A method for determining the content of rutin in iron wire tradescantia medicinal materials, decoction pieces, standard decoction and formula granules

Abstract

The present application belongs to the technical field of detection, and particularly relates to a method for determining the rutin content in iron wire through bone Chinese herbal medicine, decoction pieces, standard decoction and formula granules, comprising the following steps: preparing a test sample and a control sample, wherein the control sample is rutin; dissolving the test sample and the control sample respectively to obtain corresponding test sample solution and control sample solution; determining the test sample solution and the control sample solution respectively by using high performance liquid chromatography (HPLC) to obtain HPLC color spectrum of the test sample solution and the control sample solution; and calculating the rutin content in the test sample by using an external standard method according to the concentration of rutin in the control sample solution, the peak area of the control sample solution in the chromatogram and the peak area of the component corresponding to the control sample in the chromatogram of the test sample. The method provided by the present application can quantitatively determine the index component, i.e. rutin, in iron wire through bone Chinese herbal medicine, decoction pieces, standard decoction and formula granules, thereby providing a more scientific basis for quality control of the above-mentioned medicines.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, and in particular relates to a method for determining the rutin content in *Imperata cylindrica* herbal medicine, decoction pieces, standard decoctions, and formula granules. 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 method for determining the content of its key components to control its quality. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide a method for determining the rutin content in *Imperata cylindrica* herbal materials, processed slices, standard decoctions, and formulated granules. The method provided by this invention can quantitatively determine the indicator component—rutin—in *Imperata cylindrica* herbal materials, processed slices, standard decoctions, and formulated granules, thereby providing a more scientific basis for the quality control of the above-mentioned medicines.

[0004] This invention provides a method for determining the rutin content in *Imperata cylindrica* herbal medicine, processed slices, standard decoctions, and formulated granules, comprising the following steps:

[0005] Prepare test samples and reference standards. The test samples are *Imperata cylindrica* herbal medicine, *Imperata cylindrica* slices, *Imperata cylindrica* standard decoction, or *Imperata cylindrica* formula granules. The reference standard is rutin.

[0006] The test sample and the reference sample were dissolved separately to obtain the corresponding test sample solution and reference sample solution;

[0007] The test solution and the reference solution were determined by high performance liquid chromatography (HPLC) to obtain the corresponding HPLC chromatograms of the test solution and the reference solution. The chromatographic conditions of the HPLC were as follows: C18 column; mobile phase A was acetonitrile, mobile phase B was 0.5 wt% phosphoric acid solution, and gradient elution was used.

[0008] The rutin content in the test sample is calculated using the external standard method based on the concentration of rutin in the reference solution, the peak area of ​​the reference solution in the chromatogram, and the peak area of ​​the component in the test sample corresponding to the reference in the chromatogram.

[0009] In the determination 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 determination method provided by the present invention, the solvent for dissolving the test sample is preferably an 80 vol% ethanol solution, which has the highest dissolution efficiency; the ratio of the test sample to the solvent is (0.2-0.5) g: 25 mL, which ensures complete dissolution and extraction of the target component.

[0011] In the determination 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, which ensures complete dissolution and extraction of the target component.

[0012] In the determination 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 determination 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 preferred ratio of the amount of test sample to solvent is 0.2g:25mL.

[0014] In the determination 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 determination method provided by the present invention, the solvent for dissolving the reference standard is preferably methanol; the ratio of the amount of the reference standard to the solvent is preferably (20-30) μg:1 mL, more preferably 25 μg:1 mL.

[0016] In the determination method provided by this invention, the gradient elution process is preferably performed as follows when conducting the high-performance liquid chromatography determination:

[0017] 0–10 min, Phase A: 10–15 vol%, Phase B: 90–85 vol%;

[0018] 10–15 min, Phase A: 15–18 vol%, Phase B: 85–82 vol%.

[0019] In the determination method provided by the present invention, when performing the determination by high performance liquid chromatography, the flow rate of the mobile phase is preferably 0.35 mL / min.

[0020] 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.

[0021] In the determination method provided by the present invention, the column temperature is preferably 30°C when performing the high performance liquid chromatography determination.

[0022] In the determination method provided by the present invention, the detection wavelength is preferably 354 nm when performing the high performance liquid chromatography determination.

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

[0024] In the determination method provided by the present invention, when performing the determination by high performance liquid chromatography, the theoretical plate number calculated based on the rutin peak should not be less than 5000.

[0025] In the determination method provided by the present invention, the concentration of rutin is used as the abscissa and the corresponding chromatographic peak area is used as the ordinate. When the concentration of rutin is in the range of 3.028296 to 100.9432 μg / mL, the linear relationship is preferably y = 2.9903x - 0.4660.

[0026] Compared with existing technologies, this invention provides a method for determining the rutin content in *Tie Xian Tou Gu* herbal materials, processed slices, standard decoctions, and formulated granules. This invention uses rutin as an indicator component in *Tie Xian Tou Gu* herbal materials, processed slices, standard decoctions, and formulated granules, enabling more standardized, comprehensive, and effective quality control of these materials and preparations, thus ensuring the efficacy of the drugs. The determination method provided by this invention is time-efficient, stable, precise, reproducible, convenient, and easy to master, and has good prospects for widespread application. Attached Figure Description

[0027] 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.

[0028] Figure 1 This is a chromatogram of different mobile phases for the herbal medicine *Imperata cylindrica* provided in Example 1 of this invention;

[0029] Figure 2 This is a chromatogram of the flow rate of the herbal medicine *Imperata cylindrica* provided in Example 1 of this invention.

[0030] Figure 3 This is a column temperature chromatogram of the herbal medicine *Imperata cylindrica* provided in Example 1 of the present invention.

[0031] Figure 4 This is a chromatogram of the injection volume of the herbal medicine *Imperata cylindrica* provided in Example 1 of the present invention.

[0032] Figure 5 This is the specific experimental chromatogram provided in Embodiment 1 of the present invention;

[0033] Figure 6 This is the rutin standard curve diagram provided in Embodiment 1 of the present invention;

[0034] Figure 7 This is a chromatogram of different mobile phases for the standard decoction of *Imperata cylindrica* provided in Example 2 of the present invention;

[0035] Figure 8 This is a chromatogram of the flow rate of the standard decoction of *Imperata cylindrica* provided in Example 2 of the present invention.

