen that when the solvent volume is 50 mL, the peak shape and resolution of each chromatographic peak are good, so the solvent volume is selected as 50 mL.

[0137] 4.4) Investigation of dissolution methods

[0138] Take 1g of the powder (passed through a No. 3 sieve, batch number: YC-21), place it in a stoppered conical flask, add 50mL of 50vol% methanol solution, seal tightly, and subject the test sample to reflux, sonication (power 600W, frequency 40kHz), and boiling in water for 30 minutes respectively. Cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0139] Under the experimental conditions specified above, the test solutions treated with reflux, ultrasonication, and boiling water were subjected to chromatographic analysis, and the results are shown in the figure. Figure 9 , Figure 9 This is a graph showing the experimental results of investigating the dissolution method during the preparation of the *Imperata cylindrica* herbal sample solution provided in Example 1 of this invention. Figure 9 It can be seen that the three treatment methods have little difference in their effects on the solution of the test sample. Since the ultrasonic method is fast and simple, the ultrasonic-assisted dissolution method was determined to be the test sample dissolution method.

[0140] 5) Methodological investigation

[0141] 5.1) Chromatographic peak identification

[0142] According to the experimental conditions proposed above, prepare the test solution of *Imperata cylindrica*, the reference solution of rutin, and the reference solution of *Imperata cylindrica*. At the same time, according to the experimental conditions proposed above, prepare the negative control solution (i.e., blank solution) of iron-deficient *Imperata cylindrica* and perform chromatographic detection.

[0143] Based on the above chromatographic detection results, the characteristic chromatographic peaks of the herb *Imperata cylindrica* were located, and the results are shown below. Figures 10-12 , Figure 10 This is the chromatographic peak identification diagram of the characteristic chromatogram of the herbal medicine *Imperata cylindrica* provided in Embodiment 1 of the present invention. Figure 11This is the ultraviolet absorption spectrum of the rutin reference standard provided in Example 1 of the present invention. Figure 12 This is the ultraviolet absorption spectrum of rutin in the *Imperata cylindrica* herbal sample provided in Example 1 of this invention. The results show that peak 7 is the rutin peak. In the following methodological investigation, the rutin peak was designated as the S peak, and eight characteristic peaks in the sample were investigated.

[0144] 5.2) Precision test

[0145] Take the test solution of the herbal medicine *Imperata cylindrica* (batch number: YC-21), inject it 6 times consecutively according to the proposed experimental method, 1 μL each time, and calculate the retention time of each characteristic peak. The results are shown in Table 2.

[0146] Table 2 Precision Examination - Retention Time

[0147]

[0148] As shown in Table 2, the RSD values ​​of the retention times of each peak are 0.05% to 0.09%, indicating that the instrument has good precision.

[0149] 5.3) Repeatability test

[0150] Six samples of *Imperata cylindrica* (batch number: YC-21) were prepared and tested according to the proposed experimental method. The results are shown in Table 3.

[0151] Table 3 Repeatability Tests—Relative Retention Times of Characteristic Peaks

[0152]

[0153] As shown in Table 3, the RSD of the relative retention time of each characteristic peak is between 0.04% and 0.77%, indicating that the method has good repeatability.

[0154] 5.4) Intermediate precision test

[0155] 5.4.1) Investigation with different instruments

[0156] Based on the above-planned experimental conditions, the herbal medicine *Imperata cylindrica* (batch number: YC-21) was weighed and a test solution was prepared. The solutions were then measured on instruments 1, 2, and 3, respectively. The results are shown in the table below. Figure 13 And Table 4, Figure 13 This is a graph showing the experimental results of different instruments used to examine the herbal medicine *Iron Wire Penetrating Bone* provided in Embodiment 1 of the present invention.

[0157] Table 4. Relative retention times of characteristic peaks observed using different instruments

[0158]

[0159] pass Figure 13As shown in Table 4, when the above three instruments were used to detect the test sample, the RSD of the relative retention time of each characteristic peak was between 0.67% and 5.35%, indicating that the instruments had good durability.

[0160] 5.4.2) Investigations by different personnel and at different times

[0161] Based on the experimental conditions proposed above, different personnel (A and B) weighed out the herbal medicine of *Imperata cylindrica* (batch number: YC-21) at different times (T1 and T2) to prepare test samples and conduct tests. The results are shown in Table 5.

[0162] Table 5. Relative Retention Time of Characteristic Peaks for Different Personnel and Time Periods

[0163]

[0164] As shown in Table 5, under different sample preparation personnel and different sample preparation time conditions, the RSD of the relative retention time of each characteristic peak is between 0.06% and 0.89%, indicating good method stability.

[0165] 5.5) Durability test

[0166] 5.5.1) Column robustness test

[0167] Based on the above-planned experimental conditions, analyses were conducted using chromatographic columns 1, 2, and 3, respectively. The results are shown in the table below. Figure 14 And Table 6, Figure 14 This is a graph showing the experimental results of different chromatographic columns for the *Imperata cylindrica* herb provided in Example 1 of this invention.

[0168] Table 6. Column robustness study—relative retention times of characteristic peaks

[0169]

[0170] pass Figure 14 As shown in Table 6, when the samples were detected using the above three chromatographic columns, the RSD of the relative retention time of the characteristic peaks ranged from 1.09% to 4.33%, indicating that the chromatographic columns had good robustness.

[0171] 5.5.2) Stability Test

[0172] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 2h, 4h, 8h, 16h and 24h respectively. The results are shown in Table 7.

[0173] Table 7 Stability Study—Retention Time

[0174]

[0175] As can be seen from Table 7, the RSD of the corresponding characteristic peak retention time is between 0.67% and 1.42%, and the sample solution is relatively stable within 24 hours.

[0176] In summary, the RSD of the relative retention times of each characteristic peak meets the requirements in all the above tests, indicating that the method is effective. The above eight characteristic peaks will be included in subsequent investigations.

[0177] 6) Verification of the characteristic atlas of *Iron Wire Penetrating Bone Herb*

[0178] Using the proposed method, characteristic chromatographic analysis was performed on 21 batches of *Imperata cylindrica* herbal medicine, and the relative retention time was calculated. The results are shown below. Figure 15 And Table 8, Figure 15 These are the characteristic chromatogram verification images of 21 batches of *Imperata cylindrica* herbal medicine provided in Example 1 of this invention. Figure 15 Among them, S1~S21 are: YC-01, YC-02, YC-03, YC-04, YC-05, YC-06, YC-07, YC-08, YC-09, YC- 10. YC-11, YC-12, YC-13, YC-14, YC-15, YC-16, YC-17, YC-18, YC-19, YC-20, YC-21.

[0179] Table 821 shows the relative retention times of *Imperata cylindrica* (a type of herbal medicine).

