A detection method and application of a formula granule of prunus davidiana bean

Abstract

This invention belongs to the field of drug detection technology, specifically relating to a detection method and application for *Haloxylon ammodendron* leaf formula granules. The *Haloxylon ammodendron* leaf formula granules are made from *Haloxylon ammodendron* leaf slices through decoction, concentration, drying, and granulation. The detection method includes morphology, identification, inspection, extractives, characteristic chromatograms, and content determination. The identification method uses thin-layer chromatography; the characteristic chromatogram uses high-performance liquid chromatography, with the chlorogenic acid reference peak as the S peak, indicating four characteristic peaks; the content determination measures the content of chlorogenic acid. The detection method established by this invention improves the detection requirements for *Haloxylon ammodendron* leaf medicinal materials, slices, standard decoctions, and formula granules, contributing to the establishment of a comprehensive quality control system for various *Haloxylon ammodendron* leaf products and helping to ensure the quality and clinical efficacy of *Haloxylon ammodendron* leaf formula granules.

Description

Technical Field

[0001] This invention belongs to the field of drug detection technology, specifically relating to a detection method and application of Hantao leaf formula granules. Background Technology

[0002] Traditional Chinese medicine (TCM) granules are made from single-herb medicinal slices through one or more extraction and concentration processes, and are intended for use in TCM clinical prescriptions. They have the advantages of being stable and controllable, safe and efficient, requiring no decoction, easy to prepare, convenient to carry, and ready to take with a simple dissolving process.

[0003] Han Tao Ye, also known as Goose Palm Vine or Seven-Leaf Lotus, refers to the dried stems or leafy stems of *Schefflera leacantha* Vig., a plant belonging to the genus *Schefflera* in the family Araliaceae. It has a slightly bitter and astringent taste, and is warm in nature. It possesses properties such as dispelling wind and relieving pain, relaxing muscles and tendons, and is used for sciatica, trigeminal neuralgia, nervous headaches, and rheumatic joint pain, and is a commonly used folk remedy. The chemical components of Han Tao Ye are mainly oleanane-type pentacyclic triterpenes and their glycosides, as well as sterols, long-chain alkenes, organic acids, and volatile oils. It is mainly distributed in Guangxi, Guizhou, Yunnan, and Fujian provinces of my country.

[0004] Currently, the standard for Han Tao Ye (Han Tao leaf) medicinal materials is included in the 2003 edition of the "Guizhou Province Quality Standards for Traditional Chinese and Ethnic Medicinal Materials," which only covers morphological and microscopic identification. With the increasing clinical use of formulated granules, there is an urgent need for comprehensive, convenient, and controllable testing methods to control the quality of Han Tao Ye formulated granules. Summary of the Invention

[0005] This invention addresses the shortcomings of existing detection methods for Hantao leaf formula granules by providing a quality detection method for Hantao leaf formula granules, specifically:

[0006] The testing items for the Hantao leaf formula granules include morphology, identification, inspection, extractives, characteristic chromatograms, and content determination.

[0007] In some embodiments, the granules of the Han Tao leaf formula are: yellowish-brown granules; with a slight odor and a bitter taste.

[0008] In some embodiments, the granules of the Han Tao leaf formula are: brownish-brown granules; with a slight odor and a bitter taste.

[0009] Identification: Take 1g of *Haloxylon ammodendron* leaf granules, grind them finely, add 20ml of water, reflux for 30min, filter, add 30ml of anhydrous ethanol for precipitation, refrigerate and stand for 4h, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of methanol to obtain the test solution. Separately, take 1g of *Haloxylon ammodendron* leaf reference material and prepare a reference material solution using the same method. Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, General Chapter 0502). Apply 5μl of the test solution and 1μl of the reference material solution to the same silica gel GF254 thin-layer plate, using chloroform-ethyl acetate-formic acid (2:0.5:0.2) as the developing solvent. Develop, remove, air dry, and examine under ultraviolet light (365nm). In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference material.

[0010] Inspection: It should comply with all relevant provisions under the General Rules for Granules in the Chinese Pharmacopoeia;

[0011] Extractives: Take 2g of the Han Tao leaf formula granules, grind them into a fine powder, accurately weigh them, add 100ml of ethanol, and determine them according to the hot extraction method under the "Determination of Alcohol-soluble Extractives" section of the Chinese Pharmacopoeia.

[0012] Characteristic chromatograms: determined by high performance liquid chromatography according to the general principles of the Chinese Pharmacopoeia;

[0013] Chromatographic conditions and system suitability experiments: The chromatographic column was packed with octadecylsilane-bonded silica gel, with acetonitrile as mobile phase A and 0.1%–0.5% formic acid solution as mobile phase B for gradient elution. The flow rate was 0.8–1.2 ml / min; the column temperature was 35–40℃; the detection wavelength was 300–350 nm; and the theoretical plate number, calculated based on the chlorogenic acid peak, should be no less than 3000–8000. The gradient elution conditions were as follows:

[0014] From 0 to 5 min, mobile phase A increased from 5% to 10%, and mobile phase B increased from 95% to 90%.

[0015] 5–13 min, mobile phase A 10%, mobile phase B 90%;

[0016] 13–18 min, mobile phase A 10 → 15%, mobile phase B 90 → 85%;

[0017] 18–38 min, mobile phase A 15 → 25%, mobile phase B 85 → 75%;

[0018] The detection method includes the following operations:

[0019] Preparation of reference solution: Take 0.5-2.0g of *Prunus camphora* leaf reference material, add 25-50ml of 50% methanol, heat under reflux for 1-2 hours, cool, shake well, filter, and take the filtrate as the reference solution of the reference material; separately take appropriate amounts of neochlorogenic acid reference standard, chlorogenic acid reference standard and cryptochlorogenic acid reference standard, accurately weigh them, add methanol to prepare a mixed solution containing 20-40μg of each per ml, as the reference solution of the reference standard.

[0020] Preparation of the test solution: Take the granules of the Chinese peach leaf formula, grind them finely, take about 0.2-1g, weigh accurately, add 25-50ml of 50% methanol accurately, weigh accurately, sonicate for 30-60 minutes, cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0021] For the determination method, accurately pipette 5-10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and perform the determination according to the working conditions of high performance liquid chromatography.

[0022] The chromatogram of the test sample should show four characteristic peaks, and the retention times should correspond to the four characteristic peaks in the chromatogram of the reference medicinal material. Among them, peaks 1 to 3 should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the chlorogenic acid reference peak is the S peak. The relative retention time of peak 4 and peak S should be calculated, and the relative retention time should be within ±10% of the specified value. The specified value is: the relative retention time of peak 4 is 1.31.