[0036] Figure 9 This is a column temperature chromatogram of the standard decoction of *Imperata cylindrica* provided in Example 2 of this invention;

[0037] Figure 10 This is a chromatogram of the injection volume of the standard decoction of *Imperata cylindrica* provided in Example 2 of the present invention.

[0038] Figure 11 This is the specific experimental chromatogram provided in Embodiment 2 of the present invention;

[0039] Figure 12 This is a chromatogram of different mobile phases for the *Imperata cylindrica* formulation granules provided in Example 3 of the present invention;

[0040] Figure 13This is a chromatogram of the flow rate of the *Imperata cylindrica* formulation granules provided in Example 3 of the present invention.

[0041] Figure 14 This is a column temperature chromatogram of the *Imperata cylindrica* formula granules provided in Example 3 of the present invention.

[0042] Figure 15 This is a chromatogram of the injection volume of the *Imperata cylindrica* formula granules provided in Example 3 of the present invention.

[0043] Figure 16 This is the specific experimental chromatogram provided in Embodiment 3 of the present invention. Detailed Implementation

[0044] 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.

[0045] Example 1

[0046] Determination of rutin content in *Imperata cylindrica* herbal medicine and processed medicinal slices:

[0047] 1) Experimental instruments and materials:

[0048] High performance liquid chromatograph: Agilent ultra-high performance liquid chromatograph, Waters ultra-high performance liquid chromatograph, Thermo Fisher ultra-high performance liquid chromatograph. Unless otherwise specified, Agilent ultra-high performance liquid chromatograph is used by default.

[0049] Chromatographic column: Column 1 (Manufacturer: Waters; Model) Column 1 (C18), Column 2 (manufacturer: YMC; model: YMC-Triart C18), and Column 3 (manufacturer: Shimadzu; model: Shim-pack Velox SP-C18) are all used. All three columns use octadecylsilane-bonded silica gel as the packing material, have a column length of 100 mm, an inner diameter of 2.1 mm, and a packing material particle size of 1.7 μm. Unless otherwise specified, Column 1 is used by default.

[0050] Electronic balance, ultrapure water system, ultrasonic cleaner.

[0051] 2) Reagents and reagents:

[0052] Acetonitrile and phosphoric acid were of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0053] Rutin reference standard (China National Institutes for Food and Drug Control, batch number: 100080-202012, purity: 91.6%);

[0054] Iron Wire Penetrating Bone Herbs: Herb 1, Herb 2, Herb 3, Herb 4, Herb 5, Herb 6, Herb 7, Herb 8, Herb 9, Herb 10, Herb 11, Herb 12, Herb 13, Herb 14, Herb 15, Herb 16, Herb 17, Herb 18, Herb 19, Herb 20, Herb 21;

[0055] Iron Wire Clematis Root Slices: Slice 1, Slice 2, Slice 3, Slice 4, Slice 5, Slice 6, Slice 7, Slice 8, Slice 9, Slice 10, Slice 11, Slice 12, Slice 13, Slice 14, Slice 15, Slice 16, Slice 17, Slice 18, Slice 19, Slice 20, Slice 21.

[0056] 3) Proposed testing methods:

[0057] 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 0.35 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.

[0058] Table 1 Gradient elution program

[0059]

[0060] Preparation of reference solution: Take an appropriate amount of rutin reference standard, accurately weigh it, and add methanol to prepare a solution containing 25 μg per ml.

[0061] Preparation of the test solution: Weigh approximately 1 g of the powder (passed through a No. 3 sieve), place it in a stoppered conical flask, accurately add 50 mL of 80 vol% ethanol solution, seal tightly, weigh, sonicate (600 W power, 40 kHz frequency) for 30 minutes, cool, weigh again, replenish the lost weight with 80 vol% ethanol solution, shake well, filter, and collect the filtrate to obtain the test solution.

[0062] Assay: Accurately pipette 1 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0063] 4) Investigation of chromatographic conditions:

[0064] 4.1) Selection of mobile phase

[0065] 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 1 This is a chromatogram of different mobile phases for the *Imperata cylindrica* herb provided in Example 1 of the present invention. (The chromatogram is obtained 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 rutin content in *Imperata cylindrica*.

[0066] 4.2) Flow velocity investigation

[0067] Based on the above-specified experimental conditions, the test solution was subjected to chromatographic detection at flow rates of 0.3 mL / min, 0.35 mL / min, and 0.4 mL / min, respectively. The results are shown in the figure. Figure 2 , Figure 2 This is a chromatogram for the flow rate determination of the *Imperata cylindrica* herb provided in Example 1 of the present invention. (Through...) Figure 2 It can be seen that rutin can be effectively detected at flow rates of 0.3 to 0.4 mL / min, and the proposed flow rate is 0.35 mL / min.

[0068] 4.3) Column Temperature Investigation

[0069] 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 rutin can be effectively detected at column temperatures ranging from 25 to 35℃, and 30℃ is proposed as the detection column temperature.

[0070] 4.4) Sample volume assessment

[0071] Under the proposed chromatographic conditions, the test solution was subjected to chromatographic detection at injection volumes of 1 μL, 2 μL, and 3 μL, respectively. The results are shown in the figure. Figure 4 , Figure 4 This is a chromatogram of the injection volume investigation of the *Imperata cylindrica* herb provided in Example 1 of the present invention. (Through...) Figure 4 It can be seen that rutin can be effectively detected when the injection volume is between 1 and 3 μL, and the proposed injection volume is 1 μL.

[0072] 5) Preparation and investigation of the test solution:

[0073] 5.1) Investigation of dissolution solvent

[0074] Take approximately 1g of the medicinal powder, accurately weigh it, and place it in a stoppered conical flask. Use methanol, 30vol% methanol, 50vol% methanol, 80vol% methanol, ethanol, 30vol% ethanol, 50vol% ethanol, 80vol% ethanol, and water as extraction solvents respectively for testing. Accurately add 50mL of each solvent, seal the flask tightly, weigh it, and sonicate it (600W power, 40kHz frequency) for 30 minutes. Let it cool, weigh it again, and replenish the lost weight with the corresponding solvent. Shake well, filter it, and collect the filtrate to obtain the test solution.

[0075] Under the experimental conditions outlined above, chromatographic analysis was performed on the test solutions in different solvents, and the rutin content was calculated. The results are shown in Table 2.