[0180]

[0181]

[0182] Based on the principles of stable relative retention time, detectability in all batches of samples, and relatively high peak values, eight peaks with good repeatability were selected as characteristic peaks. As shown in Table 8, the relative retention time RSD of all eight characteristic peaks in the 21 batches of *Imperata cylindrica* herbal medicine was less than 0.1%.

[0183] The relative retention times of 21 batches of *Imperata cylindrica* herbal materials were all within ±10% of the average relative retention times of the three batches of *Imperata cylindrica* formula granules. Therefore, the average relative retention time of the three batches of *Imperata cylindrica* formula granules was taken as the specified value for *Imperata cylindrica* herbal materials, as detailed in Table 9.

[0184] Table 93 shows the relative retention time of *Imperata cylindrica* (iron-wire grass) formula granules.

[0185]

[0186] The final specification stipulates that the chromatogram of the test sample should show 8 characteristic peaks, and the retention times should correspond to the 8 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peak 7 should correspond to the retention time of the rutin reference peak. The peak corresponding to the rutin reference peak is the S peak. The relative retention times of the remaining characteristic peaks and the S peak should be calculated, and the relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.35 (peak 1), 0.44 (peak 2), 0.55 (peak 3), 0.61 (peak 4), 0.75 (peak 5), 0.80 (peak 6), 1.00 (peak 7, S), and 1.16 (peak 8).

[0187] The chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version) was used to synthesize 21 batches of *Tie Xian Tou Gu Cao* (a type of herbal medicine), and a reference characteristic chromatogram for *Tie Xian Tou Gu Cao* was established, such as... Figure 16 As shown, Figure 16 This is a comparative atlas of the characteristic atlas of the herbal medicine *Imperata cylindrica* provided in Embodiment 1 of the present invention.

[0188] 7) Verification of the characteristic chromatogram of *Imperata cylindrica* slices

[0189] Using the proposed method, characteristic chromatographic analysis was performed on 21 batches of *Clematis chinensis* slices, and the relative retention time was calculated. The results are shown below. Figure 17 And Table 10, Figure 17 These are the characteristic chromatogram verification images of 21 batches of *Imperata cylindrica* decoction pieces provided in Example 1 of this invention. Figure 17 Among them, S1~S21 are: YP-01, YP-02, YP-03, YP-04, YP-05, YP-06, YP-07, YP-08, YP-09, YP- 10. YP-11, YP-12, YP-13, YP-14, YP-15, YP-16, YP-17, YP-18, YP-19, YP-20, YP-21.

[0190] Table 1021 shows the relative retention times of *Clematis chinensis* (Tie Xian Tou Gu Cao) slices.

[0191]

[0192] Based on the principles of stable relative retention time, detectability in all batches of samples, and relatively high peak values, eight peaks with good repeatability were selected as characteristic peaks. As shown in Table 10, the relative retention time RSD of the eight characteristic peaks in all 21 batches of *Imperata cylindrica* decoction pieces was less than 0.11%.

[0193] The relative retention times of 21 batches of *Imperata cylindrica* decoction pieces were all within ±10% of the average relative retention times of 3 batches of *Imperata cylindrica* formula granules (see Table 9 for details). Therefore, the average relative retention time of 3 batches of *Imperata cylindrica* formula granules was taken as the specified value for *Imperata cylindrica* decoction pieces.

[0194] The final specification stipulates that the chromatogram of the test sample should show 8 characteristic peaks, and the retention times should correspond to the 8 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peak 7 should correspond to the retention time of the rutin reference peak. The peak corresponding to the rutin reference peak is the S peak. The relative retention times of the remaining characteristic peaks and the S peak should be calculated, and the relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.35 (peak 1), 0.44 (peak 2), 0.55 (peak 3), 0.61 (peak 4), 0.75 (peak 5), 0.80 (peak 6), 1.00 (peak 7, S), and 1.16 (peak 8).

[0195] The chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version) was used to synthesize chromatographic fingerprints of 21 batches of *Imperata cylindrica* (Tie Xian Tou Gu Cao) decoction pieces, and a reference characteristic chromatogram for *Imperata cylindrica* decoction pieces was established, such as... Figure 18 As shown, Figure 18 This is a comparative chromatogram of the characteristic chromatogram of *Imperata cylindrica* slices provided in Embodiment 1 of the present invention.

[0196] 8) Method for determining the characteristic atlas of *Imperata cylindrica* herbal materials and processed medicinal slices

[0197] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material (column length 100 mm, inner diameter 2.1 mm, packing material particle size 1.7 μm); acetonitrile was used as mobile phase A, and 0.5 wt% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 1; the flow rate was 0.3 mL per minute; the column temperature was 30 °C; the detection wavelength was 354 nm; the theoretical plate number calculated based on the rutin peak should not be less than 5000.

[0198] Preparation of reference solution: Take 0.5g of *Clematis chinensis* reference material, place it in a stoppered conical flask, add 25mL of 50vol% methanol solution, stopper tightly, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference material; separately take an appropriate amount of rutin reference standard, accurately weigh it, add methanol to prepare a solution containing 25μg of rutin per 1mL, as the reference solution of the reference standard.

[0199] Preparation of the test solution: Take 1g of the powder (passed through a No. 3 sieve), place it in a stoppered conical flask, add 50mL of 50vol% methanol solution, stopper tightly, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0200] Determination method: Accurately pipette 1 μL of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0201] The chromatogram of the test sample should show 8 characteristic peaks, and the retention times should correspond to the 8 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peak 7 should correspond to the retention time of the rutin reference peak. The peak corresponding to the rutin reference peak is the S peak. Calculate the relative retention times of the remaining characteristic peaks and the S peak. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.35 (peak 1), 0.44 (peak 2), 0.55 (peak 3), 0.61 (peak 4), 0.75 (peak 5), 0.80 (peak 6), 1.00 (peak 7, S), and 1.16 (peak 8).

[0202] Example 2

[0203] Construction of HPLC characteristic chromatograms of standard decoction of *Clematis chinensis* (Tie Xian Tou Gu Cao):

[0204] 1) Materials, reagents and instruments

[0205] Standard decoction of *Imperata cylindrica* (prepared by Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., batch numbers: BT-01, BT-02, BT-03, BT-04, BT-05, BT-06, BT-07, BT-08, BT-09, BT-10, BT-11, BT-12, BT-13, BT-14, BT-15, BT-16, BT-17, BT-18, BT-19, BT-20, BT-21);

[0206] Other materials, reagents and instruments are the same as in Example 1, and will not be described again.

