Detection method and application of schefflera kwangsiensis formula granules

Abstract

The invention belongs to the technical field of medicine detection, and particularly relates to a detection method and application of schefflera kwangsiensis formula granules, schefflera kwangsiensis formula granules are prepared by decocting schefflera kwangsiensis decoction pieces, concentrating, drying and granulating, and the detection method comprises the items of character determination, identification, inspection, extract determination, characteristic spectrum determination and content determination. According to the identification method, thin-layer chromatography is adopted for identification; the characteristic chromatogram is characterized in that a high performance liquid chromatography method is adopted, a chlorogenic acid reference substance peak is taken as an S peak, and four characteristic peaks are marked; and the content determination is to detect the content of chlorogenic acid. According to the detection method established by the invention, the detection requirements on schefflera kwangsiensis medicinal materials, decoction pieces, standard decoction and formula granules are improved, establishment of a perfect quality control system for each product of schefflera kwangsiensis is facilitated, and the quality and clinical curative effect of the schefflera kwangsiensis formula granules are guaranteed.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and specifically relates to a detection method and application of peach leaf formula granules. Background Art

[0002] Chinese medicine formula granules are granules made from single Chinese medicine pieces through one or more processes for extraction and concentration, and are used in Chinese medicine clinical prescriptions. They have the advantages of being stable and controllable, safe and efficient, no need for decoction, easy to prepare, convenient to carry, and ready to take.

[0003] Hantao leaf, also known as Schefflera vine and seven-leaf lotus, is the dried stems or stems with leaves of Schefflera leacantha (ScheffLeraLeacantha Vig.), a plant of the Araliaceae family. It is slightly bitter, astringent, and warm in nature. It has the effects of dispelling wind and relieving pain, relaxing muscles and activating collaterals, and is used for sciatica, trigeminal neuralgia, neuralgic headache, rheumatic joint pain, etc. It is a commonly used analgesic in the folk. The chemical components of Hantao leaf are mainly oleanane-type pentacyclic triterpenes and their glycosides, as well as sterols, long-chain olefins, organic acids, volatile oils and other components. It is mainly distributed in Guangxi, Guizhou, Yunnan, Fujian and other places in my country.

[0004] At present, the standard of Hantao leaf medicinal material is included in the 2003 edition of "Quality Standards of Traditional Chinese and Ethnic Chinese Medicinal Materials in Guizhou Province", which only contains properties and microscopic identification. With the increasing clinical application of formula granules, a comprehensive, convenient and controllable detection method is urgently needed to control the quality of Hantao leaf formula granules. Summary of the invention

[0005] In view of the imperfect detection methods in the prior art of Chinese peach leaf formula granules, the present invention provides a quality detection method for Chinese peach leaf formula granules, which is specifically:

[0006] The detection items of the peach leaf formula granules include properties, identification, inspection, extracts, characteristic spectra, and content determination.

[0007] In some embodiments, the properties of the peach leaf formula granules are: yellow-brown granules; slight odor, bitter taste.

[0008] In some embodiments, the properties of the peach leaf formula granules are: brown granules; slight odor, bitter taste.

[0009] Identification: Take 1g of Hantao leaf formula granules, grind them, add 20ml of water, condense and reflux for 30min, filter, add 30ml of anhydrous ethanol for alcohol precipitation, refrigerate and stand for 4h, filter, evaporate the filtrate to dryness, and add 1ml of methanol to dissolve the residue as the test solution. Take another 1g of Hantao leaf control medicinal material and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), take 5μl of the test solution and 1μl of the control medicinal material solution respectively, and spot them on the same silica gel GF254 thin layer plate, use chloroform-ethyl acetate-formic acid (2:0.5:0.2) as the developing agent, develop, take out, dry, and examine under ultraviolet light (365nm). In the chromatogram of the test sample, spots of the same color appear at the corresponding position of the chromatogram of the control medicinal material.

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

[0011] Extract: Take the formula granules of Hantao leaves, grind them into powder, take 2g, weigh accurately, add 100ml of ethanol accurately, and determine it according to the hot soaking method under the item of determination method of alcohol-soluble extract in the general rules of the Chinese Pharmacopoeia.

[0012] Characteristic spectrum: Determined by high performance liquid chromatography according to the general rules of the Chinese Pharmacopoeia;

[0013] Chromatographic conditions and system suitability experiment The chromatographic column is filled with octadecylsilane bonded silica gel, acetonitrile is used as mobile phase A, 0.1% to 0.5% formic acid solution is used as mobile phase B for gradient elution, and the flow rate is 0.8 ml to 1.2 ml per minute; the column temperature is 35 to 40°C; the detection wavelength is 300 to 350 nm, and the theoretical plate number should be not less than 3000 to 8000 calculated according to the chlorogenic acid peak; the gradient elution conditions are:

[0014] 0-5min, mobile phase A 5→10%, mobile phase B 95→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 comprises the following operations:

[0019] Preparation of reference solution Take 0.5-2.0g of Hantao leaf reference medicinal material, add 25-50ml of 50% methanol, heat and reflux for 1-2 hours, cool, shake, filter, and take the filtrate as the reference medicinal material reference solution; take appropriate amounts of neochlorogenic acid reference substance, chlorogenic acid reference substance and cryptochlorogenic acid reference substance, accurately weigh, add methanol to make a mixed solution containing 20-40μg of each per 1ml, as the reference substance reference solution;

