CN112763618B

CN112763618B - Method for identifying characteristic spectrum of hairyvein agrimony formula particles

Info

Publication number: CN112763618B
Application number: CN202011594894.3A
Authority: CN
Inventors: 周厚成; 胡昌江; 费文波; 付林; 高锐; 周维
Original assignee: Sichuan New Green Pharmaceutical Technology Development Co ltd
Current assignee: Sichuan New Green Pharmaceutical Technology Development Co ltd
Priority date: 2020-12-29
Filing date: 2020-12-29
Publication date: 2022-03-01
Anticipated expiration: 2040-12-29
Also published as: CN112763618A

Abstract

The invention discloses a method for identifying a characteristic spectrum of hairyvein agrimony formula particles, which comprises the steps of taking 0.5g of hairyvein agrimony formula particles, precisely weighing, placing in a conical flask with a plug, precisely adding an extraction solvent, weighing, extracting for 20-40min, cooling, weighing again, complementing the weight loss by the extraction solvent, shaking up, filtering, and taking a subsequent filtrate; taking a proper amount of quercetin, chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid as reference substances, precisely weighing, and adding 50% methanol to obtain solution; octadecylsilane chemically bonded silica is used as a filler, the column length is 250mm, the inner diameter is 4.6mm, and the particle size of the silica is 5 microns; a diode array detector with detection wavelength of 250-360 nm; column temperature: 20-40 ℃; flow rate: 0.8-1.2 mL/min; taking methanol-acetonitrile as a mobile phase A and 0.1% formic acid solution as a mobile phase B, and performing gradient elution; precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

Description

Method for identifying characteristic spectrum of hairyvein agrimony formula particles

Technical Field

The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for identifying a characteristic spectrum of agrimony formula granules.

Background

Herba et Gemma Agrimoniae is dry aerial part of Agrimonia pilosa Ledeb of Rosaceae. Harvesting stems and leaves in summer and autumn when they are flourishing, removing impurities, and drying. The product has a length of 50-100 cm, and is totally white and soft. The lower part of the stem is cylindrical, the diameter of the stem is 4-6 mm, the stem is reddish brown, the upper part of the stem is square cylindrical, four sides of the stem are slightly concave, the stem is greenish brown, and the stem is provided with longitudinal furrows, ridge lines and nodes; light weight, hard quality, easy breaking and hollow section. Single feathered, compound leaves growing, dark green bags, shrinking and curling; the texture is crisp and fragile; the blades are 2 in size, are alternatively grown on a blade shaft, have larger tip leaflets, are oval or oblong after being flattened, have pointed tips at the tips and wedge-shaped bases, and have sawteeth at the edges; leaf, stem, oblique oval. The general inflorescence is slender, the lower part of the calyx is cylindrical, the upper part of the calyx cylinder is provided with a hook thorn, the tip is split, and the petals are yellow. Light smell, slightly bitter taste. At present, a part of documents are used for carrying out content determination research on components of hairyvein agrimonia herb and bud formula particles, and the documents are less aiming at characteristic map qualitative research.

Disclosure of Invention

In order to solve the problems, the invention provides a method for identifying the characteristic spectrum of the agrimony formula granules.

The purpose of the invention is realized by the following technical scheme:

a method for identifying a characteristic spectrum of hairyvein agrimonia herb formula particles is characterized by comprising the following steps:

(1) and preparing a test solution: weighing herba et Gemma Agrimoniae granule 0.5g, precisely weighing, placing in conical flask with plug, precisely adding extraction solvent, weighing, extracting for 20-40min, cooling, weighing again, supplementing loss weight with extraction solvent, shaking, filtering, and collecting filtrate;

(2) and preparing a reference substance solution: a. precisely weighing herba et Gemma Agrimoniae 1.0g, placing in conical flask with plug, precisely adding extraction solvent, sealing plug, weighing, extracting for 20-40min, cooling, weighing again, supplementing with 50% methanol, shaking, filtering, and collecting filtrate; precisely weighing appropriate amount of quercetin, chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid as reference substances, and adding 50% methanol to obtain solution;

(3) and chromatographic conditions: a chromatographic column: octadecylsilane chemically bonded silica is used as a filler, the column length is 250mm, the inner diameter is 4.6mm, and the particle size of the silica is 5 microns; a detector: a diode array detector with detection wavelength of 250-360 nm; column temperature: 20-40 ℃; flow rate: 0.8-1.2 mL/min; mobile phase: taking methanol-acetonitrile as a mobile phase A and 0.1% formic acid solution as a mobile phase B, and performing gradient elution;

(4) and testing: precisely sucking 10 μ L of reference solution and sample solution respectively, injecting into liquid chromatograph, and measuring.