[0207] 2) Feature map detection method

[0208] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material (column length 100 mm, inner diameter 2.1 mm, packing material particle size 1.7 μm); acetonitrile was used as mobile phase A, and 0.5 wt% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 1; the flow rate was 0.3 mL per minute; the column temperature was 30 °C; the detection wavelength was 354 nm; the theoretical plate number calculated based on the rutin peak should not be less than 5000.

[0209] Preparation of reference solution: Take 0.5g of *Clematis chinensis* reference material, place it in a stoppered conical flask, add 25mL of 50vol% methanol solution, stopper tightly, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference material; separately take an appropriate amount of rutin reference standard, accurately weigh it, add methanol to prepare a solution containing 25μg of rutin per 1mL, as the reference solution of the reference standard.

[0210] Preparation of the test solution: Take 0.2 g of this product, place it in a stoppered conical flask, add 25 mL of 50 vol% methanol solution, stopper tightly, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0211] Determination method: Accurately pipette 1 μL of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0212] 3) Investigation of chromatographic conditions

[0213] 3.1) Selection of mobile phase

[0214] Based on the experimental conditions outlined above, the separation effects of two different mobile phases were investigated: methanol (phase A) - 0.5 vol% phosphoric acid (phase B) and acetonitrile (phase A) - 0.5 vol% phosphoric acid (phase B). The results are shown in [Figure 1]. Figure 19 , Figure 19 Chromatograms of different mobile phases for the standard decoction of *Imperata cylindrica* provided in Example 2 of this invention. (Through...) Figure 19 It can be seen that the chromatogram baseline is relatively stable and there are many chromatographic peaks under the gradient elution condition of acetonitrile-0.5 vol% phosphoric acid solution. Therefore, the gradient elution of acetonitrile-0.5 vol% phosphoric acid solution is used as the mobile phase for the determination of the characteristic chromatogram of the standard decoction of *Imperata cylindrica*.

[0215] 3.2) Wavelength Selection

[0216] Based on the above-specified experimental conditions, a diode array detector was used to perform a full-band scan of the test solution, and chromatograms of the test solution at wavelengths of 330 nm, 354 nm, and 370 nm were extracted. The results are shown in the figure. Figure 20 , Figure 20 This is a chromatogram of different wavelengths of the standard decoction of *Imperata cylindrica* provided in Example 2 of the present invention. (Through...) Figure 20 It can be seen that the chromatographic peak information is greater and the chromatographic baseline is more stable when the detection wavelength is 354nm, so the detection wavelength is determined to be 354nm.

[0217] 3.3) Column Temperature Investigation

[0218] Based on the above-specified experimental conditions, the test solution was subjected to chromatographic detection at column temperatures of 25℃, 30℃, and 35℃, respectively. The results are shown in the figure. Figure 21 And Tables 11-12, Figure 21 This is a column temperature chromatogram of the standard decoction of *Imperata cylindrica* provided in Example 2 of the present invention.

[0219] Table 11 Column Temperature Study - Retention Time

[0220]

[0221] Table 12 Column Temperature Study - Relative Retention Time

[0222]

[0223] pass Figure 21 As can be seen from Tables 11 and 12, when the column temperature is 30℃, the chromatogram peaks are more symmetrical, the separation is better, and the peaks are more complete. Therefore, the column temperature is determined to be 30℃.

[0224] 3.4) Flow velocity investigation

[0225] Based on the above-specified experimental conditions, the test solution was subjected to chromatographic detection at flow rates of 0.2 mL / min, 0.3 mL / min, and 0.4 mL / min, respectively. The results are shown in the figure. Figure 22 And Table 13, Figure 22 This is a chromatogram of the flow rate of the standard decoction of *Imperata cylindrica* provided in Example 2 of the present invention.

[0226] Table 13 Flow velocity study - relative retention time

[0227]

[0228]

[0229] pass Figure 22 As can be seen from Table 13, the chromatogram peak shape is good and the resolution is moderate when the flow rate is 0.3 mL / min, so the flow rate is determined to be 0.3 mL / min.

[0230] 3.5) Delayed Examination

[0231] Based on the above-specified experimental conditions, a delay test was conducted, and the results are shown below. Figure 23 , Figure 23 This is a delayed-effect chromatogram of the standard decoction of *Imperata cylindrica* provided in Example 2 of the present invention. (Through...) Figure 23 It can be seen that the sample has basically no useful chromatographic peaks after 30 minutes, so the sample detection time is set at 30 minutes.

[0232] 4) Preparation of the test solution

[0233] 4.1) Investigation of the dissolving solvent

[0234] Take 0.2 g of this product (batch number: BT-21) and place it in a stoppered conical flask. Dissolve it in 25 mL each of methanol, 30 vol% methanol, 50 vol% methanol, 80 vol% methanol, ethanol, 30 vol% ethanol, 50 vol% ethanol, 80 vol% ethanol, and water. Seal the flask tightly and sonicate it (600 W power, 40 kHz frequency) for 30 minutes. Let it cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0235] Under the experimental conditions outlined above, chromatographic determinations were performed on the test solutions prepared using different solvents. The results are shown below. Figure 24 , Figure 24 This is a graph showing the experimental results of investigating the dissolution solvent during the preparation of the standard decoction of *Clematis chinensis* provided in Example 2 of this invention. Figure 24 It can be seen that when the solvent is 50 vol% methanol, the peak shapes of each characteristic peak are good and the separation is moderate, so the solvent is determined to be 50 vol% methanol.

[0236] 4.2) Investigation of dissolution methods

[0237] Take 0.2g of this product (batch number: BT-21), place it in a stoppered conical flask, add 25mL of 50vol% methanol solution, stopper tightly, and reflux and sonicate the sample (power 600W, frequency 40kHz) for 30 minutes. Cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0238] Under the experimental conditions specified above, the chromatographic determinations of the reflux-treated and sonicated test solutions were performed respectively. The results are shown in the figure. Figure 25 , Figure 25 This is a graph showing the experimental results of investigating the dissolution method during the preparation of the standard decoction of *Clematis chinensis* provided in Example 2 of this invention. Figure 25 It can be seen that the two treatment methods have little difference in effect on the solution of the test sample. Since the ultrasonic method is fast and simple, the method of dissolving the test sample is determined to be ultrasonic-assisted dissolution.

[0239] 4.3) Investigation of dissolution time

[0240] Take 0.2g of this product (batch number: BT-21), place it in a stoppered conical flask, add 25mL of 50vol% methanol solution, stopper tightly, and sonicate (power 600W, frequency 40kHz) for 20 minutes, 30 minutes and 40 minutes respectively. Cool, shake well, filter, and take the filtrate to obtain the test solution.