[0023] In some embodiments, the characteristic chromatogram detection method is: high performance liquid chromatography as per the general principles of the Chinese Pharmacopoeia;

[0024] Chromatographic conditions and system suitability experiments: The chromatographic column was packed with octadecylsilane-bonded silica gel, with acetonitrile as mobile phase A and 0.2% formic acid solution as mobile phase B for gradient elution. The flow rate was 1.0 mL / min; the column temperature was 35℃; the detection wavelength was 326 nm; and the theoretical plate number, calculated based on the chlorogenic acid peak, should not be less than 8000. The gradient elution conditions were as follows:

[0025] From 0 to 5 min, mobile phase A increased from 5% to 10%, and mobile phase B increased from 95% to 90%.

[0026] 5–13 min, mobile phase A 10%, mobile phase B 90%;

[0027] 13–18 min, mobile phase A 10 → 15%, mobile phase B 90 → 85%;

[0028] 18–38 min, mobile phase A 15 → 25%, mobile phase B 85 → 75%;

[0029] The detection method includes the following operations:

[0030] Preparation of reference solution: Take 1.0 g of *Prunus persica* leaf reference material, add 25 ml of 50% methanol, heat under reflux for 2 hours, cool, shake well, filter, and take the filtrate as the reference solution of the reference material; separately take appropriate amounts of neochlorogenic acid reference standard, chlorogenic acid reference standard and cryptochlorogenic acid reference standard, accurately weigh them, add methanol to prepare a mixed solution containing 20 μg of each per 1 ml, as the reference solution of the reference standard;

[0031] Preparation of the test solution: Take the granules of the Hantao leaf formula, grind them finely, take about 0.2g, weigh accurately, add 25ml of 50% methanol accurately, weigh accurately, sonicate for 30 minutes, cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0032] Determination method: Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph, and perform the determination according to the working conditions of high performance liquid chromatography. Record the chromatograms from 0 to 38 min.

[0033] The chromatogram of the test sample should show four characteristic peaks, and the retention times should correspond to the four characteristic peaks in the chromatogram of the reference medicinal material. Among them, peaks 1 to 3 should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the chlorogenic acid reference peak is the S peak. The relative retention time of peak 4 and peak S should be calculated, and the relative retention time should be within ±10% of the specified value. The specified value is: the relative retention time of peak 4 is 1.31.

[0034] Content determination: determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020).

[0035] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A and 0.2% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 35 °C; detection wavelength 326 nm. The theoretical plate number, calculated based on the chlorogenic acid peak, should be no less than 8000.

[0036]

[0037] Preparation of the reference solution: Take an appropriate amount of chlorogenic acid reference standard, accurately weigh it, and add methanol to prepare a solution containing 20 μg per ml.

[0038] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take about 0.2g, weigh it accurately, place it in a stoppered conical flask, accurately add 25ml of 50% methanol, weigh it, sonicate it (power 250W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the weight loss with 50% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0039] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0040] Among them, the Han Tao Ye formula granules are made by taking 9000g of Han Tao Ye slices, adding water to decoct, filtering, concentrating the filtrate, drying, pulverizing, adding appropriate excipients, mixing, granulating, and making 1000g.

[0041] In some embodiments, this detection method can be applied to the detection of Chinese peach leaf medicinal materials and processed slices.

[0042] In some embodiments, the detection method can be applied to the detection of standard decoction of Prunus persica leaves.

[0043] The standard decoction of Chinese peach leaf is prepared by weighing 100g of Chinese peach leaf slices, adding 10 times the amount of water and soaking for 30 minutes, bringing it to a boil over high heat, then simmering it over low heat for 30 minutes, filtering it while hot, and cooling the filtrate in a cold water bath; for the second decoction, add 8 times the amount of water, bring it to a boil over high heat, then simmer it over low heat for 20 minutes, filtering it while hot, and cooling the filtrate in a cold water bath; the two filtrates are combined and concentrated under reduced pressure at 65℃: concentrated to a relative density of 1.04~1.10 (65℃), the concentrated solution is dispensed into vials and placed in a freeze dryer for freeze drying to obtain the final product.

[0044] Beneficial effects:

[0045] 1. This invention is a detection method for Hantao leaf formula granules. The detection method has comprehensive detection items, simple operation, high precision, strong specificity and good reproducibility.

[0046] 2. In addition to the conventional detection items, this invention adds the detection of characteristic chromatograms, which can more comprehensively reflect the types and quantities of medicinal chemical components contained in the product. In particular, the characteristic chromatograms are detected by HPLC, which has the characteristics of simple operation and good reproducibility. At the same time, the content determination method in the detection method is consistent with the chromatographic conditions used for characteristic chromatogram detection. The quantitative determination of chlorogenic acid can be carried out when detecting characteristic chromatograms, saving time and effort.

[0047] 3. Through testing, the detection method of this invention is also applicable to Litsea cubeba medicinal materials, decoction pieces, standard decoctions, etc., and has a wide range of applications. Attached Figure Description

[0048] Figure 1Figures show the effects of different developing solvents. Sample 1 is a granulated formula of *Haloxylon ammodendron* leaf, and Sample 2 is a reference herb of *Haloxylon ammodendron* leaf. The developing solvent for Figure A is: petroleum ether-ethyl acetate-formic acid (2:0.5:0.2); the developing solvent for Figure B is: cyclohexane-ethyl acetate-formic acid (2:0.5:0.2); and the developing solvent for Figure C is: chloroform-ethyl acetate-formic acid (2:0.5:0.2).

[0049] Figure 2 The specificity chromatogram of the Han Tao Ye (Han Tao Ye) formula granules is shown, where peak 1 represents neochlorogenic acid; peak 2 (S) represents chlorogenic acid; and peak 3 represents cryptochlorogenic acid.

[0050] Figure 3 Figure showing the results of the overall performance evaluation of the Han Tao Ye (Prunus persica) leaf granule formula.

[0051] Figure 4 Separation effect of different chromatographic columns for Hantao leaf formulation granules

[0052] Figure 5 The characteristic chromatogram of the Han Tao Ye (Han Tao Ye) formula granules is shown, with peak 1 indicating neochlorogenic acid; peak 2 (S) indicating chlorogenic acid; and peak 3 indicating cryptochlorogenic acid.