[0020] Preparation of the test solution Take the formula granules of Hantao leaves, grind them into powder, take about 0.2-1g, weigh accurately, add 25-50ml of 50% methanol accurately, weigh the weight, ultrasonically treat for 30-60 minutes, cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, and take the filtrate to obtain;

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

[0022] There should be 4 characteristic peaks in the chromatogram of the test sample, and the retention times of the 4 characteristic peaks in the chromatogram of the reference medicinal material should correspond to each other. 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 is calculated, and the relative retention time should be within the range of ±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 spectrum detection method is: determination according to the high performance liquid chromatography method in accordance with the general rules of the Chinese Pharmacopoeia;

[0024] Chromatographic conditions and system suitability experiment The chromatographic column is filled with octadecylsilane bonded silica gel, acetonitrile is used as mobile phase A, and 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°C; the detection wavelength is 326nm, and the theoretical plate number should be not less than 8000 calculated based on the chlorogenic acid peak; the gradient elution conditions are:

[0025] 0-5min, mobile phase A 5→10%, mobile phase B 95→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 comprises the following operations:

[0030] Preparation of reference solution: Take 1.0g of Hantao leaf reference medicinal material, add 25ml of 50% methanol, heat and reflux for 2 hours, cool, shake, filter, and take the filtrate as the reference medicinal material reference solution; take appropriate amounts of new chlorogenic acid reference substance, chlorogenic acid reference substance and cryptochlorogenic acid reference substance, accurately weigh, add methanol to make a mixed solution containing 20μg of each per 1ml, as the reference substance solution;

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

[0032] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, measure according to HPLC working conditions, and record the chromatogram from 0 to 38 min.

[0033] There should be 4 characteristic peaks in the chromatogram of the test sample, and the retention times of the 4 characteristic peaks in the chromatogram of the reference medicinal material should correspond to each other. 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 is calculated, and the relative retention time should be within the range of ±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 Rules 0512 of the Chinese Pharmacopoeia 2020 Edition).

[0035] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, 0.2% formic acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 1.0 ml per minute; the column temperature was 35°C; the detection wavelength was 326 nm. The theoretical plate number calculated based on the chlorogenic acid peak should be no less than 8000.

[0036]

[0037] Preparation of reference solution: Take an appropriate amount of chlorogenic acid reference substance, weigh it accurately, and add methanol to make a solution containing 20 μg per 1 ml.

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

[0039] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0040] Among them, the Hantao leaf formula granules are taken from 9000g of Hantao leaf slices, boiled in water, filtered, concentrated the filtrate, dried, crushed, added with appropriate amount of auxiliary materials, mixed and granulated to make 1000g.

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

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

[0043] Among them, the standard decoction of Chinese peach leaves is to take 100g of Chinese peach leaf slices, weigh it, add 10 times water to soak for 30 minutes, boil it with high heat first, then simmer it with low heat for 30 minutes, filter it while hot, and cool the filtrate in a cold water bath; add 8 times water for the second decoction, boil it with high heat, then simmer it with low heat for 20 minutes, filter it while hot, and cool the filtrate in a cold water bath; combine the two filtrates and concentrate them under reduced pressure at 65°C: concentrate to a relative density of 1.04~1.10 (65°C), divide the concentrate into vials, place them in a freeze dryer, and freeze-dry them.

[0044] Beneficial effects:

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

[0046] 2. The present invention also adds the detection of characteristic spectra on the basis of conventional detection items, which can more comprehensively reflect the types and quantities of chemical components of medicinal materials contained in the product. In particular, the characteristic spectra adopts HPLC detection method, 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 spectra detection. The quantitative determination of chlorogenic acid can be carried out when detecting the characteristic spectra, which saves time and effort.

[0047] 3. Through experiments, the detection method of the present invention is also applicable to Litsea cubeba medicinal materials, decoction pieces, standard decoctions, etc., and has a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1The following are the investigation diagrams of different developing agents, where sample 1 is the formula granules of Chinese peach leaves, and sample 2 is the control medicinal material of Chinese peach leaves; the developing agent of Figure A is: petroleum ether-ethyl acetate-formic acid (2:0.5:0.2), the developing agent of Figure B is: cyclohexane-ethyl acetate-formic acid (2:0.5:0.2), and the developing agent of Figure C is: chloroform-ethyl acetate-formic acid (2:0.5:0.2);

[0049] Figure 2 This is a graph showing the specificity of the characteristic spectrum of Hantao leaf formula granules, where Peak 1: Neochlorogenic acid; Peak 2 (S): Chlorogenic acid; Peak 3: Cryptochlorogenic acid

[0050] Figure 3 This is the result of the overall investigation of the Hantaoye formula granules

[0051] Figure 4 This is the separation effect diagram of different chromatographic columns of Hantao leaf formula particles

[0052] Figure 5 This is the reference characteristic spectrum of Hantao leaf formula granules, where peak 1: neochlorogenic acid; peak 2 (S): chlorogenic acid; peak 3: cryptochlorogenic acid

[0053] Figure 6 The specificity investigation diagram of the content determination of Hantao leaf formula granules