[0076] Table 2. Analytical results of different dissolving solvents

[0077]

[0078] As can be seen from Table 2, 80 vol% ethanol has the highest dissolution efficiency, so 80% ethanol is used as its solvent.

[0079] 5.2) Investigation of dissolution methods

[0080] Take about 1g of the medicinal powder, accurately weigh it, place it in a stoppered conical flask, accurately add 50mL of 80vol% ethanol, seal tightly, weigh it, and then reflux and sonicate (power 600W, frequency 40kHz) for 30 minutes. After cooling, weigh it again, and make up the lost weight with 80vol% ethanol. Shake well, filter, and take the filtrate to obtain the test solution.

[0081] Under the experimental conditions proposed above, the reflux and ultrasonically treated test solutions were subjected to chromatographic analysis, and the rutin content was calculated. The results are shown in Table 3.

[0082] Table 3. Analytical results of different dissolution methods

[0083]

[0084] As can be seen from Table 3, the dissolution efficiency of reflux and ultrasound is not significantly different. Since the ultrasound method is fast and simple, the dissolution method of the test sample is determined to be ultrasound-assisted dissolution.

[0085] 5.3) Decomposition time study

[0086] Take about 1g of the medicinal powder, accurately weigh it, place it in a stoppered conical flask, accurately add 50mL of 80vol% ethanol solution, seal tightly, weigh it, and sonicate it (power 600W, frequency 40kHz) for 20 minutes, 30 minutes, and 40 minutes respectively for observation. After cooling, weigh it again, make up the weight loss with 80vol% ethanol, shake well, filter, and take the filtrate to obtain the test solution.

[0087] Under the experimental conditions proposed above, the test solutions with different dissolution times were subjected to chromatographic detection, and the rutin content was calculated. The results are shown in Table 4.

[0088] Table 4. Analytical results for different dissolution times

[0089]

[0090] As can be seen from Table 4, when the dissolution time is 30 minutes, the sample can be fully extracted. Therefore, the dissolution time of the test sample is determined to be 30 minutes.

[0091] 5.4) Investigation of Solvent Addition Amount

[0092] Take about 1g of the medicinal powder, accurately weigh it, and place it in a stoppered conical flask. Accurately add 25mL, 50mL, and 100mL of 80vol% ethanol respectively for testing. Seal the flask tightly, weigh it, and sonicate it (600W power, 40kHz frequency) for 30 minutes. Let it cool, weigh it again, and make up the weight loss with 80vol% ethanol. Shake well, filter it, and take the filtrate to obtain the test solution.

[0093] Under the experimental conditions specified above, the test solutions with different solvent additions were subjected to chromatographic detection, and the rutin content was calculated. The results are shown in Table 5.

[0094] Table 5. Analytical results for different solvent addition amounts

[0095]

[0096] As can be seen from Table 5, when the sample amount is 1g, the solvent addition amount is 50mL to ensure complete dissolution and extraction of the target component. Therefore, the solvent addition amount is determined to be 50mL.

[0097] 6) Methodological examination:

[0098] 6.1) Specificity Experiment

[0099] The test solution, reference solution, and negative control solution (i.e., blank solution) were prepared according to the prescribed method, and chromatographic detection was performed. The results are as follows: Figure 5 As shown, Figure 5 This is a specific experimental chromatogram provided in Embodiment 1 of the present invention. (Through...) Figure 5It can be seen that the chromatogram of the negative solution does not interfere with the determination of the target peak, indicating that the method has good specificity.

[0100] 6.2) Precision test

[0101] Under the experimental conditions specified above, the reference solution was injected six times consecutively, the peak area of ​​rutin was recorded, and the RSD value was calculated. The results are shown in Table 6.

[0102] Table 6. Precision test results

[0103]

[0104] As shown in Table 6, the peak area RSD of rutin in the precision study was 0.3%, indicating that the injection precision of this method is good.

[0105] 6.3) Linear Relationship

[0106] Take an appropriate amount of rutin reference standard and place it in a 50 mL volumetric flask. Dissolve it in methanol to prepare a solution containing 100.9432 μg per mL. Then dilute it to solutions containing 3.028296 μg, 6.056592 μg, 12.113184 μg, 30.28296 μg, 50.4716 μg, and 100.9432 μg per mL, respectively.

[0107] Under the experimental conditions specified above, 1 μL of the above solution was precisely injected into the liquid chromatograph, and the peak area was analyzed. A response curve was plotted with rutin content (X) on the x-axis and peak area (Y) on the y-axis. The results are shown in Table 7 and... Figure 6 , Figure 6 This is the rutin standard curve provided in Embodiment 1 of the present invention.

[0108] Table 7. Results of Standard Curve Analysis

[0109]

[0110] The results showed that when the rutin concentration ranged from 3.028296 to 100.9432 μg / ml, the linear relationship was y = 2.9903x - 0.4660, R0 2 =0.9999. This indicates a good linear relationship within the concentration range of 3.028296–100.9432 μg / ml.

[0111] 6.4) Repeatability Experiment

[0112] Take approximately 1g of the herbal powder, accurately weigh 6 portions, and prepare the test solution by the same operator according to the established method; perform chromatographic detection on the test solution under the above-specified experimental conditions, and calculate the rutin content. The results are shown in Table 8:

[0113] Table 8 Results of Repeatability Experiments

[0114]

[0115]

[0116] As shown in Table 8, the RSD value of rutin content is 0.5%, indicating that the method has good repeatability.

[0117] 6.5) Accuracy Test

[0118] Take approximately 0.5 g of the medicinal powder with a known content (rutin content 0.109%), make 6 portions, accurately weigh them, and accurately add a certain amount of rutin reference standard to each portion. Prepare and determine the test solution according to the proposed method, and calculate the recovery rate (results are shown in Table 9). The calculation formula is as follows:

[0119]

[0120] Table 9 Results of Rutin Recovery Experiment

[0121]

[0122] As shown in Table 9, the average recovery rate of rutin was 103.1%, and the RSD value was 1.7%, indicating that the method had good accuracy.

[0123] 6.6) Investigation with different instruments

[0124] Based on the above-specified experimental conditions, the powder of *Imperata cylindrica* was accurately weighed, and a test solution was prepared. The solution was then analyzed using an Agilent, Waters, or Thermo Fisher ultra-high performance liquid chromatograph, and the rutin content was calculated. The results are shown in Table 10.