[0241] Under the experimental conditions outlined above, chromatographic determinations were performed on the test solutions subjected to ultrasonic treatment for different times. The results are shown in the table below. Figure 26 , Figure 26This is a graph showing the experimental results of investigating the dissolution time during the preparation of the standard decoction of *Clematis chinensis* provided in Example 2 of this invention. Figure 26 It can be seen that the chromatogram peak shape and resolution are better when the dissolution time is 30 min, so the dissolution time is determined to be 30 min.

[0242] 4.4) Investigation of the amount of solvent added

[0243] Take 0.2g of this product (batch number: BT-21), place it in a stoppered conical flask, add 15mL, 25mL and 50mL of 50vol% methanol solution respectively, stopper tightly, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0244] Under the experimental conditions specified above, chromatographic determinations were performed on the test solutions with different solvent addition amounts. The results are shown in the figure. Figure 27 , Figure 27 This is a graph showing the experimental results regarding the amount of solvent added during the preparation of the standard decoction of *Clematis chinensis* provided in Example 2 of this invention. Figure 27 It can be seen that when the solvent volume is 25 mL, the peak shape and resolution of each chromatographic peak are better, so the solvent volume is selected as 25 mL.

[0245] 5) Methodological investigation

[0246] 5.1) Chromatographic peak identification

[0247] According to the experimental conditions proposed above, prepare the test solution of the standard decoction of *Hedyotis diffusa*, the reference solution of rutin, and the reference solution of *Hedyotis diffusa* reference material. At the same time, according to the experimental conditions proposed above, prepare the negative control solution (i.e., blank solution) of the iron-deficient *Hedyotis diffusa* standard decoction and perform chromatographic detection.

[0248] Based on the above chromatographic detection results, the characteristic peaks of the standard decoction of *Clematis chinensis* were located, and the results are shown in the figure. Figures 28-30 , Figure 28 This is the chromatographic peak identification diagram of the characteristic chromatogram of the standard decoction of *Imperata cylindrica* provided in Example 2 of the present invention. Figure 29 This is the ultraviolet absorption spectrum of the rutin reference standard provided in Example 2 of the present invention. Figure 30 This is the ultraviolet absorption spectrum of rutin in the standard decoction of *Clematis chinensis* provided in Example 2 of this invention. The results show that peak 7 is the rutin peak. In the following methodological investigation, the rutin peak was designated as the S peak, and the eight characteristic peaks in the sample were investigated.

[0249] 5.2) Precision test

[0250] Take the test solution of the standard decoction of *Clematis chinensis* (batch number: BT-21), inject it 6 times consecutively according to the proposed experimental method, 1 μL each time, and calculate the retention time of each characteristic peak. The results are shown in Table 14.

[0251] Table 14 Precision Examination - Retention Time

[0252]

[0253] As shown in Table 14, the RSD values ​​of the retention times of each peak are 0.09% to 0.19%, indicating that the instrument has good precision.

[0254] 5.3) Repeatability test

[0255] Six portions of the standard decoction of *Imperata cylindrica* (batch number: BT-21) were accurately weighed and prepared and measured according to the proposed experimental method. The results are shown in Table 15.

[0256] Table 15 Repeatability Tests—Relative Retention Times of Characteristic Peaks

[0257]

[0258]

[0259] As shown in Table 15, the RSD of the relative retention time of each characteristic peak is between 0.07% and 0.49%, indicating that the method has good repeatability.

[0260] 5.4) Intermediate precision test

[0261] 5.4.1) Investigation with different instruments

[0262] Based on the above-planned experimental conditions, standard decoction of *Clematis chinensis* (batch number: BT-21) was weighed and used to prepare test solutions. These solutions were then measured on instruments 1, 2, and 3, respectively. The results are shown in the table below. Figure 31 And Table 16, Figure 31 This is a graph showing the experimental results of the standard decoction of *Imperata cylindrica* provided in Example 2 of this invention, examined using different instruments.

[0263] Table 16 Relative Retention Time of Characteristic Peaks Observed with Different Instruments

[0264]

[0265] pass Figure 31 As shown in Table 16, when the above three instruments were used to detect the test sample, the RSD of the relative retention time of each characteristic peak was between 0.63% and 5.28%, indicating that the instruments had good durability.

[0266] 5.4.2) Investigations by different personnel and at different times

[0267] Based on the experimental conditions proposed above, different personnel (A and B) weighed the standard decoction of *Imperata cylindrica* (batch number: BT-21) at different times (T1 and T2) to prepare test samples and conduct tests. The results are shown in Table 17.

[0268] Table 17 Relative Retention Time of Characteristic Peaks for Different Personnel and Time Periods

[0269]

[0270] As shown in Table 17, under different sample preparation personnel and different sample preparation time conditions, the RSD of the relative retention time of each characteristic peak is between 0.08% and 0.58%, indicating good method stability.

[0271] 5.5) Durability test

[0272] 5.5.1) Column robustness test

[0273] Based on the above-planned experimental conditions, analyses were conducted using chromatographic columns 1, 2, and 3, respectively. The results are shown in the table below. Figure 32 And Table 18, Figure 32 This is a graph showing the experimental results of different chromatographic columns for the standard decoction of *Imperata cylindrica* provided in Example 2 of this invention.

[0274] Table 18 Column Robustness Study—Relative Retention Times of Characteristic Peaks

[0275]

[0276] pass Figure 32 As shown in Table 18, when the samples were detected using the above three chromatographic columns, the RSD of the relative retention time of the characteristic peaks ranged from 1.09% to 4.33%, indicating that the chromatographic columns had good robustness.

[0277] 5.5.2) Stability Test

[0278] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 2h, 4h, 8h, 16h and 24h respectively. The results are shown in Table 19.

[0279] Table 19 Stability Study—Retention Time

[0280]

[0281] As can be seen from Table 19, the RSD of the corresponding characteristic peak retention time is between 0.70% and 1.46%, and the sample solution is relatively stable within 24 hours.

[0282] In summary, the RSD of the relative retention times of each characteristic peak meets the requirements in all the above tests, indicating that the method is effective. The above eight characteristic peaks will be included in subsequent investigations.

[0283] 6) Determination of characteristic peaks and establishment of reference spectra

[0284] 6.1) Verification results of 21 batches of standard decoction of *Imperata cylindrica*

[0285] Using the proposed method, characteristic chromatographic analysis was performed on 21 batches of standard decoctions of *Imperata cylindrica*, and the relative retention times were calculated. The results are shown below. Figure 33 And Table 20, Figure 33 These are the characteristic chromatogram verification diagrams of 21 batches of *Imperata cylindrica* standard decoction provided in Example 2 of this invention. Figure 33 Among them, S1~S21 are: BT-01, BT-02, BT-03, BT-04, BT-05, BT-06, BT-07, BT-08, BT-09, BT-10, BT-11, BT-12, BT-13, BT-14, BT-15, BT-16, BT-17, BT-18, BT-19, BT-20, BT-21.