[0054] Figure 7 This is the reference characteristic spectrum of the medicinal material of Hantao leaf, where Peak 1: neochlorogenic acid; Peak 2: chlorogenic acid; Peak 3: cryptochlorogenic acid

[0055] Figure 8 This is the reference characteristic spectrum of the standard decoction of Hantao leaves, where Peak 2 (S): Chlorogenic acid DETAILED DESCRIPTION

[0056] In order to make the technical scheme and technical effect of the present invention clearer, the technical scheme in the embodiment of the present invention is described clearly and completely. The embodiments described below are part of the embodiments of the present invention, rather than all of the embodiments. In combination with the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0057] Example 1 Establishment of TLC Identification Method for Peach Leaf Formula Granules

[0058] The present invention uses Chinese peach leaf control medicinal material as a positive control and uses chloroform-ethyl acetate-formic acid (2:0.5:0.2) as a developing agent to investigate the thin layer identification method.

[0059] 1.1 Solution preparation

[0060] (1) Preparation of test solution

[0061] Take 1g of Hantao leaf granules, grind them into powder, add 20mL of water, condense and reflux for 30min, filter, add 30mL of anhydrous ethanol to precipitate, 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 medicinal material solution

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

[0064] (3) Preparation of negative solution

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

[0066] 1.2 Chromatographic conditions

[0067] Carrier: Silica gel G254 thin layer plate

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

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

[0070] 1.3 Methodological considerations

[0071] 1.3.1 Developing agent selection

[0072] ① Petroleum ether-ethyl acetate-formic acid (2:0.5:0.2), ② Cyclohexane-ethyl acetate-formic acid (2:0.5:0.2), ③ Chloroform-ethyl acetate-formic acid (2:0.5:0.2) were used as developing agents for development. After development was complete, the samples were observed under 365nm fluorescence. The results showed that the petroleum ether and cyclohexane spots were piled up, the separation was incomplete, and the main spots were not obvious. Chloroform had better separation, and the main spots were clear and bright. Therefore, chloroform-ethyl acetate-formic acid (2:0.5:0.2) was selected as the developing agent for the thin layer identification of Hantao leaf formula particles. See for details. Figure 1 .

[0073] 1.3.2 Sampling quantity inspection

[0074] 1μL, 5μL, 10μL of the test solution and 0.5μL, 1μL, 1.5μL of the Hantao leaf control medicinal material solution were respectively taken and spotted on the same silica gel G254 thin layer plate, developed according to the above thin layer chromatography conditions, taken out, dried, and placed under ultraviolet light (365nm) for inspection. Results According to the separation and clarity of the spots, the Hantao leaf formula granules thin layer identification was determined. 5μL of the Hantao leaf formula granules test solution and 1μL of the Hantao leaf control medicinal material solution were taken for development.

[0075] 1.3.3 Specificity inspection: 10 μl of each of the test solution, negative solution and control solution of Hantao leaf formula granules were taken and spotted on the same silica gel G254 thin layer plate, developed according to the above thin layer chromatography conditions, taken out, dried, and placed under ultraviolet light (365nm) for inspection. Results In the chromatogram of the test sample, spots of the same color appeared at the corresponding position of the chromatogram of the control medicinal material. There was no interference from the negative solution at the corresponding position, and the specificity was good.

[0076] 1.3.4 Durability inspection:

[0077] 1.3.4.1 Investigation at different temperatures and humidities: 5.0 μL of the test solution and 1 μL of the control medicinal solution of Chinese peach leaves were taken and spotted on the same silica gel G254 thin layer plate, and chloroform-ethyl acetate-formic acid (2:0.5:0.2) was used as the developing agent. The plates were developed at low temperature (10°C), normal temperature (25°C), normal humidity (40%), and high humidity (80%), respectively. The plates were taken out, dried, and placed under ultraviolet light at 365 nm for inspection. Results The temperature and humidity had no obvious effect on the separation and clarity of the Chinese peach leaf samples, and the durability was good at different temperatures and humidities.

[0078] 1.3.4.2 Inspection of thin layer plates from different manufacturers: 5.0 μL of the test solution and 1.0 μL of the control medicinal material solution were respectively taken and spotted on silica gel G254 thin layer plates from different manufacturers (Qingdao Ocean Silica G254 Plate, Yantai Chemical Silica G254 Plate, Merck Silica G254 Plate), and chloroform-ethyl acetate-formic acid (2:0.5:0.2) was used as the developing agent. The plates were taken out, dried, and placed under ultraviolet light at 365 nm for inspection. Results Using silica gel G254 thin layer plates from different manufacturers, the test solution of Hantaoye Formula Granules showed spots of roughly the same color at the corresponding position of the control medicinal material solution, and the separation effect was relatively good. The results showed that thin layer plates from different manufacturers had no significant effect on the identification of Hantaoye Formula Granules.

[0079] 1.3.4.3 Durability test by different personnel: Different inspectors, at different times, used the same method to prepare the test sample and control medicinal material solutions, and took 5.0 μL of the test sample solution and 1.0 μL of the control medicinal material solution, respectively, and spotted them on the same silica gel G254 thin layer plate, developed with chloroform-ethyl acetate-formic acid (2:0.5:0.2) as the developing agent, took out, dried, and placed under ultraviolet light at 365 nm for inspection. Results Different inspectors used the same inspection method, which had no significant effect on the thin layer identification, and the thin layer identification method had good durability.