[0125] Table 10 Experimental results using different instruments

[0126]

[0127] As shown in Table 10, the RSD value of the results measured by the Agilent, Waters, and Thermo Fisher ultra-high performance liquid chromatographs was 1.5%, indicating that the instruments used in this method have good durability.

[0128] 6.7) Investigations by different personnel and at different times

[0129] Based on the above-planned experimental conditions, different personnel (A, B) precisely weighed the powder of *Imperata cylindrica* at different times (I, II) to prepare test samples, and the rutin content was determined and calculated. The results are shown in Table 11.

[0130] Table 11 Results of the investigation of different personnel and time periods

[0131]

[0132] As shown in Table 11, the RSD value of the rutin content determination result was 0.4%, indicating that the intermediate precision of this method was good.

[0133] 6.8) Column robustness test

[0134] Based on the above-specified experimental conditions, the powder of *Imperata cylindrica* was accurately weighed to prepare the test sample. The results were investigated on chromatographic columns 1, 2, and 3, and are shown in Table 12.

[0135] Table 12 Results of column robustness test

[0136]

[0137] As can be seen from Table 12, the analytical chromatographic parameters of different columns are good. Among the six measurement results, the RSD value of rutin is 2.9%, indicating that the column of this method has good durability.

[0138] 6.9) Stability test

[0139] Based on the above-specified experimental conditions, the powder of *Imperata cylindrica* was accurately weighed to prepare the test sample. The peak area of ​​rutin was measured at 0h, 2h, 4h, 8h, 16h, and 24h, respectively. The results are shown in Table 13.

[0140] Table 13 Stability test results

[0141]

[0142] As can be seen from Table 13, under the experimental conditions, the RSD value of the rutin peak area was 0.4%, and the test solution showed good stability within 24 hours.

[0143] 7) Verification of herbal medicines and processed medicinal slices containing iron wire:

[0144] Based on the above-mentioned experimental conditions, the rutin content of 21 batches of *Imperata cylindrica* herbal medicine and decoction pieces was verified. The results are shown in Tables 14 and 15.

[0145] Table 1421 Verification of Rutin Content in Herbal Extracts of *Imperata cylindrica*

[0146]

[0147] Table 1521 Verification of Rutin Content in Batches of *Clematis chinensis* Medicinal Slices

[0148]

[0149]

[0150] As can be seen from Tables 14-15, the method for determining the rutin content in *Tie Xian Tou Gu* herbal medicine and its processed form can effectively detect the rutin content, proving that the method is feasible.

[0151] 8) Determination of the method for determining rutin content in *Imperata cylindrica* herbal medicines and processed medicinal slices:

[0152] 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 0.35 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.

[0153] Preparation of reference solution: Take an appropriate amount of rutin reference standard, accurately weigh it, and add methanol to prepare a solution containing 25 μg per ml.

[0154] Preparation of the test solution: Weigh approximately 1 g of the powder (passed through a No. 3 sieve), place it in a stoppered conical flask, accurately add 50 mL of 80 vol% ethanol solution, seal tightly, weigh, sonicate (600 W power, 40 kHz frequency) for 30 minutes, cool, weigh again, replenish the lost weight with 80 vol% ethanol solution, shake well, filter, and collect the filtrate to obtain the test solution.

[0155] Assay: Accurately pipette 1 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0156] Example 2

[0157] Determination of rutin content in standard decoction of *Clematis chinensis* (Tie Xian Tou Gu Cao):

[0158] 1) Experimental instruments and materials:

[0159] High performance liquid chromatograph: Agilent ultra-high performance liquid chromatograph, Waters ultra-high performance liquid chromatograph, Thermo Fisher ultra-high performance liquid chromatograph. Unless otherwise specified, Agilent ultra-high performance liquid chromatograph is used by default.

[0160] Chromatographic column: Column 1 (Manufacturer: Waters; Model) Column 1 (C18), Column 2 (manufacturer: YMC; model: YMC-Triart C18), and Column 3 (manufacturer: Shimadzu; model: Shim-pack Velox SP-C18) are all used. All three columns use octadecylsilane-bonded silica gel as the packing material, have a column length of 100 mm, an inner diameter of 2.1 mm, and a packing material particle size of 1.7 μm. Unless otherwise specified, Column 1 is used by default.

[0161] Electronic balance, ultrapure water system, ultrasonic cleaner.

[0162] 2) Reagents and reagents:

[0163] Acetonitrile and phosphoric acid were of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0164] Rutin reference standard (China National Institutes for Food and Drug Control, batch number: 100080-202012, purity: 91.6%);

[0165] Standard decoctions of *Imperata cylindrica*: Standard Decoction 1, Standard Decoction 2, Standard Decoction 3, Standard Decoction 4, Standard Decoction 5, Standard Decoction 6, Standard Decoction 7, Standard Decoction 8, Standard Decoction 9, Standard Decoction 10, Standard Decoction 11, Standard Decoction 12, Standard Decoction 13, Standard Decoction 14, Standard Decoction 15, Standard Decoction 16, Standard Decoction 17, Standard Decoction 18, Standard Decoction 19, Standard Decoction 20, Standard Decoction 21.

[0166] 3) Proposed testing methods:

[0167] 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 0.35 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.

[0168] Preparation of reference solution: Take an appropriate amount of rutin reference standard, accurately weigh it, and add methanol to prepare a solution containing 25 μg per ml.

[0169] Preparation of the test solution: Weigh approximately 0.2 g of this product accurately, place it in a stoppered conical flask, accurately add 25 mL of 80 vol% ethanol solution, stopper tightly, weigh, sonicate (600 W power, 40 kHz frequency) for 30 minutes, cool, weigh again, replenish the lost weight with 80 vol% ethanol solution, shake well, filter, and collect the filtrate to obtain the test solution.

[0170] Assay: Accurately pipette 1 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0171] 4) Investigation of chromatographic conditions:

[0172] 4.1) Selection of mobile phase

[0173] 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 7 , Figure 7 This is a chromatogram of different mobile phases for the standard decoction of *Imperata cylindrica* provided in Example 2 of the present invention. (The chromatogram is obtained through...) Figure 7 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 rutin content in the standard decoction of *Imperata cylindrica*.