[0286] Table 2021 Relative Retention Time of Standard Decoction of *Clematis chinensis* (Tie Xian Tou Gu Cao)

[0287]

[0288]

[0289] Based on the principles of stable relative retention time, detectability in all batches of samples, and relatively high peak values, eight peaks with good repeatability were selected as characteristic peaks. According to the methodological investigation results and the validation results of 21 batches of standard decoctions, the theoretical plate number calculated based on the rutin peak should be no less than 5000.

[0290] 6.2) Establishment of limits for relative retention time

[0291] Table 21 summarizes the methodological examination items and validation results:

[0292] Table 21 Summary of RSD% for Methodological Results — Relative Retention Time / Retention Time

[0293]

[0294] As can be seen from Table 21, the relative retention times of each characteristic peak are stable and within ±10% of the average value. Therefore, the specified range of the relative retention times of each peak is determined to be ±10%.

[0295] The relative retention times of the 21 batches of *Imperata cylindrica* standard decoction were all within ±10% of the average relative retention times of the 3 batches of *Imperata cylindrica* formula granules (see Table 9 for details). Therefore, the average relative retention time of the 3 batches of *Imperata cylindrica* formula granules was taken as the specified value for the *Imperata cylindrica* standard decoction.

[0296] The final specification stipulates that the chromatogram of the test sample should show 8 characteristic peaks, and the retention times should correspond to the 8 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peak 7 should correspond to the retention time of the rutin reference peak. The peak corresponding to the rutin reference peak is the S peak. The relative retention times of the remaining characteristic peaks and the S peak should be calculated, and the relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.35 (peak 1), 0.44 (peak 2), 0.55 (peak 3), 0.61 (peak 4), 0.75 (peak 5), 0.80 (peak 6), 1.00 (peak 7, S), and 1.16 (peak 8).

[0297] The chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version) was used to synthesize 21 batches of *Imperata cylindrica* standard decoction, and a reference characteristic chromatogram of the *Imperata cylindrica* standard decoction was established, such as... Figure 34 As shown, Figure 34 This is a comparative chromatogram of the characteristic chromatogram of the standard decoction of *Imperata cylindrica* provided in Example 2 of the present invention.

[0298] 7) Determination of the characteristic chromatogram method for standard decoction of *Imperata cylindrica*

[0299] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material (column length 100 mm, inner diameter 2.1 mm, packing material particle size 1.7 μm); acetonitrile was used as mobile phase A, and 0.5 wt% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 1; the flow rate was 0.3 mL per minute; the column temperature was 30 °C; the detection wavelength was 354 nm; the theoretical plate number calculated based on the rutin peak should not be less than 5000.

[0300] Preparation of reference solution: Take 0.5g of *Clematis chinensis* reference material, place it in a stoppered conical flask, add 25mL of 50vol% methanol solution, stopper tightly, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference material; separately take an appropriate amount of rutin reference standard, accurately weigh it, add methanol to prepare a solution containing 25μg of rutin per 1mL, as the reference solution of the reference standard.

[0301] Preparation of the test solution: Take 0.2 g of this product, place it in a stoppered conical flask, add 25 mL of 50 vol% methanol solution, stopper tightly, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0302] Determination method: Accurately pipette 1 μL of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0303] The chromatogram of the test sample should show 8 characteristic peaks, and the retention times should correspond to the 8 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peak 7 should correspond to the retention time of the rutin reference peak. The peak corresponding to the rutin reference peak is the S peak. Calculate the relative retention times of the remaining characteristic peaks and the S peak. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.35 (peak 1), 0.44 (peak 2), 0.55 (peak 3), 0.61 (peak 4), 0.75 (peak 5), 0.80 (peak 6), 1.00 (peak 7, S), and 1.16 (peak 8).

[0304] Example 3

[0305] Construction of HPLC characteristic chromatograms of *Imperata cylindrica* formula granules:

[0306] 1) Materials, reagents and instruments

[0307] Iron Wire Toutugucao Formula Granules (Prepared by Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., Batch No.: KL-01, KL-02, KL-03);

[0308] Other materials, reagents and instruments are the same as in Example 1, and will not be described again.

[0309] 2) Feature map detection method

[0310] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material (column length 100 mm, inner diameter 2.1 mm, packing material particle size 1.7 μm); acetonitrile was used as mobile phase A, and 0.5 wt% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 1; the flow rate was 0.3 mL per minute; the column temperature was 30 °C; the detection wavelength was 354 nm; the theoretical plate number calculated based on the rutin peak should not be less than 5000.

[0311] Preparation of reference solution: Take 0.5g of *Clematis chinensis* reference material, place it in a stoppered conical flask, add 25mL of 50vol% methanol solution, stopper tightly, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference material; separately take an appropriate amount of rutin reference standard, accurately weigh it, add methanol to prepare a solution containing 25μg of rutin per 1mL, as the reference solution of the reference standard.

[0312] Preparation of the test solution: Take 0.2 g of this product, grind it finely, place it in a stoppered conical flask, add 25 mL of 50% methanol solution, stopper tightly, treat it with ultrasonic waves (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0313] Determination method: Precisely pipette 1 μL each of the reference solution and the test solution, inject them into the liquid chromatograph for determination to obtain the results.

[0314] 3) Investigation of chromatographic conditions

[0315] 3.1) Selection of mobile phase

[0316] Based on the above-mentioned determined experimental conditions, investigate the separation effects of 2 different mobile phases, namely: methanol (phase A) - 0.5% phosphoric acid (phase B), acetonitrile (phase A) - 0.5% phosphoric acid (phase B). The results are shown in Figure 35 , Figure 35 the chromatograms of different mobile phases of the Herba Phrymae leptostachyae formula granules provided in Example 3 of the present invention. It can be seen from Figure 35 that under the gradient elution conditions of acetonitrile - 0.5% phosphoric acid solution, the chromatogram baseline is relatively stable and there are many chromatographic peaks. Therefore, the gradient elution of acetonitrile - 0.5% phosphoric acid solution is used as the mobile phase for the determination method of the characteristic chromatogram of the Herba Phrymae leptostachyae formula granules.

[0317] 3.2) Selection of wavelength

[0318] Based on the above-mentioned determined experimental conditions, use a diode array detector to perform a full wavelength scan on the test solution, and separately extract the chromatograms of the test solution at wavelengths of 330 nm, 354 nm, and 370 nm. The results are shown in Figure 36 , Figure 36 the chromatograms of different wavelengths of the Herba Phrymae leptostachyae formula granules provided in Example 3 of the present invention. It can be seen from Figure 36 that when the detection wavelength is 354 nm, the chromatographic peak information amount is larger and the chromatogram baseline is more stable. Therefore, the detection wavelength is determined to be 354 nm.