[0080] The thin-layer chromatography identification method of the present invention has good specificity and durability after methodological investigation, and can be used for thin-layer chromatography identification of Hantao leaf formula granules.

[0081] Example 2 Establishment of a characteristic spectrum detection method for Chinese peach leaf formula granules

[0082] Determined by high performance liquid chromatography (General Rules 0512 of the Chinese Pharmacopoeia 2020 Edition).

[0083] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, 0.2% formic acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 1.0 ml per minute; the column temperature was 35°C; the detection wavelength was 326 nm. The theoretical plate number calculated based on the chlorogenic acid peak should be no less than 8000.

[0084]

[0085] Preparation of reference solution Take 1.0g of Hantao leaf reference medicinal material, place it in a stoppered conical flask, add 25ml of 50% methanol, heat and reflux for 2 hours, cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution. Take appropriate amounts of neochlorogenic acid reference substance, chlorogenic acid reference substance and cryptochlorogenic acid reference substance, accurately weigh them, add methanol to make a mixed solution containing 20μg of each per 1ml, as the reference substance reference solution.

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

[0087] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0088] There should be 4 characteristic peaks in the chromatogram of the test sample, and the retention time should correspond to the 4 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 1 to 3 should correspond to the retention time 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 is calculated, and the relative retention time should be within the range of ±10% of the specified value. The specified value is: 1.31 (peak 4).

[0089] Example 3 Methodological verification of the characteristic spectrum detection method of peach leaf formula granules

[0090] The practicability and durability of the characteristic spectrum detection method of Example 2 were verified from the aspects of specificity, integrity, precision, repeatability, solution stability, durability, etc. of the detection method.

[0091] In the examples, all raw reagents and materials are commercially available, and the experimental methods without specific conditions are conventional methods and conventional conditions well known in the art, or are based on the conditions recommended by the instrument manufacturer.

[0092] 3.1 Instruments, reagents and drugs 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 1 / 10,000 balance (METTLER TOLEDO, Switzerland); AE-240 1 / 100,000 balance (METTLER TOLEDO, Switzerland); micropipette (Eppendorf, Germany); KQ-300DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); SYG-1-7 electric constant temperature 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 drugs: chlorogenic acid (batch number: 110753-202119, purity: 96.3%, China Food and Drug Inspection Institutes); neochlorogenic acid, cryptochlorogenic acid (batch number: AZCC2416, AFCE3004, purity: 99.96%, 99.69%, Chengdu Aifa Biotechnology Co., Ltd.); Hantao leaf control medicinal material (batch number: 121575-201503, China Food and Drug Inspection Institutes).

[0096] 3.2 Exclusivity Investigation

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

[0098] The results showed that the test sample chromatogram had the same chromatographic peak at the corresponding retention time as the reference chromatogram, and there was no interference from the blank solvent, indicating that the method had good specificity.

[0099] 3.3 Overall Examination

[0100] Take an appropriate amount of Hantao leaf formula granules, grind them into powder, take about 0.2g, prepare the test solution according to the test solution preparation method under Example 2, on the basis of the chromatographic conditions under Example 2, maintain the elution gradient at the initial ratio, extend the elution time by one time, record the chromatogram, and the results are shown in Figure 3 .

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

[0102] 3.4 Precision investigation

[0103] Take an appropriate amount of Hantao leaf formula granules, grind them into powder, take about 0.2g, prepare the test solution according to the test solution preparation method under Example 2, and perform sampling analysis according to the chromatographic conditions under Example 2. The same test solution is sampled 6 times continuously. 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 the same sample was injected six times continuously, 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 0.01-0.10%, and the relative peak area was in the range of 0.18-0.45%, all less than 1%, indicating that the instrument had good precision.

[0105] 3.5 Repeatability study

[0106] Take an appropriate amount of Hantao leaf formula granules, grind them into powder, take about 0.2g, weigh accurately, prepare the test solution according to the test solution preparation method in Example 2, and conduct 6 parallel injections and analysis according to the chromatographic conditions in Example 2. Take 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.

[0107] The results showed that 6 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 had good repeatability.

[0108] 3.6 Stability investigation

[0109] Take an appropriate amount of Hantao leaf formula granules, grind them into powder, take about 0.2g, prepare the test solution according to the test solution preparation method under Example 2, inject 10μL at 0, 2, 4, 8, 12, and 24h after sample preparation according to the chromatographic conditions under Example 2, and record the chromatogram. 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 the characteristic peaks and the S peak were in the range of 0.02-0.13%, and the relative peak areas were in the range of 0.13-0.37%, both less than 1%, indicating that the test solution was stable within 24 hours.

[0111] 3.7 Durability test

[0112] 3.7.1 Chromatographic column inspection:

[0113] 1: ACE Excel 5C18-PFP (ACE, 4.6×250mm, 5μm); 2: Hypesil BDS C18 (Elite, 4.6×250mm, 5μm); 3: XB-C18 (Welch, 4.6×250mm, 5μm) three chromatographic columns were used to analyze the Hantao leaf formula granule samples. The results are shown in Figure 4 .