[0053] Figure 6 Specificity investigation diagram for the determination of content in Han Tao leaf granules

[0054] Figure 7 The chromatogram of the medicinal material *Prunus camphora* leaves is shown. Peak 1: neochlorogenic acid; Peak 2: chlorogenic acid; Peak 3: cryptochlorogenic acid.

[0055] Figure 8 The characteristic chromatogram of the standard decoction of Prunus persica leaf is shown, in which peak 2 (S) is chlorogenic acid. Detailed Implementation

[0056] To make the technical solutions and effects of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. The embodiments described below are some embodiments of the present invention, but not all embodiments. In conjunction with 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.

[0057] Example 1: Establishment of a thin-layer chromatography method for identifying granules of *Haloxylon ammodendron* leaf extract.

[0058] This invention uses *Prunus persica* leaves as a positive control and employs chloroform-ethyl acetate-formic acid (2:0.5:0.2) as the developing solvent to investigate the thin-layer chromatography identification method.

[0059] 1.1 Solution Preparation

[0060] (1) Preparation of the test solution

[0061] Take 1g of Hantao leaf formula granules, grind them into a fine powder, add 20mL of water, reflux for 30min, filter, add 30mL of anhydrous ethanol for alcohol precipitation, refrigerate and stand for 4h, filter, evaporate the filtrate to dryness, add 1mL of methanol to dissolve the residue, and use it as the test solution.

[0062] (2) Preparation of control herbal solution

[0063] Take 1g of Chinese peach leaves as reference material, add 20mL of water, reflux for 30min, filter, add 30mL of anhydrous ethanol for alcohol precipitation, refrigerate and stand for 4h, filter, evaporate the filtrate to dryness, add 1mL of methanol to dissolve the residue, and use it as the reference material solution.

[0064] (3) Preparation of negative solution

[0065] Take 1g of maltodextrin and prepare a negative solution according to the test solution preparation method.

[0066] 1.2 Chromatographic conditions

[0067] Carrier: Silicone G254 thin film plate

[0068] Developing solvent: chloroform-ethyl acetate-formic acid (2:0.5:0.2)

[0069] Inspection: Examine under ultraviolet light (365nm).

[0070] 1.3 Methodological Examination

[0071] 1.3.1 Selection of developing solvent

[0072] The developing solvents were ① petroleum ether-ethyl acetate-formic acid (2:0.5:0.2), ② cyclohexane-ethyl acetate-formic acid (2:0.5:0.2), and ③ chloroform-ethyl acetate-formic acid (2:0.5:0.2). After complete development, the samples were observed under 365 nm fluorescence. The results showed that petroleum ether and cyclohexane spots accumulated and were incompletely separated, with indistinct main spots. Chloroform showed better separation, with clear and bright main spots. Therefore, chloroform-ethyl acetate-formic acid (2:0.5:0.2) was selected as the developing solvent for the thin-layer identification of the Hantao leaf formula granules. See details. Figure 1 .

[0073] 1.3.2 Sampling Quantity Investigation

[0074] 1 μL, 5 μL, and 10 μL of the test solution and 0.5 μL, 1 μL, and 1.5 μL of *Prunus persica* leaf reference material solution were respectively spotted onto the same silica gel G254 thin-layer plate. The plates were developed under the above-described thin-layer chromatographic conditions. The plates were then removed, air-dried, and examined under a UV lamp (365 nm). Based on the separation and clarity of the spots, the thin-layer identification of *Prunus persica* leaf formula granules was determined. 5 μL of the *Prunus persica* leaf formula granule test solution and 1 μL of the *Prunus persica* leaf reference material solution were then developed.

[0075] 1.3.3 Specificity Examination: 10 μl each of the test solution, negative control solution, and reference herbal solution of *Haloxylon ammodendron* leaf granules were spotted onto the same silica gel G254 thin-layer plate. The plates were developed under the aforementioned thin-layer chromatographic conditions, removed, air-dried, and examined under a UV lamp (365 nm). Results showed that the test sample chromatogram exhibited spots of the same color at the corresponding positions as the reference herbal solution chromatogram. The negative control solution showed no interference at the corresponding positions, indicating good specificity.

[0076] 1.3.4 Durability assessment:

[0077] 1.3.4.1 Temperature and Humidity Testing: 5.0 μL of the test solution and 1 μL of the *Prunus persica* leaf reference solution were spotted onto the same silica gel G254 thin-layer plate. Using chloroform-ethyl acetate-formic acid (2:0.5:0.2) as the developing solvent, the plates were developed under low temperature (10℃), room temperature (25℃), normal humidity (40%), and high humidity (80%) conditions. The plates were then removed, air-dried, and examined under a UV lamp at 365 nm. Results showed that temperature and humidity had no significant effect on the resolution and clarity of the *Prunus persica* leaf samples, indicating good durability under different temperature and humidity conditions.

[0078] 1.3.4.2 Investigation of Thin-Layer Plates from Different Manufacturers: 5.0 μL of the test solution and 1.0 μL of the reference herbal solution were respectively spotted onto silica gel G254 thin-layer plates from different manufacturers (Qingdao Ocean Silica G254 plate, Yantai Chemical Silica G254 plate, and Merck Silica G254 plate). The developing solvent was chloroform-ethyl acetate-formic acid (2:0.5:0.2). The plates were then removed, dried, and examined under a UV lamp at 365 nm. Results showed that using silica gel G254 thin-layer plates from different manufacturers, the test solution of the *Hantaoye* leaf formula granules showed spots of approximately the same color at the corresponding positions in the reference herbal solution, and the separation effect was good in all cases. The results indicate that thin-layer plates from different manufacturers have no significant effect on the identification of *Hantaoye* leaf formula granules.

[0079] 1.3.4.3 Durability Study by Different Personnel: Different personnel prepared the test sample and control drug solutions using the same method at different times. 5.0 μL of the test sample solution and 1.0 μL of the control drug solution were respectively spotted onto the same silica gel G254 thin-layer plate. The plates were developed using chloroform-ethyl acetate-formic acid (2:0.5:0.2) as the developing solvent. After development, the plates were removed, dried, and examined under a UV lamp at 365 nm. Results showed that the use of the same test method by different personnel had no significant impact on the thin-layer identification, indicating that the method has good durability.

[0080] The thin-layer chromatography identification method of this invention has been examined methodologically and has good specificity and robustness, and can be used for thin-layer chromatography identification of Chinese peach leaf formula granules.

[0081] Example 2: Establishment of a method for detecting the characteristic spectra of Prunus persica leaf granules

[0082] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0083] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A and 0.2% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 35 °C; detection wavelength 326 nm. The theoretical plate number, calculated based on the chlorogenic acid peak, should be no less than 8000.