[0319] 3.3) Investigation of column temperature

[0320] Based on the above-mentioned determined experimental conditions, perform chromatographic detection on the test solution at column temperatures of 25 °C, 30 °C, and 35 °C respectively. The results are shown in Figure 37 and Table 22 - 23, Figure 37 which are the chromatograms of the investigation of column temperature of the Herba Phrymae leptostachyae formula granules provided in Example 3 of the present invention.

[0321] Table 22 Investigation of column temperature - retention time

[0322]

[0323] Table 23 Column Temperature Study - Relative Retention Time

[0324]

[0325] pass Figure 37 As can be seen from Tables 22 and 23, when the column temperature is 30℃, the chromatogram peaks are more symmetrical, the separation is better, and the peaks are more complete. Therefore, the column temperature is determined to be 30℃.

[0326] 3.4) Flow velocity investigation

[0327] Based on the above-specified experimental conditions, the test solution was subjected to chromatographic detection at flow rates of 0.2 mL / min, 0.3 mL / min, and 0.4 mL / min, respectively. The results are shown in the figure. Figure 38 And Tables 24-25, Figure 38 This is a chromatogram of the flow rate of the *Imperata cylindrica* formulation granules provided in Example 3 of the present invention.

[0328] Table 24 Flow velocity assessment - retention time

[0329]

[0330]

[0331] Table 25 Flow velocity study - relative retention time

[0332]

[0333] pass Figure 38 As can be seen from Tables 24-25, the chromatogram peak shape is good and the resolution is moderate when the flow rate is 0.3 mL / min, so the flow rate is determined to be 0.3 mL / min.

[0334] 3.5) Delayed Examination

[0335] Based on the above-specified experimental conditions, a delay test was conducted, and the results are shown below. Figure 39 , Figure 39 This is a delayed-action chromatogram of the *Imperata cylindrica* formulation granules provided in Example 3 of the present invention. (Through...) Figure 39 It can be seen that the sample has basically no useful chromatographic peaks after 30 minutes, so the sample detection time is set at 30 minutes.

[0336] 4) Preparation of the test solution

[0337] 4.1) Investigation of the dissolving solvent

[0338] Take 0.2g of this product (batch number: KL-01), grind it into a fine powder, place it in a stoppered conical flask, and dissolve it separately in 25mL each of methanol, 30vol% methanol, 50vol% methanol, 80vol% methanol, ethanol, 30vol% ethanol, 50vol% ethanol, 80vol% ethanol, and water. Seal the flask tightly, sonicate it (power 600W, frequency 40kHz) for 30 minutes, cool it, shake it well, filter it, and take the filtrate to obtain the test solution.

[0339] Under the experimental conditions outlined above, chromatographic determinations were performed on the test solutions prepared using different solvents. The results are shown below. Figure 40 , Figure 40 This is a graph showing the experimental results of investigating the dissolution solvent during the preparation of the test solution of the *Imperata cylindrica* (Imperata cylindrica) formula granules provided in Example 3 of this invention. Figure 40 It can be seen that when the solvent is 50 vol% methanol, the peak shapes of each characteristic peak are good and the separation is moderate, so the solvent is determined to be 50 vol% methanol.

[0340] 4.2) Investigation of dissolution methods

[0341] Take 0.2g of this product (batch number: KL-01), grind it into a fine powder, place it in a stoppered conical flask, add 25mL of 50vol% methanol solution, stopper tightly, and reflux and sonicate the sample (power 600W, frequency 40kHz) for 30 minutes. Cool, shake well, filter, and take the filtrate to obtain the test solution.

[0342] Under the experimental conditions specified above, the chromatographic determinations of the reflux-treated and sonicated test solutions were performed respectively. The results are shown in the figure. Figure 41 , Figure 41 This is a graph showing the experimental results of investigating the dissolution method during the preparation of the test sample solution of the *Imperata cylindrica* (Imperata cylindrica) formula granules provided in Example 3 of this invention. Figure 41 It can be seen that the two treatment methods have little difference in effect on the solution of the test sample. Since the ultrasonic method is fast and simple, the method of dissolving the test sample is determined to be ultrasonic-assisted dissolution.

[0343] 4.3) Investigation of dissolution time

[0344] Take 0.2g of this product (batch number: KL-01), grind it into a fine powder, place it in a stoppered conical flask, add 25mL of 50vol% methanol solution, seal tightly, and sonicate (power 600W, frequency 40kHz) for 20 minutes, 30 minutes, and 40 minutes respectively. Cool, shake well, filter, and take the filtrate to obtain the test solution.

[0345] Under the experimental conditions outlined above, chromatographic determinations were performed on the test solutions subjected to ultrasonic treatment for different times. The results are shown in the table below. Figure 42 , Figure 42This is a graph showing the experimental results of investigating the dissolution time during the preparation of the test sample solution of *Imperata cylindrica* (Imperata cylindrica) formula granules provided in Example 3 of this invention. Figure 42 It can be seen that the chromatogram peak shape and resolution are better when the dissolution time is 30 min, so the dissolution time is determined to be 30 min.

[0346] 4.4) Investigation of the amount of solvent added

[0347] Take 0.2g of this product (batch number: KL-01), grind it into a fine powder, place it in a stoppered conical flask, add 15mL, 25mL and 50mL of 50vol% methanol solution respectively, stopper tightly, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0348] Under the experimental conditions specified above, chromatographic determinations were performed on the test solutions with different solvent addition amounts. The results are shown in the figure. Figure 43 , Figure 43 This is a graph showing the experimental results of investigating the amount of solvent added during the preparation of the test sample solution of *Imperata cylindrica* (Imperata cylindrica) formula granules provided in Example 3 of this invention. Figure 43 It can be seen that when the solvent volume is 25 mL, the peak shape and resolution of each chromatographic peak are better, so the solvent volume is selected as 25 mL.

[0349] 5) Methodological investigation

[0350] 5.1) Chromatographic peak identification

[0351] According to the experimental conditions proposed above, prepare the test solution of *Imperata cylindrica* formula granules, the reference solution of rutin, and the reference solution of *Imperata cylindrica* reference medicinal material. At the same time, according to the experimental conditions proposed above, prepare the negative control solution (i.e., blank solution) of iron-deficient *Imperata cylindrica* formula granules and perform chromatographic detection.

[0352] Based on the above chromatographic detection results, the characteristic peaks of the *Imperata cylindrica* formula granules were located, and the results are shown below. Figures 44-46 , Figure 44 This is the chromatographic peak identification diagram of the characteristic granules of *Imperata cylindrica* provided in Example 3 of the present invention. Figure 45 This is the ultraviolet absorption spectrum of the rutin reference standard provided in Example 3 of the present invention. Figure 46 This is the UV absorption spectrum of rutin in the *Imperata cylindrica* (Imperata cylindrica) formula granules provided in Example 3 of this invention. The results show that peak 7 is the rutin peak. In the following methodological investigation, the rutin peak was designated as the S peak, and the eight characteristic peaks in the sample were investigated.