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

[0115] 3.7.2 Flow rate investigation

[0116] The ACE Excel 5C18-PFP (column length 250mm, inner diameter 4.6mm, particle size 5μm) chromatographic column was used to investigate the sample separation effect at flow rates of 0.9mL / min, 1.0mL / min, and 1.1mL / min. The results showed that the analysis method had good separation of each characteristic peak when the flow rate varied in the range of 0.9-1.1mL / min. Smaller flow rate changes can meet the system suitability requirements, and 1.0mL / min was used in subsequent studies.

[0117] 3.7.3 Column temperature investigation

[0118] Three temperatures of 30℃, 35℃ and 40℃ were investigated respectively. The results showed that the peak separation was good when the column temperature was between 30℃ and 40℃, and the durability was good within the range of 30℃ to 40℃. According to the peak elution of the sample, 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 powder, take 0.2g, prepare the test solution according to the test solution preparation method under Example 2, inject and measure according to the chromatographic conditions under Example 2, record the spectrum, and the results are shown in the table below.

[0121] Relative retention time of characteristic spectra of three batches of Hantaoye formula granules

[0122]

[0123] The HPLC characteristic spectra of the three batches of Hantaoye formula granules were matched using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System", and the No. 2 chlorogenic acid peak was used as the reference peak S. The reference characteristic spectrum of the Hantaoye formula granules was generated by the average method. Figure 5 .

[0124] The final standard for the characteristic spectrum of Hantao leaves is: the test sample chromatogram should show 4 characteristic peaks, and the retention time should correspond to the 4 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 1 to 3 should correspond to the retention time 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 is calculated, and the relative retention time should be within the range of ±10% of the specified value. The specified value is: 1.31 (peak 4).

[0125] Example 4 Investigation of the pretreatment method of the test sample in the method for determining the content of the peach leaf formula granules

[0126] The extraction solvent, extraction method and extraction time of the sample pretreatment in the content determination of Hantao leaf formula granules were investigated to determine the best sample pretreatment method.

[0127] 4.1 Investigation of different extraction solvents

[0128] Take an appropriate amount of Hantao leaf formula granules, grind them finely, take 0.2g, put them in a stoppered conical flask, parallel 4 groups, accurately add 30% methanol, 50% methanol, 70% methanol, methanol 25mL each, weigh the weight, respectively, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, let cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, and take the filtrate. Accurately aspirate 10μL of each test solution, inject it into the liquid chromatograph, measure it according to the chromatographic conditions of Example 2, and calculate the content of chlorogenic acid. The results are shown in the table below:

[0129]

[0130] The results showed that when 50% methanol and 70% methanol were used as the extraction solvent, the content of chlorogenic acid was higher, and 50% methanol was finally selected as the extraction solvent.

[0131] 4.2 Investigation of different extraction methods

[0132] Take an appropriate amount of Hantao leaf formula granules, grind them into powder, take 0.2g, put them in a stoppered conical flask, accurately add 25mL of 50% methanol, seal it tightly, weigh it, ultrasonically treat it (power 250W, frequency 40kHz), heat it under reflux, and shake it for 30 minutes, let it cool, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate. Accurately take 10μL of each test solution, inject it into the liquid chromatograph, and measure it according to the chromatographic conditions of Example 2 to calculate the content of chlorogenic acid. The results are shown in the following table:

[0133]

[0134] The results showed that the content of chlorogenic acid in ultrasonic extraction and reflux extraction was not much different. Considering its convenience and speed, ultrasonic extraction was finally selected.

[0135] 4.3 Investigation of different extraction times

[0136] Take an appropriate amount of Hantao leaf formula granules, grind them into powder, take 0.2g, put them in a stoppered conical bottle, accurately add 25mL of 50% methanol, seal it tightly, weigh it, sonicate it (power 250W, frequency 40kHz) for 15min, 30min, 45min, 60min, take it out, let it cool, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate. Accurately take 10μL of each test solution, inject it into the liquid chromatograph, and determine it according to the chromatographic conditions of Example 2 to calculate the content of chlorogenic acid.

[0137]

[0138] The results showed that after the extraction time was 30 minutes, the chlorogenic acid content did not change much, indicating that the extraction was complete in 30 minutes. Therefore, the extraction time was considered to be 30 minutes.

[0139] 4.4 Determination of test solution preparation

[0140] According to 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 into powder, take 0.2g, put it in a stoppered conical flask, accurately add 25mL of 50% methanol, weigh the weight, ultrasonically treat (power 250W, frequency 40kHz) for 30min, cool, make up 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 peach leaf formula granules

[0142] Determined by high performance liquid chromatography (General Rules 0512 of the Chinese Pharmacopoeia 2020 Edition).

[0143] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, 0.2% formic acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 1.0 ml per minute; the column temperature was 35°C; the detection wavelength was 326 nm. The theoretical plate number calculated based on the chlorogenic acid peak should be no less than 8000.

[0144]

[0145] Preparation of reference solution: Take an appropriate amount of chlorogenic acid reference substance, weigh it accurately, and add methanol to make a solution containing 20 μg per 1 ml.

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

[0147] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0148] Example 6 Methodological Verification of the Content Determination Method of Peach Leaf Formula Granules

[0149] The practicability and accuracy of the content determination method of Example 5 were verified from the aspects of the linear range, precision, repeatability, solution stability, and sample recovery rate of the detection method.