[0084]

[0085] Preparation of the reference solution: Take 1.0 g of *Prunus persica* leaf reference material, place it in a stoppered conical flask, add 25 ml of 50% methanol, heat under reflux for 2 hours, cool, shake well, filter, and collect the filtrate as the reference solution. Separately, take appropriate amounts of neochlorogenic acid reference standard, chlorogenic acid reference standard, and cryptochlorogenic acid reference standard, accurately weigh them, and add methanol to prepare a mixed solution containing 20 μg of each per ml, as the reference solution.

[0086] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take about 0.2g, weigh it accurately, place it in a stoppered conical flask, accurately add 25ml of 50% methanol, weigh it, sonicate it (power 250W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the weight loss with 50% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0087] The determination method involves precisely pipetting 10 μl each of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0088] The chromatogram of the test sample should show four characteristic peaks, which should correspond to the retention times of the four characteristic peaks in the chromatogram of the reference medicinal material. Peaks 1 to 3 should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the chlorogenic acid reference peak is designated as peak S. The relative retention time of peak 4 and peak S should be calculated and should be within ±10% of the specified value. The specified value is 1.31 (peak 4).

[0089] Example 3: Methodological validation of the characteristic spectrum detection method for Han Tao leaf formula granules

[0090] The practicality and robustness of the feature map detection method in Example 2 were verified by examining the specificity, integrity, precision, repeatability, solution stability, and robustness of the detection method.

[0091] In the embodiments, all original reagent materials are commercially available, and experimental methods without specific conditions are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0092] 3.1 Instruments, reagents, and reagents used in the examples

[0093] Instruments: Ultimate 3000 high-performance liquid chromatograph (ThermoFisher Scientific, USA, including system controller, infusion pump, degassing assembly, low-pressure gradient assembly, autosampler, column oven, temperature-controlled sample chamber, UV-DAD detector, Chromeleon 7 chromatography data workstation); EL-204 0.001% balance (METTLER TOLEDO, Switzerland); AE-240 0.001% balance (METTLER TOLEDO, Switzerland); micropipette (Eppendorf, Germany); KQ-300DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); SYG-1-7 electric thermostatic water bath (Tianjin Test Instrument Co., Ltd.).

[0094] Reagents: Acetonitrile, methanol (chromatographic grade, Sinopharm Chemical Reagent Co., Ltd.); anhydrous formic acid (analytical grade, Aladdin Reagent).

[0095] Test reagents: chlorogenic acid (batch number: 110753-202119, purity: 96.3%, China National Institutes for Food and Drug Control); neochlorogenic acid and cryptochlorogenic acid (batch numbers: AZCC2416 and AFCE3004, purity: 99.96% and 99.69%, respectively, Chengdu Efa Biotechnology Co., Ltd.); Prunus persica leaf reference material (batch number: 121575-201503, China National Institutes for Food and Drug Control).

[0096] 3.2 Specificity Examination

[0097] Take blank solvent (50% methanol), reference solution, and Hantao leaf granule test solution, and inject them according to the chromatographic conditions under "Example 2". Record the chromatograms. The results are shown in [Figure 2]. Figure 2 .

[0098] The results showed that the chromatogram of the test sample had the same chromatographic peak at the corresponding retention time as the chromatogram of the reference sample, and the blank solvent did not interfere, indicating that the method has good specificity.

[0099] 3.3 Holistic Examination

[0100] Take an appropriate amount of *Prunus persica* leaf granules, grind them finely, and take about 0.2 g. Prepare a test solution according to the method for preparing the test solution in Example 2. Based on the chromatographic conditions in Example 2, maintain the elution gradient at the initial ratio, but double the elution time. Record the chromatogram. The results are shown in [Figure 2]. Figure 3 .

[0101] The results showed that no obvious chromatographic peaks eluted after the initial gradient elution, indicating that the chromatographic conditions basically met the principle of maximizing information content.

[0102] 3.4 Precision Examination

[0103] Take an appropriate amount of *Prunus persica* leaf granules, grind them finely, and take about 0.2 g. Prepare a test solution according to the method for preparing the test solution in Example 2. Inject the sample for analysis under the chromatographic conditions in Example 2. Inject the same test solution six times consecutively. Using the chlorogenic acid chromatographic peak as the reference S peak, calculate the relative retention time and relative peak area of ​​each characteristic peak and the S peak, and calculate the RSD.

[0104] The results showed that when the same sample was injected six times consecutively, with the chlorogenic acid chromatographic peak as the reference S peak, the relative retention time RSD values ​​of each characteristic peak and the S peak were in the range of 0.01 to 0.10%, and the relative peak area was in the range of 0.18 to 0.45%, all less than 1%, indicating that the instrument precision was good.

[0105] 3.5 Repeatability Test

[0106] Take an appropriate amount of *Prunus persica* leaf granules, grind them finely, and accurately weigh approximately 0.2 g. Prepare a test solution according to the method for preparing the test solution in Example 2. Perform six parallel analyses and inject the solutions under the chromatographic conditions described in Example 2. Using the chlorogenic acid peak as the reference S peak, calculate the relative retention time and relative peak area of ​​each characteristic peak relative to the S peak, and calculate the RSD.

[0107] The results showed that when six samples were prepared in parallel from the same batch, with the chlorogenic acid chromatographic peak as the reference S peak, the relative retention time RSD values ​​of each characteristic peak and the S peak were in the range of 0.06-0.38%, and the relative peak area was in the range of 0.28-1.06%, all less than 3%, indicating that the method has good repeatability.

[0108] 3.6 Stability Test

[0109] Take an appropriate amount of *Prunus persica* leaf granules, grind them finely, and take about 0.2 g. Prepare a test solution according to the method for preparing the test solution in Example 2. Inject 10 μL of the solution at 0, 2, 4, 8, 12, and 24 h after sample preparation, and record the chromatograms. Using the chlorogenic acid chromatographic peak as the reference S peak, calculate the relative retention time and relative peak area of ​​each characteristic peak and the S peak, and calculate the RSD.

[0110] The results showed that within 24 hours, the RSD values ​​of the relative retention times of each characteristic peak and the S peak were in the range of 0.02 to 0.13%, and the relative peak areas were in the range of 0.13 to 0.37%, all less than 1%, indicating that the test solution was stable within 24 hours.