[0353] 5.2) Precision test

[0354] Take the test solution of *Imperata cylindrica* formula granules (batch number: KL-01), inject it 6 times consecutively according to the proposed experimental method, 1 μL each time, and calculate the retention time of each characteristic peak. The results are shown in Table 26.

[0355] Table 26 Precision Examination - Retention Time

[0356]

[0357] As shown in Table 26, the RSD values ​​of the retention times of each peak are 0.12% to 0.30%, indicating that the instrument has good precision.

[0358] 5.3) Repeatability test

[0359] Six portions of the Iron Thread Herb Formula Granules (batch number: KL-01) were prepared and tested according to the proposed experimental method. The results are shown in Table 27.

[0360] Table 27 Repeatability Tests—Relative Retention Time of Characteristic Peaks

[0361]

[0362]

[0363] As shown in Table 27, the relative retention time (RSD) of the six samples ranged from 0.04% to 0.38%, indicating that the method has good repeatability.

[0364] 5.4) Intermediate precision test

[0365] 5.4.1) Investigation with different instruments

[0366] Based on the above-established experimental conditions, three portions of the *Imperata cylindrica* formula granules (batch number: KL-01) were weighed and used to prepare test solutions. The solutions were then measured on instruments 1, 2, and 3, respectively. The results are shown below. Figure 47 And Table 28, Figure 47 This is a graph showing the experimental results of the Iron Wire Thorn Grass Formula Granules provided in Embodiment 3 of the present invention, examined using different instruments.

[0367] Table 28 Relative Retention Time of Characteristic Peaks Observed with Different Instruments

[0368]

[0369] pass Figure 47 As shown in Table 28, when the above three instruments were used to detect the test sample, the RSD of the relative retention time of each characteristic peak was between 0.63% and 5.24%, indicating that the instruments had good durability.

[0370] 5.4.2) Investigations by different personnel and at different times

[0371] Based on the experimental conditions proposed above, different personnel (A and B) weighed the Iron Thread Herb Formula Granules (batch number: KL-01) at different times (T1 and T2) to prepare test samples and conduct tests. The results are shown in Table 29.

[0372] Table 29 Relative Retention Time of Characteristic Peaks for Different Personnel and Time Periods

[0373]

[0374] As shown in Table 29, under different sample preparation personnel and different sample preparation times, the RSD of the relative retention times of each characteristic peak ranged from 0.00% to 0.33%, indicating that the method has good intermediate precision.

[0375] 5.5) Durability test

[0376] 5.5.1) Column robustness test

[0377] Based on the above-planned experimental conditions, analyses were conducted using chromatographic columns 1, 2, and 3, respectively. The results are shown in the table below. Figure 48 And Table 30, Figure 48 This is a graph showing the experimental results of different chromatographic columns for the *Imperata cylindrica* formulation granules provided in Example 3 of this invention.

[0378] Table 30 Column Robustness Study—Relative Retention Times of Characteristic Peaks

[0379]

[0380] pass Figure 48 As shown in Table 30, when the samples were detected using the above three chromatographic columns, the RSD of the relative retention time of the characteristic peaks ranged from 1.09% to 4.33%, indicating that the chromatographic columns had good robustness.

[0381] 5.5.2) Stability Test

[0382] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 2h, 4h, 8h, 16h and 24h respectively. The results are shown in Table 31.

[0383] Table 31 Stability Study—Retention Time

[0384]

[0385] As can be seen from Table 31, the RSD of the corresponding characteristic peak retention time is between 0.62% and 1.50%, and the sample solution is relatively stable within 24 hours.

[0386] In summary, the RSD of the relative retention times of each characteristic peak meets the requirements in all the above tests, indicating that the method is effective. The above eight characteristic peaks will be included in subsequent investigations.

[0387] 6) Determination of characteristic peaks and establishment of reference spectra

[0388] 6.1) Validation results of three batches of *Imperata cylindrica* (iron-wire grass) formula granules

[0389] Using the proposed method, characteristic chromatographic analysis was performed on three batches of *Imperata cylindrica* (Tie Xian Tou Gu Cao) formula granules, and the relative retention time was calculated. The results are shown below. Figure 49 And Table 32, Figure 49 These are the characteristic spectral verification diagrams of three batches of *Imperata cylindrica* formula granules provided in Example 3 of this invention.

[0390] Table 323 shows the relative retention times of the formula granules for *Imperata cylindrica*.

[0391]

[0392] Based on the principles of relatively stable retention time, detectability in all batches of samples, and relatively high peak values, eight robust peaks (peaks 1, 2, 3, 4, 5, 6, 7, and 8) were selected as characteristic peaks. Based on the methodological investigation results and the particle validation results of three batches, the theoretical plate number calculated using the rutin peak value was determined to be no less than 5000.

[0393] 6.2) Establishment of limits for relative retention time

[0394] Table 33 summarizes the methodological examination items and validation results:

[0395] Table 33 Summary of RSD% for Methodological Results—Relative Retention Time / Retention Time

[0396]

[0397]

[0398] As can be seen from Table 33, the relative retention times of each characteristic peak are stable and within ±10% of the average value. Therefore, the specified range of the relative retention times of each peak is determined to be ±10%.

[0399] The final specification stipulates that the chromatogram of the test sample should show 8 characteristic peaks, and the retention times should correspond to the 8 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peak 7 should correspond to the retention time of the rutin reference peak. The peak corresponding to the rutin reference peak is the S peak. The relative retention times of the remaining characteristic peaks and the S peak should be calculated, and the relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.35 (peak 1), 0.44 (peak 2), 0.55 (peak 3), 0.61 (peak 4), 0.75 (peak 5), 0.80 (peak 6), 1.00 (peak 7, S), and 1.16 (peak 8).

[0400] The chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version) was used to synthesize three batches of *Imperata cylindrica* formula granules, and a reference characteristic chromatogram for the *Imperata cylindrica* formula granules was established, such as... Figure 50 As shown, Figure 50 This is a comparative chromatogram of the characteristic chromatogram of the *Imperata cylindrica* formula granules provided in Example 3 of the present invention.

[0401] 7) Determination of the characteristic chromatogram method for the formulation granules of *Imperata cylindrica*

[0402] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material (column length 100 mm, inner diameter 2.1 mm, packing material particle size 1.7 μm); acetonitrile was used as mobile phase A, and 0.5 wt% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 1; the flow rate was 0.3 mL per minute; the column temperature was 30 °C; the detection wavelength was 354 nm; the theoretical plate number calculated based on the rutin peak should not be less than 5000.