[0174] 4.2) Flow velocity investigation

[0175] Based on the above-specified experimental conditions, the test solution was subjected to chromatographic detection at flow rates of 0.3 mL / min, 0.35 mL / min, and 0.4 mL / min, respectively. The results are shown in the figure. Figure 8 , Figure 8 This is a chromatogram showing the flow rate of the standard decoction of *Imperata cylindrica* provided in Example 2 of this invention. (Through...) Figure 8 It can be seen that rutin can be effectively detected at flow rates of 0.3 to 0.4 mL / min, and the proposed flow rate is 0.35 mL / min.

[0176] 4.3) Column Temperature Investigation

[0177] 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 9 , Figure 9 This is a column temperature chromatogram of the standard decoction of *Imperata cylindrica* provided in Example 2 of this invention. (The text repeats itself here.) Figure 9 It can be seen that rutin can be effectively detected at column temperatures ranging from 25 to 35℃, and 30℃ is proposed as the detection column temperature.

[0178] 4.4) Sample volume assessment

[0179] Under the proposed chromatographic conditions, the test solution was subjected to chromatographic detection at injection volumes of 1 μL, 2 μL, and 3 μL, respectively. The results are shown in the figure. Figure 10 , Figure 10This is a chromatogram showing the injection volume investigation of the standard decoction of *Imperata cylindrica* provided in Example 2 of this invention. (Through...) Figure 10 It can be seen that rutin can be effectively detected when the injection volume is between 1 and 3 μL, and the proposed injection volume is 1 μL.

[0180] 5) Preparation and investigation of the test solution:

[0181] 5.1) Investigation of dissolution solvent

[0182] Take approximately 0.2 g of the standard decoction powder, accurately weigh it, and place it in a stoppered conical flask. Use methanol, 30 vol% methanol, 50 vol% methanol, 80 vol% methanol, ethanol, 30 vol% ethanol, 50 vol% ethanol, 80 vol% ethanol, and water as extraction solvents respectively for testing. Accurately add 25 mL of each solvent, seal the flask tightly, weigh it, and sonicate it (600 W power, 40 kHz frequency) for 30 minutes. Let it cool, weigh it again, and replenish the lost weight with the corresponding solvent. Shake well, filter it, and take the filtrate to obtain the test solution.

[0183] Under the experimental conditions proposed above, chromatographic analysis was performed on the test solutions in different solvents, and the rutin content was calculated. The results are shown in Table 16.

[0184] Table 16 Analytical results of different dissolving solvents

[0185]

[0186] As can be seen from Table 16, 80 vol% ethanol has the highest dissolution efficiency, so 80% ethanol is used as its solvent.

[0187] 5.2) Investigation of dissolution methods

[0188] Take approximately 0.2g of standard decoction powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of 80vol% ethanol, seal tightly, weigh it, and then reflux and sonicate (600W power, 40kHz frequency) for 30 minutes. After cooling, weigh it again, replenish the lost weight with 80vol% ethanol, shake well, filter, and collect the filtrate to obtain the test solution.

[0189] Under the experimental conditions proposed above, the reflux and ultrasonically treated test solutions were subjected to chromatographic analysis, and the rutin content was calculated. The results are shown in Table 17.

[0190] Table 17 Analytical results of different dissolution methods

[0191]

[0192] As can be seen from Table 17, the dissolution efficiency of reflux and ultrasound is not significantly different. Since the ultrasound method is fast and simple, the dissolution method of the test sample is determined to be ultrasound-assisted dissolution.

[0193] 5.3) Decomposition time study

[0194] Take approximately 0.2g of standard decoction powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of 80vol% ethanol solution, seal tightly, weigh it, and sonicate it (power 600W, frequency 40kHz) for 20 minutes, 30 minutes, and 40 minutes respectively for observation. After cooling, weigh it again, replenish the lost weight with 80vol% ethanol, shake well, filter, and take the filtrate to obtain the test solution.

[0195] Under the experimental conditions proposed above, the test solutions with different dissolution times were subjected to chromatographic detection, and the rutin content was calculated. The results are shown in Table 18.

[0196] Table 18 Analytical results for different dissolution times

[0197]

[0198] As can be seen from Table 18, when the dissolution time is 30 minutes, the sample can be fully extracted. Therefore, the dissolution time of the test sample is determined to be 30 minutes.

[0199] 5.4) Investigation of Solvent Addition Amount

[0200] Take approximately 0.2 g of standard decoction powder, accurately weigh it, and place it in a stoppered conical flask. Accurately add 15 mL, 25 mL, and 50 mL of 80 vol% ethanol respectively for testing. Seal the flask tightly, weigh it, and sonicate it (600 W power, 40 kHz frequency) for 30 minutes. Let it cool, weigh it again, and replenish the lost weight with 80 vol% ethanol. Shake well, filter it, and take the filtrate to obtain the test solution.

[0201] Under the experimental conditions proposed above, the test solutions with different solvent addition amounts were subjected to chromatographic detection, and the rutin content was calculated. The results are shown in Table 19.

[0202] Table 19 Analytical results for different solvent addition amounts

[0203]

[0204] As can be seen from Table 19, when the sample amount is 0.2g, the amount of solvent added is 25mL to ensure complete dissolution and extraction of the target component. Therefore, the amount of solvent added is determined to be 25mL.

[0205] 6) Methodological examination:

[0206] 6.1) Specificity Experiment

[0207] The test solution, reference solution, and negative control solution (i.e., blank solution) were prepared according to the prescribed method, and chromatographic detection was performed. The results are as follows: Figure 11 As shown, Figure 11 This is a specific experimental chromatogram provided in Embodiment 2 of the present invention. (Through...) Figure 11 It can be seen that the chromatogram of the negative solution does not interfere with the determination of the target peak, indicating that the method has good specificity.

[0208] 6.2) Precision test

[0209] The results are shown in Table 6 of Example 1 and will not be repeated here.

[0210] 6.3) Linear Relationship

[0211] The results are shown in Table 7 of Example 1 and Figure 6 I will not go into details.