[0111] 3.7 Durability Test

[0112] 3.7.1 Column analysis:

[0113] Use 1: ACE Excel 5C18-PFP (ACE, 4.6×250mm, 5μm); 2: Hypesil BDS C18 (Elite, 4.6×250mm, 5μm); 3: Three chromatographic columns, XB-C18 (Welch, 4.6×250mm, 5μm), were used to analyze samples of Hantao leaf granules. Results are shown below. Figure 4 .

[0114] The results showed that good separation of the peach leaf samples could be obtained using chromatographic columns from three different brands. Subsequent studies selected an ACE Excel 5C18-PFP column (250 mm length, 4.6 mm inner diameter, 5 μm particle size).

[0115] 3.7.2 Flow velocity investigation

[0116] An ACE Excel 5C18-PFP column (250 mm column length, 4.6 mm inner diameter, 5 μm particle size) was used to investigate the sample separation performance at flow rates of 0.9 mL / min, 1.0 mL / min, and 1.1 mL / min. The results showed that the analytical method achieved good separation of all characteristic peaks within a flow rate range of 0.9–1.1 mL / min. Small flow rate variations were sufficient to meet the system suitability requirements; a flow rate of 1.0 mL / min was used in subsequent studies.

[0117] 3.7.3 Column Temperature Investigation

[0118] The results were investigated at three temperatures: 30℃, 35℃, and 40℃. The results showed good peak separation at column temperatures between 30℃ and 40℃, and good robustness within this temperature range. Based on the sample peak elution, 35℃ was selected as the detection temperature.

[0119] 3.8 Sample Determination

[0120] Take an appropriate amount of 3 batches of Hantao leaf formula granules, grind them into a fine powder, take 0.2g, and prepare a test solution according to the test solution preparation method in Example 2. Inject the sample and measure it according to the chromatographic conditions in Example 2, record the chromatogram, and the results are shown in the table below.

[0121] Relative retention times of characteristic spectra of three batches of Hantao leaf formula granules

[0122]

[0123] The HPLC characteristic chromatograms of three batches of Hantao leaf formula granules were matched using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine". Using peak 2 (chlorogenic acid) as the reference peak S, a control chromatogram was generated using the mean method to establish a control characteristic chromatogram for Hantao leaf formula granules. (See attached image.) Figure 5 .

[0124] The final standard for the characteristic chromatogram of *Prunus persica* leaves was determined as follows: the test sample chromatogram should show four characteristic peaks, and the retention times should correspond to the four characteristic peaks in the reference chromatogram of the reference medicinal material. Peaks 1 to 3 should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the chlorogenic acid reference peak is designated as peak S. The relative retention time between peak 4 and peak S should be calculated and should be within ±10% of the specified value. The specified value is 1.31 (peak 4).

[0125] Example 4: Investigation of the pretreatment method of test sample in the method for determining the content of *Haloxylon ammodendron* leaf granules

[0126] The extraction solvent, extraction method, and extraction time for the pretreatment of samples in the determination of the content of *Haloxylon ammodendron* leaf granules were investigated to determine the optimal sample pretreatment method.

[0127] 4.1 Investigation of different extraction solvents

[0128] Take an appropriate amount of *Prunus persica* leaf granules, grind them finely, and take 0.2g. Place the granules in a stoppered conical flask. Perform four parallel trials, accurately adding 25mL each of 30% methanol, 50% methanol, 70% methanol, and methanol. Weigh the flasks and sonicate each solution (250W power, 40kHz frequency) for 30 minutes. After cooling, weigh the flasks again. Make up the lost weight with the appropriate solvent, shake well, filter, and collect the filtrate. Accurately pipette 10μL of each test solution and inject it into a liquid chromatograph. Determine the chlorogenic acid content according to the chromatographic conditions of Example 2. The results are shown in the table below:

[0129]

[0130] The results showed that the chlorogenic acid content was higher when 50% methanol or 70% methanol was used as the extraction solvent, and 50% methanol was ultimately chosen as the extraction solvent.

[0131] 4.2 Examination of different extraction methods

[0132] Take an appropriate amount of *Prunus persica* leaf granules, grind them finely, take 0.2g, place them in a stoppered conical flask, accurately add 25mL of 50% methanol, seal tightly, weigh, and then sonicate (250W power, 40kHz frequency), heat under reflux, and shake to extract for 30 minutes. Cool, weigh again, and replenish the lost weight with 50% methanol. Shake well, filter, and collect the filtrate. Accurately pipette 10μL of each test solution and inject it into a liquid chromatograph. Determine the chlorogenic acid content according to the chromatographic conditions of Example 2. The results are shown in the table below:

[0133]

[0134] The results showed that the chlorogenic acid content was not significantly different between ultrasonic extraction and reflux extraction. Considering the convenience and speed, ultrasonic extraction was ultimately chosen as the extraction method.

[0135] 4.3 Examination of different extraction times

[0136] Take an appropriate amount of *Haloxylon ammodendron* leaf granules, grind them finely, take 0.2g, place them in a stoppered conical flask, accurately add 25mL of 50% methanol, seal tightly, weigh, and perform sound treatment (power 250W, frequency 40kHz) for 15min, 30min, 45min, and 60min respectively. Remove, cool, weigh again, replenish the lost weight with 50% methanol, shake well, filter, and collect the filtrate. Accurately pipette 10μL of each test solution and inject it into a liquid chromatograph. Determine the chlorogenic acid content according to the chromatographic conditions of Example 2.

[0137]

[0138] The results showed that the chlorogenic acid content did not change significantly after an extraction time of 30 minutes, indicating that extraction was complete within 30 minutes. Therefore, an extraction time of 30 minutes was chosen.

[0139] 4.4 Determination of the preparation of the test solution

[0140] Based on the results of the pretreatment investigation experiment, the preparation method of the test solution can be determined as follows: Take an appropriate amount of Hantao leaf formula granules, grind them finely, take 0.2g, place them in a stoppered conical flask, accurately add 25mL of 50% methanol, weigh the contents, sonicate (power 250W, frequency 40kHz) for 30min, cool, replenish the lost weight with 50% methanol, shake well, filter, and take the filtrate as the test solution.

[0141] Example 5: Establishment of a method for determining the content of *Haloxylon ammodendron* leaf granules.

[0142] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0143] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A and 0.2% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 35 °C; detection wavelength 326 nm. The theoretical plate number, calculated based on the chlorogenic acid peak, should be no less than 8000.

[0144]

[0145] Preparation of the reference solution: Take an appropriate amount of chlorogenic acid reference standard, accurately weigh it, and add methanol to prepare a solution containing 20 μg per ml.