[0403] Preparation of reference solution: Take 0.5g of *Clematis chinensis* reference material, place it in a stoppered conical flask, add 25mL of 50vol% methanol solution, stopper tightly, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference material; separately take an appropriate amount of rutin reference standard, accurately weigh it, add methanol to prepare a solution containing 25μg of rutin per 1mL, as the reference solution of the reference standard.

[0404] Preparation of the test solution: Take 0.2g of this product, grind it into a fine powder, place it in a stoppered conical flask, add 25mL of 50vol% methanol solution, stopper tightly, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0405] Determination method: Accurately pipette 1 μL of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0406] The chromatogram of the test sample should show 8 characteristic peaks, and the retention times should correspond to the 8 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peak 7 should correspond to the retention time of the rutin reference peak. The peak corresponding to the rutin reference peak is the S peak. Calculate the relative retention times of the remaining characteristic peaks and the S peak. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.35 (peak 1), 0.44 (peak 2), 0.55 (peak 3), 0.61 (peak 4), 0.75 (peak 5), 0.80 (peak 6), 1.00 (peak 7, S), and 1.16 (peak 8).

[0407] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing HPLC characteristic chromatograms of *Imperata cylindrica* herbal medicine, processed slices, standard decoctions, and formulated granules, characterized in that, Includes the following steps: Prepare test samples, which are herbal medicine of *Imperata cylindrica*, sliced ​​*Imperata cylindrica*, standard decoction of *Imperata cylindrica*, or formula granules of *Imperata cylindrica*. The test sample is dissolved to obtain a test sample solution; the solvent for dissolution is a 50 vol% methanol solution, and the dissolution is carried out under ultrasonic assistance. The test solution was analyzed by high performance liquid chromatography to obtain the corresponding HPLC characteristic chromatogram of the test sample; The chromatographic conditions for the high-performance liquid chromatography (HPLC) method are as follows: C18 column; mobile phase A is acetonitrile, mobile phase B is 0.5 wt% phosphoric acid solution, gradient elution; mobile phase flow rate is 0.3 mL / min; detection wavelength is 354 nm; injection volume is 1 μL; column temperature is 30 ℃; theoretical plate number calculated based on rutin peak is not less than 5000. The gradient elution specifically refers to: 0~9min, Phase A: 4~8 vol%, Phase B: 96~92 vol% 9~16min, Phase A: 8~13 vol%, Phase B: 92~87 vol% 16~20min, Phase A: 13~15 vol%, Phase B: 87~85 vol% 20-25 min, Phase A: 15-13 vol%, Phase B: 85-87 vol% 25-30 min, Phase A: 13 vol%, Phase B: 87 vol%.

2. The construction method according to claim 1, characterized in that, The ratio of the sample to the solvent is (0.2~0.5) g: 25 mL; the ultrasonic assistance power is 580~620 W, the frequency is 35~45 kHz, and the time is 25~35 min.

3. The construction method according to claim 1, characterized in that, It also includes the following steps: Dissolve the reference herb *Clematis chinensis* to obtain a reference herb solution; dissolve rutin to obtain a reference standard solution. The reference solutions of the reference medicinal materials and the reference standard were determined by high performance liquid chromatography to obtain the chromatograms of the reference materials; and the components of the HPLC characteristic chromatograms of the test sample were identified based on the chromatograms of the reference materials.

4. The construction method according to claim 3, characterized in that, It also includes the following steps: The similarity of the HPLC characteristic chromatogram of *Imperata cylindrica* was evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, resulting in an HPLC standard characteristic chromatogram of *Imperata cylindrica* consisting of eight characteristic peaks. In this HPLC standard characteristic chromatogram, peak 7, corresponding to the rutin reference peak, is peak S. The relative retention times of each characteristic peak and peak S were calculated, and the relative retention times were within ±10% of a specified value. The specified value is: 0.35 (peak 1), 0.44 (peak 2), 0.55 (peak 3), 0.61 (peak 4), 0.75 (peak 5), 0.80 (peak 6), 1.00 (peak 7), 1.16 (peak 8).

5. The construction method according to claim 3, characterized in that, It also includes the following steps: The similarity of the HPLC characteristic chromatograms of *Imperata cylindrica* slices was evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, resulting in an HPLC standard characteristic chromatogram of *Imperata cylindrica* slices consisting of 8 characteristic peaks. In this HPLC standard characteristic chromatogram, peak 7, corresponding to the rutin reference peak, is peak S. The relative retention times of each characteristic peak and peak S were calculated, and the relative retention times were within ±10% of a specified value. The specified value is: 0.35 (peak 1), 0.44 (peak 2), 0.55 (peak 3), 0.61 (peak 4), 0.75 (peak 5), 0.80 (peak 6), 1.00 (peak 7), 1.16 (peak 8).

6. The construction method according to claim 3, characterized in that, It also includes the following steps: The similarity of the HPLC characteristic chromatograms of the *Imperata cylindrica* standard decoction was evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system. An HPLC standard characteristic chromatogram of the *Imperata cylindrica* standard decoction consisting of eight characteristic peaks was obtained. In the HPLC standard characteristic chromatogram, peak 7, corresponding to the rutin reference peak, is peak S. The relative retention times of each characteristic peak and peak S were calculated. The relative retention times were within ±10% of a specified value. The specified value is: 0.35 (peak 1), 0.44 (peak 2), 0.55 (peak 3), 0.61 (peak 4), 0.75 (peak 5), 0.80 (peak 6), 1.00 (peak 7), 1.16 (peak 8).

7. The construction method according to claim 3, characterized in that, It also includes the following steps: The similarity of the HPLC characteristic chromatograms of *Imperata cylindrica* formula granules was evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, resulting in an HPLC standard characteristic chromatogram of the *Imperata cylindrica* formula granules consisting of 8 characteristic peaks. In the HPLC standard characteristic chromatogram, peak 7, corresponding to the rutin reference peak, is peak S. The relative retention times of each characteristic peak and peak S were calculated, and the relative retention times were within ±10% of a specified value. The specified value is: 0.35 (peak 1), 0.44 (peak 2), 0.55 (peak 3), 0.61 (peak 4), 0.75 (peak 5), 0.80 (peak 6), 1.00 (peak 7), 1.16 (peak 8).

8. A method for identifying *Imperata cylindrica* herbal medicine, processed slices, standard decoctions, and formulated granules, characterized in that... The HPLC characteristic chromatograms obtained by the construction method described in any one of claims 1 to 7 are used as the basis for identifying the medicinal materials, decoction pieces, standard decoctions, and formula granules of *Imperata cylindrica*.

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