[0212] 6.4) Repeatability Experiment

[0213] Take approximately 0.2g of standard decoction powder, accurately weigh 6 portions, and prepare the test solution by the same operator according to the established method; perform chromatographic detection on the test solution under the above-specified experimental conditions, and calculate the rutin content. The results are shown in Table 20:

[0214] Table 20 Results of Repeatability Experiments

[0215]

[0216] As shown in Table 20, the RSD value of rutin content is 0.8%, indicating that the method has good repeatability.

[0217] 6.5) Accuracy Test

[0218] Six portions of a standard decoction powder with a known content (rutin content 2.50 mg / g) were accurately weighed. A certain amount of rutin reference standard was accurately added to each portion. The test solutions were prepared and determined according to the prescribed method. The recovery rate was calculated, and the results are shown in Table 21.

[0219] Table 21 Results of Rutin Recovery Experiment

[0220]

[0221] As shown in Table 21, the average recovery rate of rutin was 96.0%, indicating that the method had good accuracy.

[0222] 6.6) Investigation with different instruments

[0223] Based on the above-specified experimental conditions, the standard powder of *Clematis chinensis* was accurately weighed to prepare a test solution. The solution was then analyzed using an Agilent, Waters, or Thermo Fisher ultra-high performance liquid chromatograph, and the rutin content was calculated. The results are shown in Table 22.

[0224] Table 22 Experimental Results with Different Instruments

[0225]

[0226] As shown in Table 22, the RSD value of the results measured by the Agilent, Waters, and Thermo Fisher ultra-high performance liquid chromatographs was 1.5%, indicating that the instruments used in this method have good durability.

[0227] 6.7) Investigations by different personnel and at different times

[0228] Based on the above-planned experimental conditions, different personnel (A, B) accurately weighed the standard powder of *Clematis chinensis* decoction at different times (I, II) to prepare test samples, and the rutin content was determined and calculated. The results are shown in Table 23.

[0229] Table 23 Results of the survey on different personnel and time periods

[0230]

[0231] As can be seen from Table 23, the RSD value of the rutin content determination result is 0.8%, indicating that the intermediate precision of this method is good.

[0232] 6.8) Column robustness test

[0233] Based on the above-specified experimental conditions, the standard powder of *Clematis chinensis* was accurately weighed to prepare the test sample. The results were investigated on chromatographic columns 1, 2, and 3, and are shown in Table 24.

[0234] Table 24 Results of column robustness test

[0235]

[0236]

[0237] As can be seen from Table 24, the analytical chromatographic parameters of different columns are good. Among the six measurement results, the RSD value of rutin is 2.2%, indicating that the column of this method has good durability.

[0238] 6.9) Stability test

[0239] Based on the above-specified experimental conditions, the standard powder of *Clematis chinensis* was accurately weighed to prepare the test sample. The peak area of ​​rutin was measured at 0h, 2h, 4h, 8h, 16h, and 24h, respectively. The results are shown in Table 25.

[0240] Table 25 Stability test results

[0241]

[0242] As can be seen from Table 25, under the experimental conditions, the RSD value of the rutin peak area was 0.4%, and the test solution showed good stability within 24 hours.

[0243] 7) Verification of the standard decoction of *Imperata cylindrica*:

[0244] Based on the above-mentioned experimental conditions, the rutin content of 21 batches of *Imperata cylindrica* standard decoction was verified, and the results are shown in Table 26.

[0245] Table 2621 Verification of Rutin Content in Standard Decoction of *Clematis chinensis* (Tie Xian Tou Gu Cao)

[0246]

[0247] As can be seen from Table 26, the method for determining the rutin content in the standard decoction of *Imperata cylindrica* can effectively detect the rutin content in the standard decoction, proving that the method is feasible.

[0248] 8) Determination of the method for determining rutin content in the standard decoction of *Hedyotis diffusa*:

[0249] 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 0.35 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.

[0250] Preparation of reference solution: Take an appropriate amount of rutin reference standard, accurately weigh it, and add methanol to prepare a solution containing 25 μg per ml.

[0251] Preparation of the test solution: Weigh approximately 0.2 g of this product accurately, place it in a stoppered conical flask, accurately add 25 mL of 80 vol% ethanol solution, stopper tightly, weigh, sonicate (600 W power, 40 kHz frequency) for 30 minutes, cool, weigh again, replenish the lost weight with 80 vol% ethanol solution, shake well, filter, and collect the filtrate to obtain the test solution.

[0252] Assay: Accurately pipette 1 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0253] Example 3

[0254] Determination of rutin content in *Imperata cylindrica* formula granules:

[0255] 1) Experimental instruments and materials:

[0256] High performance liquid chromatograph: Agilent ultra-high performance liquid chromatograph, Waters ultra-high performance liquid chromatograph, Thermo Fisher ultra-high performance liquid chromatograph. Unless otherwise specified, Agilent ultra-high performance liquid chromatograph is used by default.

[0257] Chromatographic column: Column 1 (Manufacturer: Waters; Model) Column 1 (C18), Column 2 (manufacturer: YMC; model: YMC-Triart C18), and Column 3 (manufacturer: Shimadzu; model: Shim-pack Velox SP-C18) are all used. All three columns use octadecylsilane-bonded silica gel as the packing material, have a column length of 100 mm, an inner diameter of 2.1 mm, and a packing material particle size of 1.7 μm. Unless otherwise specified, Column 1 is used by default.

[0258] Electronic balance, ultrapure water system, ultrasonic cleaner.

[0259] 2) Reagents and reagents:

[0260] Acetonitrile and phosphoric acid were of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0261] Rutin reference standard (China National Institutes for Food and Drug Control, batch number: 100080-202012, purity: 91.6%);

[0262] Iron Wire and Bone-Penetrating Herb Formula Granules: Formula Granule 1, Formula Granule 2, Formula Granule 3.

[0263] 3) Proposed testing methods:

[0264] 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 0.35 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.

[0265] Preparation of reference solution: Take an appropriate amount of rutin reference standard, accurately weigh it, and add methanol to prepare a solution containing 25 μg per ml.

[0266] Preparation of test solution: Take an appropriate amount of this product, powder it. Weigh accurately about 0.2 g and place it in a stoppered conical flask. Accurately add 25 mL of 80 vol% ethanol, stopper tightly, weigh, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, cool, weigh again, and replenish the lost weight with 80 vol% ethanol. Shake well, filter, and take the consecutive filtrate, which is the test solution.