[0146] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take about 0.2g, weigh it accurately, place it in a stoppered conical flask, accurately add 25ml of 50% methanol, weigh it, sonicate it (power 250W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the weight loss with 50% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0147] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0148] Example 6: Methodological Validation of the Method for Determining the Content of Peach Leaf Formula Granules

[0149] The practicality and accuracy of the content determination method in Example 5 were verified by examining the linear range, precision, repeatability, solution stability, and recovery rate of the detection method.

[0150] In this embodiment, the instruments, reagents, and reagents used are the same as in Example 3.

[0151] 6.1 Exclusivity

[0152] Take the *Haloxylon ammodendron* leaf granules and prepare a test solution according to the method described in Example 5, "Preparation of Test Solution"; use 50% methanol as a blank solvent solution. Take the above test solution, blank solvent, and chlorogenic acid reference standard and determine them according to the chromatographic conditions of Example 5. See... Figure 6 .

[0153] The results show that the chromatogram of the test sample has the same chromatographic peak at the corresponding retention time as that of the reference sample, and there is no interference from the blank solvent, indicating that the method has good specificity.

[0154] 6.2 Linearity Examination

[0155] Accurately weigh chlorogenic acid reference standard and place it in a 100 mL volumetric flask. Add methanol to prepare a stock solution containing 116.5 μg of chlorogenic acid reference standard per mL. Accurately measure 0.1 mL, 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL, and 5.0 mL of the above mixed stock solution and place them in separate 10 mL volumetric flasks. Add methanol to the mark and mix well to obtain working solutions of reference standards at different concentrations. Accurately pipette 10 μL of each working solution of reference standards at different concentrations and determine the peak areas sequentially according to the chromatographic conditions in Example 5. Plot the peak areas as the ordinate (y) and the reference standard concentration as the abscissa (x), and construct a standard curve.

[0156] The results showed that the linear regression equation for chlorogenic acid was: y = 0.4365x - 0.0989, R0 2 =0.9987. This indicates a good linear relationship between chlorogenic acid concentration and peak area within the concentration range of 1.17–58.26 μg / mL.

[0157] 6.3 Precision Test

[0158] Take an appropriate amount of Chinese peach leaf granules, grind them into a fine powder, take 0.2g, and prepare a test solution according to the test solution preparation method in Example 5. Inject the sample for analysis under the chromatographic conditions in Example 5, inject the sample 6 times consecutively, record the chromatographic peak area, and calculate the RSD.

[0159] The results showed that when the same sample solution was injected repeatedly 6 times, the RSD value of the chlorogenic acid peak area was 0.39%, which is less than 1%, indicating that the instrument has good precision.

[0160] 6.4 Repeatability Test

[0161] Take an appropriate amount of *Hantao hainanensis* leaf granules, grind them finely, and take 0.2 g. Prepare six parallel portions of the solution according to the method for preparing the test solution in Example 5. Inject the solution under the chromatographic conditions in Example 5, record the peak area, and calculate the content and RSD. The results are shown in the table below:

[0162]

[0163] The results showed that when six parallel sample solutions were injected, the RSD value of chlorogenic acid content was 2.38%, which was less than 3%, indicating that the method had good repeatability.

[0164] 6.5 Stability Test

[0165] Take an appropriate amount of *Prunus persica* leaf granules, grind them finely, and take 0.2 g. Prepare a test solution according to the method for preparing the test solution in Example 5. Inject the solution under the chromatographic conditions in Example 5 at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h, and record the peak area. Calculate the peak area and RSD respectively.

[0166] The results showed that when the test solution was injected at 0, 2, 4, 8, 12, and 24 hours, the RSD value of the chlorogenic acid peak area was 1.92%, which was less than 2%, indicating that the method had good stability within 24 hours.

[0167] 6.6 Recovery Rate Test

[0168] Take an appropriate amount of particulate sample with a known content (chlorogenic acid content: 1.79 mg / g), grind it finely, and take 0.1 g in six parallel portions, accurately weighing each portion. Add a reference solution containing the same amount of chlorogenic acid as the 0.1 g sample to each portion. Prepare the spiking and recovery test solution according to the test solution preparation method in Example 5. Inject 10 μL of each solution under the chromatographic conditions in Example 5. Calculate the recovery rate and RSD using the following formulas. The results are shown in the table below:

[0169]

[0170]

[0171] The results showed that the recovery rates of chlorogenic acid were all between 95.48% and 101.13%, and the RSD value of the chlorogenic acid recovery rate was 2.43%, which was less than 3%, indicating good recovery results.

[0172] 6.7 Sample Determination

[0173] Take an appropriate amount of three batches of Hantao leaf formula granules, grind them into a fine powder, take 0.2g, and prepare a test solution according to the "Preparation Method of Test Solution" in Example 5. Inject the sample according to the chromatographic conditions in Example 5 to determine the content of chlorogenic acid. The results are shown in the table below.

[0174]

[0175] Example 7: Establishment of Characteristic Spectrum and Content Determination Method for Prunus persica Leaf Medicinal Material

[0176] [Characteristic chromatogram] Determined by high performance liquid chromatography (General Chapter 0512 of the 2020 edition of the Chinese Pharmacopoeia).

[0177] Chromatographic conditions and system suitability tests are the same as those in the [Assay] section.

[0178] Preparation of the reference solution: Take about 1g of *Prunus persica* leaf reference material and place it in a stoppered conical flask. Add 25ml of 50% methanol, heat under reflux for 2 hours, remove, cool, shake well, filter, and collect the filtrate as the reference solution. Separately, take an appropriate amount of chlorogenic acid reference standard, accurately weigh it, and add methanol to prepare a solution containing 40μg of chlorogenic acid per ml, as the reference solution.

[0179] The preparation of the test solution is the same as in the [Assay] section.

[0180] The assay involves precisely injecting 10 μl each of the reference medicinal material and the test solution into a liquid chromatograph and measuring the solution.

[0181] The chromatogram of the test sample should show four characteristic peaks, and the retention times should correspond to the four characteristic peaks in the chromatogram of the reference medicinal material. The retention time of peak 2 should correspond to the retention time of the chlorogenic acid reference peak. The peak corresponding to the chlorogenic acid reference peak is peak S. Calculate the relative retention times of peaks 1, 3, and 4 with peak S. These relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.65 (peak 1), 1.08 (peak 3), and 1.27 (peak 4). See the characteristic chromatogram of *Prunus persica* leaf for reference. Figure 7 .