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

[0151] 6.1 Exclusivity

[0152] Take the Hantao leaf formula granules and prepare the test solution according to the method of "Preparation of the test solution" in Example 5; take 50% methanol as the blank solvent solution. Take the above test solution, blank solvent, and chlorogenic acid reference substance and measure them according to the chromatographic conditions of Example 5. Figure 6 .

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

[0154] 6.2 Linear Investigation

[0155] Accurately weigh the chlorogenic acid reference substance, place it in a 100mL volumetric flask, and add methanol to make a stock solution containing 116.5μg of chlorogenic acid reference substance per 1mL. Accurately measure 0.1mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL, and 5.0mL of the above-mentioned reference substance mixed stock solution and place them in 10mL volumetric flasks, add methanol to the scale, and shake well to obtain reference substance application solutions of different concentrations. Accurately draw 10μL of each reference substance application solution of different concentrations, and measure it in turn according to the chromatographic conditions under the top of Example 5, and record the chromatographic peak area. Use the peak area as the ordinate (y), the reference substance concentration as the abscissa (x), and draw a standard curve.

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

[0157] 6.3 Precision test

[0158] Take an appropriate amount of Hantao leaf formula granules, grind them into powder, take 0.2g, and prepare the test solution according to the test solution preparation method under Example 5. According to the chromatographic conditions under Example 5, perform sample injection analysis, inject 6 times continuously, record the chromatographic peak area, and calculate RSD.

[0159] The results showed that the same sample solution was injected six times, and the RSD value of the chlorogenic acid peak area was 0.39%, less than 1%, indicating that the instrument had good precision.

[0160] 6.4 Repeatability test

[0161] Take an appropriate amount of Hantao leaf formula granules, grind them into powder, take 0.2g, and prepare 6 parallel portions. According to the preparation method of the test solution in Example 5, prepare the test solution. According to the chromatographic conditions in Example 5, perform sample analysis, record the chromatographic peak area, and calculate its content and RSD. The results are shown in the following table:

[0162]

[0163] The results showed that when 6 test solutions were injected in parallel, 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 Hantao leaf formula granules, grind them into powder, take 0.2g, and prepare the test solution according to the test solution preparation method in Example 5. According to the chromatographic conditions in Example 5, inject and analyze at 0h, 2h, 4h, 8h, 12h, and 24h, and record the chromatographic peak area. Calculate the peak area and RSD respectively.

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

[0167] 6.6 Recovery test

[0168] Take an appropriate amount of a granule sample with a known content (chlorogenic acid content: 1.79 mg / g), grind it, take 0.1 g, and weigh six parallel portions accurately. Add a reference solution of chlorogenic acid with the same content as 0.1 g of the sample, respectively, and prepare a sample recovery test solution according to the test solution preparation method under Example 5. According to the chromatographic conditions under Example 5, inject 10 μL respectively, and calculate the recovery rate and RSD with the following formula. The results are shown in the following table:

[0169]

[0170]

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

[0172] 6.7 Sample determination

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

[0174]

[0175] Example 7 Establishment of the characteristic spectrum and content determination method of Chinese peach leaf medicinal material

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

[0177] Chromatographic conditions and system suitability test are the same as [Content determination]

[0178] Preparation of reference solution Take about 1g of Chinese peach leaf reference medicinal material, place it in a stoppered conical flask, add 25ml of 50% methanol, heat and reflux for 2 hours, take it out, cool it, shake it well, filter it, and take the filtrate as the reference medicinal material reference solution. Take an appropriate amount of chlorogenic acid reference substance, accurately weigh it, add methanol to make a solution containing 40μg of chlorogenic acid per 1ml, which is used as the reference substance solution.

[0179] Preparation of test solution is the same as that of [Content determination]

[0180] The determination method is to accurately aspirate 10μl of the control medicinal material reference and the test sample solution, inject them into the liquid chromatograph, and determine the result.

[0181] There should be 4 characteristic peaks in the chromatogram of the test sample, and the retention time should correspond to the 4 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 the S peak. The relative retention time of peak 1, peak 3, peak 4 and S peak is calculated. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.65 (peak 1), 1.08 (peak 3), 1.27 (peak 4). The reference characteristic spectrum of Hantao leaf medicinal material is shown in Figure 7 .

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

[0183] Chromatographic conditions and system suitability test: The chromatographic column was filled with octadecylsilane bonded silica gel (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, 0.2% formic acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 1.0 ml per minute; the column temperature was 35°C; the detection wavelength was 326 nm. The theoretical plate number calculated based on the chlorogenic acid peak should be no less than 8000.

[0184]

[0185] Preparation of reference solution: Take an appropriate amount of chlorogenic acid reference substance, weigh it accurately, and add methanol to make a solution containing 40 μg per 1 ml.

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

[0187] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0188] Example 8 Establishment of characteristic spectrum and content determination method of Chinese peach leaf slices

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

[0190] Chromatographic conditions and system suitability test are the same as [Content determination]

[0191] Preparation of reference solution Take about 1g of Chinese peach leaf reference medicinal material, place it in a stoppered conical flask, add 25ml of 50% methanol, heat and reflux for 2 hours, take it out, cool it, shake it well, filter it, and take the filtrate as the reference medicinal material reference solution. Take an appropriate amount of chlorogenic acid reference substance, accurately weigh it, add methanol to make a solution containing 40μg of chlorogenic acid per 1ml, which is used as the reference substance solution.