[0267] Determination method: Accurately pipette 1 μL each of the reference solution and the test solution, inject them into the liquid chromatograph for determination, and the result can be obtained.

[0268] 4.1) Selection of mobile phase

[0269] Based on the above-mentioned proposed experimental conditions, the separation effects of 2 different mobile phases were investigated, namely: methanol (phase A) - 0.5 vol% phosphoric acid (phase B), acetonitrile (phase A) - 0.​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​

[0275] Under the above-mentioned established chromatographic conditions, the test solution was chromatographically detected under the conditions of injecting 1 μL, 2 μL, and 3 μL respectively, and the results are shown in Figure 15 , Figure 15 which is the chromatogram for investigating the injection volume of the Herba Phrymae formula granules provided in Example 3 of the present invention. It can be seen from Figure 15 that when the injection volume is between 1 and 3 μL, rutin can be effectively detected, and the established injection volume is 1 μL.

[0276] 5) Investigation on the preparation of the test solution:

[0277] 5.1) Investigation on the dissolution solvent

[0278] An appropriate amount of the formula granules was taken, ground fine, about 0.2 g was taken, accurately weighed, placed in a stoppered conical flask, and investigated with extraction solvents methanol, 30 vol% methanol, 50 vol% methanol, 80 vol% methanol, ethanol, 30 vol% ethanol, 50 vol% ethanol, 80 vol% ethanol, and water respectively, 25 mL was accurately added respectively, stoppered, weighed, ultrasonicated (power 600 W, frequency 40 kHz) for 30 minutes, cooled, weighed again, the lost weight was made up with the corresponding solvent, shaken well, filtered, and the subsequent filtrate was taken to obtain the test solution.

[0279] According to the above-mentioned established experimental conditions, the test solutions of different solvents were chromatographically detected respectively, and the rutin content was calculated. The results are shown in Table 27:

[0280] Table 27 Analysis results of different dissolution solvents

[0281]

[0282] It can be seen from Table 27 that the dissolution efficiency of 80 vol% ethanol is the highest, and 80% ethanol is used as its solvent.

[0283] 5.2) Investigation on the dissolution method

[0284] An appropriate amount of the formula granules was taken, ground fine, about 0.2 g was taken, accurately weighed, placed in a stoppered conical flask, 25 mL of 80 vol% ethanol was accurately added, stoppered, weighed, refluxed and ultrasonicated (power 600 W, frequency 40 kHz) respectively for 30 minutes, cooled, weighed again, the lost weight was made up with 80 vol% ethanol, shaken well, filtered, and the subsequent filtrate was taken to obtain the test solution.

[0285] According to the above-mentioned established experimental conditions, the test solutions of reflux and ultrasonic treatment were chromatographically detected respectively, and the rutin content was calculated. The results are shown in Table 28:

[0286] Table 28 Analysis results of different dissolution methods

[0287]

[0288] As can be seen from Table 28, the dissolution efficiency of reflux and ultrasound is not significantly different. Since the ultrasound method is fast and simple, the dissolution method of the test sample is determined to be ultrasound-assisted dissolution.

[0289] 5.3) Decomposition time study

[0290] Take an appropriate amount of the formula granules, grind them into a fine powder, take about 0.2g, weigh it accurately, place it in a stoppered conical flask, accurately add 25mL of 80vol% ethanol solution, seal tightly, weigh it, and sonicate it (power 600W, frequency 40kHz) for 20 minutes, 30 minutes, and 40 minutes respectively for observation. After cooling, weigh it again, make up the weight loss with 80vol% ethanol, shake well, filter, and take the filtrate to obtain the test solution.

[0291] Under the experimental conditions proposed above, the test solutions with different dissolution times were subjected to chromatographic detection, and the rutin content was calculated. The results are shown in Table 29.

[0292] Table 29 Analytical results for different dissolution times

[0293]

[0294] As can be seen from Table 29, when the dissolution time is 30 minutes, the sample can be fully extracted. Therefore, the dissolution time of the test sample is determined to be 30 minutes.

[0295] 5.4) Investigation of Solvent Addition Amount

[0296] Take an appropriate amount of the formula granules, grind them into a fine powder, take about 0.2g, weigh it accurately, and place it in a stoppered conical flask. Accurately add 15mL, 25mL, and 50mL of 80vol% ethanol respectively for testing. Seal the flask tightly, weigh it, and sonicate it (power 600W, frequency 40kHz) for 30 minutes. Let it cool, weigh it again, and make up the weight loss with 80vol% ethanol. Shake well, filter it, and take the filtrate to obtain the test solution.

[0297] Under the experimental conditions specified above, the test solutions with different solvent additions were subjected to chromatographic detection, and the rutin content was calculated. The results are shown in Table 30.

[0298] Table 30 Analysis results for different solvent addition amounts

[0299]

[0300] As can be seen from Table 30, when the sample amount is 0.2g, the solvent addition amount of 25mL can ensure complete dissolution and extraction of the target component. Therefore, the solvent addition amount is determined to be 25mL.

[0301] 6) Methodological examination:

[0302] 6.1) Specificity Experiment

[0303] The test solution, reference solution, and negative control solution (i.e., blank solution) were prepared according to the prescribed method, and chromatographic detection was performed. The results are as follows: Figure 16 As shown, Figure 16 This is a specific experimental chromatogram provided in Embodiment 3 of the present invention. (Through...) Figure 16 It can be seen that the chromatogram of the negative solution does not interfere with the determination of the target peak, indicating that the method has good specificity.

[0304] 6.2) Precision test

[0305] The results are shown in Table 6 of Example 1 and will not be repeated here.

[0306] 6.3) Linear Relationship

[0307] The results are shown in Table 7 of Example 1 and Figure 6 I will not go into details.

[0308] 6.4) Repeatability Experiment

[0309] Take an appropriate amount of the formula granules, grind them finely, and accurately weigh 6 portions (approximately 0.2g each). Prepare the test solution using the same operator according to the established method. Perform chromatographic analysis on the test solution under the above-determined experimental conditions, and calculate the rutin content. The results are shown in Table 31.

[0310] Table 31 Results of Repeatability Experiments

[0311]

[0312] As shown in Table 31, the RSD value of rutin content is 0.2%, indicating that the method has good repeatability.