[0182] [Content Determination] Chlorogenic acid was determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0183] Chromatographic conditions and system suitability tests were performed using an octadecylsilane-bonded silica column (250 mm length, 4.6 mm inner diameter, 5 μm particle size); acetonitrile as mobile phase A and 0.2% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 35 °C; detection wavelength 326 nm. The theoretical plate number, calculated based on the chlorogenic acid peak, should be no less than 8000.

[0184]

[0185] Preparation of the reference solution: Take an appropriate amount of chlorogenic acid reference standard, accurately weigh it, and add methanol to prepare a solution containing 40 μg per ml.

[0186] Preparation of the test solution: Take about 1g of Prunus persica leaf powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 50% methanol, weigh it, heat under reflux for 2 hours, cool it, weigh it again, make up the lost weight with 50% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0187] The assay involves precisely pipetting 10 μl of both the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0188] Example 8: Establishment of Characteristic Spectrum and Content Determination Method for Prunus persica Leaf Slices

[0189] [Characteristic chromatogram] Determined by high performance liquid chromatography (General Chapter 0512 of the 2020 edition of the Chinese Pharmacopoeia).

[0190] Chromatographic conditions and system suitability tests are the same as those in the [Assay] section.

[0191] Preparation of the reference solution: Take about 1g of *Prunus persica* leaf reference material and place it in a stoppered conical flask. Add 25ml of 50% methanol, heat under reflux for 2 hours, remove, cool, shake well, filter, and collect the filtrate as the reference solution. Separately, take an appropriate amount of chlorogenic acid reference standard, accurately weigh it, and add methanol to prepare a solution containing 40μg of chlorogenic acid per ml, as the reference solution.

[0192] The preparation of the test solution is the same as in the [Assay] section.

[0193] The assay involves precisely injecting 10 μl each of the reference medicinal material and the test solution into a liquid chromatograph and measuring the solution.

[0194] The chromatogram of the test sample should show four characteristic peaks, and the retention times should correspond to the four characteristic peaks in the chromatogram of the reference medicinal material. The retention time of peak 2 should correspond to the retention time of the chlorogenic acid reference peak. The peak corresponding to the chlorogenic acid reference peak is peak S. Calculate the relative retention times of peaks 1, 3, and 4 with peak S. These relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.65 (peak 1), 1.09 (peak 3), and 1.26 (peak 4).

[0195] [Content Determination] Chlorogenic acid was determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0196] Chromatographic conditions and system suitability tests were performed using an octadecylsilane-bonded silica column (250 mm length, 4.6 mm inner diameter, 5 μm particle size); acetonitrile as mobile phase A and 0.2% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 35 °C; detection wavelength 326 nm. The theoretical plate number, calculated based on the chlorogenic acid peak, should be no less than 8000.

[0197]

[0198] Preparation of the reference solution: Take an appropriate amount of chlorogenic acid reference standard, accurately weigh it, and add methanol to prepare a solution containing 40 μg per ml.

[0199] Preparation of the test solution: Take about 1g of Chinese peach leaf powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 50% methanol, weigh it, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool it, weigh it again, add 50% methanol to make up the lost weight, shake it well, filter it, and take the filtrate to obtain the test solution.

[0200] The assay involves precisely pipetting 10 μl of both the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0201] Example 9: Establishment of Characteristic Chromatography and Content Determination Method for Standard Decoction of Prunus persica Leaf

[0202] [Characteristic chromatogram] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0203] Chromatographic conditions and system suitability tests are the same as those for the [Content Determination] chlorogenic acid section.

[0204] Preparation of the reference solution: Take about 1g of *Prunus persica* leaf reference material and place it in a stoppered conical flask. Add 25ml of 50% methanol, heat under reflux for 2 hours, remove, cool, shake well, filter, and collect the filtrate as the reference solution. Separately, take an appropriate amount of chlorogenic acid reference standard, accurately weigh it, and add methanol to prepare a solution containing 30μg of chlorogenic acid per ml, as the reference solution.

[0205] The preparation of the test solution is the same as in the [Assay] section.

[0206] The determination method involves precisely pipetting 10 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0207] The chromatogram of the test sample should show four characteristic peaks, and the retention times should correspond to the four characteristic peaks in the chromatogram of the reference medicinal material. The retention time of peak 2 should correspond to the retention time of the chlorogenic acid reference peak. The peak corresponding to the chlorogenic acid reference peak is peak S. Calculate the relative retention times of peaks 1, 3, and 4 with peak S. These relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.65 (peak 1), 1.09 (peak 3), and 1.25 (peak 4). See the characteristic chromatogram of the standard decoction of *Prunus persica* leaves for reference. Figure 8 .

[0208] [Content Determination] Chlorogenic acid was determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0209] Chromatographic conditions and system suitability tests were performed using octadecyl-bonded silane silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 35 °C; detection wavelength 326 nm. The theoretical plate number, calculated based on the chlorogenic acid peak, should be no less than 8000.

[0210]

[0211]

[0212] Preparation of the reference solution: Take an appropriate amount of chlorogenic acid reference standard, accurately weigh it, and add 50% methanol to prepare a solution containing 30 μg per ml.

[0213] Preparation of the test solution: Take an appropriate amount of standard decoction of Prunus persica leaves, grind it into a fine powder, take about 0.2g, weigh it accurately, place it in a stoppered conical flask, accurately add 25ml of 50% methanol, weigh it, sonicate it (power 250W, frequency 40kHz) for 30 minutes, cool it, weigh it again, add 50% methanol to make up the weight loss, shake it well, filter it, and take the filtrate to obtain the test solution.