[0192] Preparation of test solution is the same as that of [Content determination]

[0193] The determination method is to accurately aspirate 10μl of the control medicinal material reference and the test sample solution, inject them into the liquid chromatograph, and determine the result.

[0194] There should be 4 characteristic peaks in the chromatogram of the test sample, and the retention time should correspond to the 4 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 the S peak. The relative retention time of peak 1, peak 3, peak 4 and S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: 0.65 (peak 1), 1.09 (peak 3), 1.26 (peak 4).

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

[0196] Chromatographic conditions and system suitability test: The chromatographic column was filled with octadecylsilane bonded silica gel (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, 0.2% formic acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 1.0 ml per minute; the column temperature was 35°C; the detection wavelength was 326 nm. The theoretical plate number calculated based on the chlorogenic acid peak should be no less than 8000.

[0197]

[0198] Preparation of reference solution: Take an appropriate amount of chlorogenic acid reference substance, weigh it accurately, and add methanol to make a solution containing 40 μg per 1 ml.

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

[0200] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0201] Example 9 Establishment of characteristic spectrum and content determination method of standard decoction of Chinese peach leaves

[0202] [Characteristic spectrum] Determined by high performance liquid chromatography (General Rules 0512 of the Chinese Pharmacopoeia 2020 Edition).

[0203] Chromatographic conditions and system suitability test are the same as those for chlorogenic acid.

[0204] Preparation of reference solution Take about 1g of Chinese peach leaf reference medicinal material, place it in a stoppered conical flask, add 25ml of 50% methanol, heat and reflux for 2 hours, take it out, cool it, shake it well, filter it, and take the filtrate as the reference medicinal material reference solution. Take an appropriate amount of chlorogenic acid reference substance, accurately weigh it, add methanol to make a solution containing 30μg of chlorogenic acid per 1ml, which is used as the reference substance solution.

[0205] Preparation of test solution is the same as that of [Content determination]

[0206] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0207] There should be 4 characteristic peaks in the chromatogram of the test sample, and the retention time should correspond to the 4 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 the S peak. The relative retention time of peak 1, peak 3, peak 4 and S peak is calculated. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.65 (peak 1), 1.09 (peak 3), 1.25 (peak 4). The reference characteristic spectrum of the standard decoction of Hantao leaves is shown in Figure 8 .

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

[0209] Chromatographic conditions and system suitability test: Octadecyl bonded silane silica gel was used as filler (column length was 250 mm, inner diameter was 4.6 mm, particle size was 5 μm); acetonitrile was used as mobile phase A, 0.1% formic acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 1.0 ml per minute; the column temperature was 35°C; the detection wavelength was 326 nm. The theoretical plate number calculated based on the chlorogenic acid peak should be no less than 8000.

[0210]

[0211]

[0212] Preparation of reference solution: Take an appropriate amount of chlorogenic acid reference substance, weigh it accurately, and add 50% methanol to make a solution containing 30 μg per 1 ml.

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

[0214] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

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

Claims

1. A method for detecting a granule of a Chinese peach leaf formula, wherein the granule is prepared by boiling, concentrating, drying, crushing and granulating Chinese peach leaf pieces; the detection method comprises properties, identification, inspection, extract, characteristic spectrum and content determination, and is characterized in that: The characteristic spectrum and content determination detection method is to use high performance liquid chromatography for detection. The working conditions of the high performance liquid chromatography are: the chromatographic column is filled with octadecylsilane bonded silica gel, acetonitrile is used as mobile phase A, and 0.1% to 0.5% formic acid solution is used as mobile phase B for gradient elution, and the flow rate is 0.8 to 1.2 ml per minute; the column temperature is 35 to 40°C; the detection wavelength is 300 to 350 nm, and the theoretical plate number calculated according to the chlorogenic acid peak should be not less than 3000 to 8000; the gradient elution conditions are: 0-5min, 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 content determination detection method further comprises preparing a reference solution or preparing a control solution, preparing a test solution and injecting a sample for determination, wherein: Preparation of reference solution Take 0.5-2.0g of Hantao leaf reference medicinal material, add 25-50ml of 50% methanol, heat and reflux for 1-2 hours, cool, shake, filter, and take the filtrate as the reference medicinal material reference solution; take appropriate amounts of neochlorogenic acid reference substance, chlorogenic acid reference substance and cryptochlorogenic acid reference substance, accurately weigh, add methanol to make a mixed solution containing 20-40μg of each per 1ml, as the reference substance reference solution; Preparation of reference solution Take an appropriate amount of chlorogenic acid reference substance, weigh accurately, and add methanol to make a solution containing 20-40 μg per 1 ml. Preparation of the test solution: Take the formula granules of Hantao leaves, grind them into powder, take about 0.2-1g, weigh accurately, add 25-50ml of 50% methanol accurately, weigh the weight, ultrasonically treat for 30-60 minutes, cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, and take the filtrate to obtain; Determination method: Accurately pipette 5-10 μl of reference solution or control solution and test solution respectively, inject into liquid chromatograph, and determine according to HPLC working conditions.