[0313] 6.5) Accuracy Test

[0314] Six portions of 0.1 g of the formula granules with a known content (rutin content 1.25 mg / g) were accurately weighed. A certain amount of rutin reference standard was accurately added to each portion. The test solutions were prepared and determined according to the prescribed method. The recovery rate was calculated, and the results are shown in Table 32.

[0315] Table 32 Results of Rutin Recovery Experiment

[0316]

[0317] As shown in Table 32, the average recovery rate of rutin was 96.9%, and the RSD value was 1.1%, indicating that the method had good accuracy.

[0318] 6.6) Investigation with different instruments

[0319] Based on the above-specified experimental conditions, the granules of *Clematis chinensis* were accurately weighed to prepare a test solution. The solution was then analyzed using an Agilent, Waters, or Thermo Fisher ultra-high performance liquid chromatograph, and the rutin content was calculated. The results are shown in Table 33.

[0320] Table 33 Experimental results using different instruments

[0321]

[0322] As shown in Table 33, the RSD value of the results measured by the Agilent, Waters, and Thermo Fisher ultra-high performance liquid chromatographs was 2.0%, indicating that the instruments used in this method have good durability.

[0323] 6.7) Investigations by different personnel and at different times

[0324] Based on the above-planned experimental conditions, different personnel (A, B) accurately weighed the *Imperata cylindrica* formula granules at different times (I, II) to prepare test samples, and the rutin content was measured and calculated. The results are shown in Table 34.

[0325] Table 34 Results of the survey on different personnel and time periods

[0326]

[0327] As can be seen from Table 34, the RSD value of the rutin content determination result is 0.9%, indicating that the intermediate precision of this method is good.

[0328] 6.8) Column robustness test

[0329] Based on the above-specified experimental conditions, the granules of *Imperata cylindrica* were accurately weighed to prepare the test sample. The results were investigated on chromatographic columns 1, 2, and 3, and are shown in Table 35.

[0330] Table 35 Results of column durability testing

[0331]

[0332] As can be seen from Table 35, the analytical chromatographic parameters of different columns are good. Among the six measurement results, the RSD value of rutin is 3.3%, indicating that the column of this method has good durability.

[0333] 6.9) Stability test

[0334] Based on the above-specified experimental conditions, the granules of *Clematis chinensis* were accurately weighed to prepare the test sample. The peak area of ​​rutin was measured at 0h, 2h, 4h, 8h, 16h, and 24h, respectively. The results are shown in Table 36.

[0335] Table 36 Stability test results

[0336]

[0337] As can be seen from Table 36, under the experimental conditions, the RSD value of the rutin peak area was 0.5%, and the test solution showed good stability within 24 hours.

[0338] 7) Verification of the standard decoction of *Imperata cylindrica*:

[0339] Based on the above-mentioned experimental conditions, the rutin content of three batches of *Imperata cylindrica* formula granules was verified, and the results are shown in Table 37:

[0340] Verification of Rutin Content in Batch 373 of Iron-flecked Herb Formula Granules

[0341]

[0342] As can be seen from Table 37, the method for determining the rutin content of *Imperata cylindrica* formula granules can effectively detect the rutin content of *Imperata cylindrica* formula granules, proving that the method is feasible.

[0343] 8) Determination of the method for determining the rutin content in *Imperata cylindrica* formula granules:

[0344] 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 0.35 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.

[0345] Preparation of reference solution: Take an appropriate amount of rutin reference standard, accurately weigh it, and add methanol to prepare a solution containing 25 μg per ml.

[0346] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take about 0.2 g, weigh it accurately, place it in a stoppered conical flask, accurately add 25 mL of 80 vol% ethanol solution, stopper tightly, weigh it, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 80 vol% ethanol solution, shake well, filter it, and take the filtrate to obtain the test solution.

[0347] Assay: Accurately pipette 1 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0348] 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 determining the rutin content in *Imperata cylindrica* herbal medicine, processed slices, standard decoctions, and formulated granules, characterized in that, Includes the following steps: Prepare test samples and reference standards. The test samples are *Imperata cylindrica* herbal medicine, *Imperata cylindrica* slices, *Imperata cylindrica* standard decoction, or *Imperata cylindrica* formula granules. The reference standard is rutin. The test sample and the reference sample were dissolved separately to obtain corresponding test sample solutions and reference sample solutions; the solvent for dissolving the test sample was an 80 vol% ethanol solution; the ratio of the test sample to the solvent was (0.2~0.5) g: 25 mL; the dissolution of the test sample was carried out under ultrasonic assistance; the solvent for dissolving the reference sample was methanol. The test solution and the reference solution were determined by high performance liquid chromatography (HPLC) to obtain the corresponding HPLC chromatograms. The chromatographic conditions of the HPLC were as follows: C18 column; mobile phase A was acetonitrile, mobile phase B was 0.5 wt% phosphoric acid solution, gradient elution; detection wavelength was 354 nm. The gradient elution specifically refers to: 0~10min, Phase A: 10~15 vol%, Phase B: 90~85 vol% 10-15 min, Phase A: 15-18 vol%, Phase B: 85-82 vol% The rutin content in the test sample is calculated using the external standard method based on the concentration of rutin in the reference solution, the peak area of ​​the reference solution in the chromatogram, and the peak area of ​​the component in the test sample corresponding to the reference in the chromatogram.

2. The determination method according to claim 1, characterized in that, The ultrasound-assisted power is 580~620W, the frequency is 35~45kHz, and the duration is 25~35min.

3. The determination method according to claim 1, characterized in that, The ratio of the reference standard to the solvent is (20~30) μg: 1 mL.

4. The determination method according to claim 1, characterized in that, The chromatographic conditions for the high performance liquid chromatography method also include: a mobile phase flow rate of 0.35 mL / min.

5. The determination method according to claim 1, characterized in that, The chromatographic conditions for the high performance liquid chromatography method also include: a column temperature of 30°C.

6. The determination method according to claim 1, characterized in that, The chromatographic conditions for the high performance liquid chromatography method also include: an injection volume of 1 μL; and a theoretical plate number of not less than 5000 calculated based on the rutin peak.

7. The determination method according to claim 1, characterized in that, Plotting rutin concentration on the x-axis and the corresponding chromatographic peak area on the y-axis, the linear relationship was y = 2.9903x - 0.4660 when the rutin concentration ranged from 3.028296 to 100.9432 μg / mL.

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