[0214] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0215] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A detection method of Han Tao Ye formula granules, the Han Tao Ye formula granules are made by decocting, concentrating, drying, crushing and granulating Han Tao Ye decoction pieces; the detection method comprises characteristics, identification, inspection, extract, characteristic map, content determination, characterized in that, The characteristic spectrum and the content determination detection method are detected by high performance liquid chromatography, the working conditions of the high performance liquid chromatography are as follows: the chromatographic column is filled with octadecylsilane bonded silica gel, acetonitrile is used as mobile phase A, 0.1-0.5% formic acid solution is used as mobile phase B for gradient elution, the flow rate is 0.8-1.2 ml per minute; the column temperature is 35-40 DEG C; the detection wavelength is 300-350 nm, the theoretical plate number calculated according to the green acid peak should be not less than 3000-8000; the gradient elution conditions are as follows: ​ 0-5 min, mobile phase A 5→10%, mobile phase B 95→90%; 5-13 min, mobile phase A 10%, mobile phase B 90%; 13-18 min, mobile phase A 10→15%, mobile phase B 90→85%; 18-38 min, mobile phase A 15→25%, mobile phase B 85→75%; The characteristic spectrum and the content determination detection method further comprise preparing reference solution or preparing control solution, preparing test solution and sampling determination, wherein: The preparation of reference solution: 0.5-2.0 g of Han peach leaf control medicinal material is taken, 25-50 ml of 50% methanol is added, heating reflux is carried out for 1-2 hours, it is cooled, shaken up, filtered, the filtered solution is taken as the control medicinal material reference solution; additionally, new green acid control product, green acid control product and cryptogreen acid control product are taken in proper amount, accurately weighed, methanol is added to prepare a mixed solution containing 20-40 ug per 1 ml, which is used as the control product reference solution; The preparation of control solution: green acid control product is taken in proper amount, accurately weighed, methanol is added to prepare a solution containing 20-40 ug per 1 ml, which is obtained; The preparation of test solution: Han peach leaf formula granules are finely ground, about 0.2-1 g is taken, accurately weighed, 25-50 ml of 50% methanol is accurately added, the weight is determined, ultrasonic treatment is carried out for 30-60 minutes, it is cooled, the weight is determined again, the lost weight is made up with 50% methanol, shaken up, filtered, the filtered solution is taken, which is obtained; The determination method: 5-10 ul of reference solution or control solution and test solution is accurately taken respectively, injected into the liquid chromatograph, the determination is carried out according to the working conditions of high performance liquid chromatography; The test chromatogram of the characteristic spectrum should present four characteristic peaks, which should correspond to the retention time of the four characteristic peaks in the reference chromatogram of the control medicinal material, wherein peak 1-peak 3 should correspond to the retention time of the corresponding control product peaks, the peak corresponding to the green acid control product peak is S peak, the relative retention time of peak 4 and S peak should be within ±10% of the specified value; the specified value is that the relative retention time of peak 4 is 1.

31.

2. The detection method according to claim 1, characterized in that, The working conditions of the high performance liquid chromatography are as follows: octadecylsilane bonded silica gel is used as the filler, acetonitrile is used as mobile phase A, 0.2% formic acid solution is used as mobile phase B for gradient elution, the flow rate is 1.0 ml per minute; the column temperature is 35 DEG C; the detection wavelength is 326 nm, the theoretical plate number calculated according to the green acid peak should be not less than 8000; The preparation method of the reference solution is as follows: 1.0 g of Han Tao Ye control medicinal material is taken, 25 ml of 50% methanol is added, heated to reflux for 2 hours, cooled, shaken up, filtered, and the filtrate is taken as the control medicinal material reference solution; additionally, a mixed solution containing 20 μg per 1 ml of neochlorogenic acid control, chlorogenic acid control and cryptochlorogenic acid control is prepared as the control reference solution; The preparation method of the test solution is as follows: Han Tao Ye formula granules are taken, finely ground, about 0.2 g is taken, precisely weighed, 25 ml of 50% methanol is precisely added, the weight is determined, ultrasonic treatment is performed for 30 minutes, cooled, the weight is determined again, the lost weight is made up with 50% methanol, shaken up, filtered, and the filtrate is taken.

3. The method of claim 1, wherein The determination method of the characteristic spectrum is as follows: 10 μl of the reference solution and the test solution is precisely taken respectively, injected into the liquid chromatograph, and determined according to the high performance liquid chromatography working condition.

4. The assay of any one of claims 1 to 3, wherein, The determination method of the content is as follows: 10 μl of the control solution and the test solution is precisely taken respectively, injected into the liquid chromatograph, and determined according to the high performance liquid chromatography working condition, and the chromatogram is recorded, and the content of chlorogenic acid is calculated by the external standard method.

5. The assay of any one of claims 1 to 3, wherein the assay is a competitive assay. The identification method is as follows: 0.5-2 g of Han Tao Ye formula granules is finely ground, 20-50 ml of water is added, refluxed for 30-60 min, filtered, 30-50 ml of ethanol is added for alcohol precipitation, filtered, the filtrate is evaporated to dryness, 1 ml of methanol is added to dissolve the residue, and the solution is taken as the test solution; additionally, 0.5-2 g of Han Tao Ye control medicinal material is taken, and the control medicinal material solution is prepared by the same method; according to the thin layer chromatography test in the Chinese Pharmacopoeia, 2-10 μl of the test solution and 1-5 μl of the control medicinal material solution is taken, spotted on the same silica gel GF254 thin layer plate, developed with trichloromethane-ethyl acetate-formic acid=2:0.5:0.2 as the developing agent, taken out, dried, and observed under a 365 nm ultraviolet light; the same color spots appear in the test chromatogram at the positions corresponding to the control medicinal material chromatogram.

6. The detection method according to claim 5, characterized in that, The identification method is as follows: 1 g of Han Tao Ye formula granules is finely ground, 20 ml of water is added, refluxed for 30 min, filtered, 30 ml of anhydrous ethanol is added for alcohol precipitation, refrigerated for 4 h, filtered, the filtrate is evaporated to dryness, 1 ml of methanol is added to dissolve the residue, and the solution is taken as the test solution; additionally, 1 g of Han Tao Ye control medicinal material is taken, and the control medicinal material solution is prepared by the same method; according to the thin layer chromatography test in the Chinese Pharmacopoeia, 5 μl of the test solution and 1 μl of the control medicinal material solution is taken, spotted on the same silica gel GF254 thin layer plate, developed with trichloromethane-ethyl acetate-formic acid=2:0.5:0.2 as the developing agent, taken out, dried, and observed under a 365 nm ultraviolet light; the same color spots appear in the test chromatogram at the positions corresponding to the control medicinal material chromatogram.

7. The assay of any one of claims 1 to 3, wherein the assay is a competitive assay. The extract detection method is as follows: Han Tao Ye formula granules are finely ground, 2 g is precisely weighed, 100 ml of ethanol is precisely added, and the hot soaking method in the alcohol-soluble extract determination method in the Chinese Pharmacopoeia is used for determination.

8. The assay of any one of claims 1 to 3, wherein, The Han peach leaf formula granules are prepared by taking Han peach leaf decoction pieces, adding water to decoct, filtering, concentrating the filtrate into a clear paste, drying or drying, crushing, adding pharmaceutically acceptable adjuvants, mixing, and granulating.

9. Use of the detection method according to any one of claims 1 to 8, characterized in that, The detection method is applied to the quality detection of Han peach leaf medicinal materials, Han peach leaf decoction pieces, Han peach leaf standard decoction, and Han peach leaf formula granules.

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