2. The detection method according to claim 1, characterized in that: The working conditions of the high performance liquid chromatography method are: using octadecylsilane bonded silica gel as a filler, acetonitrile as a mobile phase A, and 0.2% formic acid solution as a mobile phase B for gradient elution, with a flow rate of 1.0 ml per minute; the column temperature is 35° C.; the detection wavelength is 326 nm, and the theoretical plate number calculated based on the chlorogenic acid peak should be no less than 8000; The preparation method of the reference solution is as follows: take 1.0 g of Chinese peach leaf reference medicinal material, add 25 ml of 50% methanol, heat and reflux for 2 hours, cool, shake, filter, and take the filtrate as the reference medicinal material reference solution; take appropriate amounts of new chlorogenic acid reference substance, chlorogenic acid reference substance and cryptochlorogenic acid reference substance, accurately weigh them, add methanol to make a mixed solution containing 20 μg of each per 1 ml, as the reference substance reference solution; The preparation method of the test solution is as follows: take the formula granules of Chinese peach leaves, grind them into powder, take about 0.2g, accurately weigh, accurately add 25ml of 50% methanol, weigh the weight, ultrasonically treat for 30 minutes, cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, and take the filtrate to obtain the solution.

3. The detection method according to claim 1, characterized in that: The characteristic spectrum is determined by: accurately taking 10 μl of the reference solution and the test solution, respectively, injecting them into a liquid chromatograph, and determining them according to the working conditions of a high performance liquid chromatography.

4. The detection method according to any one of claims 1 to 3, characterized in that: The test sample chromatogram of the characteristic spectrum should present 4 characteristic peaks, and the retention times of the 4 characteristic peaks in the chromatogram of the control medicinal material reference substance should correspond to each other, among which peaks 1 to 3 should correspond to the retention times of the corresponding reference substance peaks, and the peak corresponding to the chlorogenic acid reference substance peak is the S peak. The relative retention time of peak 4 and peak S is calculated, and the relative retention time should be within the range of ±10% of the specified value; the specified value is: the relative retention time of peak 4 is 1.

31.

5. The detection method according to claim 1, characterized in that: The content determination method is: accurately aspirate 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, measure according to the working conditions of the high performance liquid chromatography, record the chromatogram, and calculate the content of chlorogenic acid by the external standard method.

6. The detection method according to claim 1, characterized in that: The identification method is as follows: take 0.5-2g of Chinese peach leaf formula granules, grind them into powder, add 20-50ml of water, reflux for 30-60min, filter, add 30-50ml of ethanol for alcohol precipitation, filter, evaporate the filtrate to dryness, add 1ml of methanol to dissolve the residue as the test solution; take another 0.5-2g of Chinese peach leaf control medicinal material, and prepare the control medicinal material solution in the same way; according to the thin layer chromatography test of the Chinese Pharmacopoeia, respectively absorb 2-10μl of the test solution and 1-5μl of the control medicinal material solution, spot them on the same silica gel GF254 thin layer plate, use chloroform-ethyl acetate-formic acid = 2:0.5:0.2 as the developing agent, develop, take out, dry, and examine under a 365nm ultraviolet lamp; in the test sample chromatogram, spots of the same color appear at the corresponding positions of the control medicinal material chromatogram.

7. The detection method according to claim 6, characterized in that: The identification method is as follows: take 1g of Chinese peach leaf formula granules, grind them into powder, add 20ml of water, reflux for 30min, filter, add 30ml of anhydrous ethanol for alcohol precipitation, refrigerate for 4h, filter, evaporate the filtrate to dryness, add 1ml of methanol to dissolve the residue as the test solution; take another 1g of Chinese peach leaf control medicinal material, and prepare the control medicinal material solution in the same way; according to the thin layer chromatography test of the Chinese Pharmacopoeia, respectively absorb 5μl of the test solution and 1μl of the control medicinal material solution, spot them on the same silica gel GF254 thin layer plate, use chloroform-ethyl acetate-formic acid = 2:0.5:0.2 as the developing agent, develop, take out, dry, and examine under a 365nm ultraviolet lamp; in the test sample chromatogram, spots of the same color appear at the corresponding positions of the control medicinal material chromatogram.

8. The detection method according to claim 1, characterized in that: The extract detection method is as follows: take the Hantao leaf formula granules, grind them into powder, take 2g, accurately weigh, accurately add 100ml of ethanol, and determine it by the hot soaking method under the alcohol-soluble extract determination method of the Chinese Pharmacopoeia.

9. The detection method according to any one of claims 1 to 8, characterized in that: The Hantao leaf formula granules are prepared by taking Hantao leaf slices, decocting with water, filtering, concentrating the filtrate into a clear paste, drying or drying, crushing, adding pharmaceutically acceptable excipients, mixing, and granulating. Each 1g of the formula granules is equivalent to 9.0g of Hantao leaf slices.

10. Use of the detection method according to claims 1 to 8, characterized in that: The detection method is used in the quality detection of Chinese peach leaf medicinal materials, Chinese peach leaf decoction pieces, Chinese peach leaf standard decoction, and Chinese peach leaf formula granules.

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