Preferably, in step (1), the sample extraction solvent is methanol or 50% methanol or 70% methanol or water or 50% ethanol.

Preferably, in the step (1), the extraction mode is reflux extraction or ultrasonic extraction.

Preferably, in the step (1), the extraction time is 20min or 30min or 40 min.

Preferably, in the step (2), the reference substance extraction solvent is 50% methanol.

Preferably, in the step (2), a proper amount of quercetin, chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid reference substances are precisely weighed, and 50% methanol is added to prepare a solution containing 30 mug of each 1mL, so as to obtain the product.

Preferably, in step (3), the chromatographic conditions are as follows: a chromatographic column: octadecylsilane chemically bonded silica is used as a filler, the column length is 250mm, the inner diameter is 4.6mm, and the particle size of the silica is 5 microns; a detector: a diode array detector, wherein the detection wavelength is 324nm, and the number of theoretical plates is not less than 5000 according to the calculation of a quercitrin peak; column temperature: 30 ℃; flow rate: 1.0 mL/min; mobile phase: taking methanol-acetonitrile as a mobile phase A and 0.1% formic acid solution as a mobile phase B, and performing gradient elution; the gradient elution procedure was:

the mobile phase A rises from 8% (v/v) to 10% (v/v) within 0-5 min;

5-20min, the mobile phase A rises from 10% (v/v) to 16% (v/v);

20-25min, keeping the mobile phase A at 16% (v/v);

the mobile phase A rises from 16% (v/v) to 23% (v/v) within 25-30 min;

30-50min, the mobile phase A rises from 23% (v/v) to 30% (v/v);

the mobile phase A is increased from 30% (v/v) to 32% (v/v) within 50-55 min;

the mobile phase A rises from 32% (v/v) to 55% (v/v) within 55-70 min.

Preferably, the characteristic spectrum should exhibit 10 characteristic peaks, wherein peak 1 should have the same retention time as the corresponding reference peak, wherein the peak corresponding to the chlorogenic acid reference is the S1 peak, the relative retention times of peaks 1-6 and the S1 peak are calculated to be within ± 5% of the specified values, and the specified values for each peak are: peak 20.882, peak 31.000, peak 41.099, peak 51.444, peak 61.483; the peak corresponding to the quercitrin reference was the S2 peak, and the relative retention times of peaks 7-10 and S2 were calculated to be within ± 5% of the specified value between the relative retention, which was peak 70.972, peak 81.000, peak 91.023, and peak 101.457.

The beneficial effects of this technical scheme are as follows:

(1) the HPLC characteristic spectrum detection method can comprehensively reflect the types and the amounts of chemical components contained in the agrimony formula particles, and can more effectively embody the integrity and the comprehensive action of the components in the agrimony formula particles.

(2) According to the HPLC characteristic spectrum detection method of the agrimony formula particles, corresponding agrimony reference medicinal materials and chemical reference substances are used as references, the chemical component corresponding relation between the agrimony formula particles and the agrimony reference medicinal materials is shown on the one hand, and the method is simple, good in stability and high in precision; on the other hand, the property of the agrimony formula particle is possibly similar to that of other traditional Chinese medicine formula particles, and whether the agrimony formula particle is the agrimony cannot be identified from the property of the agrimony formula particle.

Drawings

FIG. 1 is a graph of the ultraviolet absorption spectrum of quercetin;

FIG. 2 is a graph of the ultraviolet absorption spectrum of chlorogenic acid;

FIG. 3 is a diagram of the UV absorption spectrum of neochlorogenic acid;

FIG. 4 is a graph of the UV absorption spectrum of cryptochlorogenic acid;

FIG. 5 is a chromatogram of different wavelengths of granule of herba Agrimoniae;

FIG. 6 is a chromatogram of agrimony formula granules at different column temperatures;

FIG. 7 is a chromatogram of Agrimonia pilosa formula granules at different flow rates;

FIG. 8 is a chromatogram of a granule formulation of Agrimonia pilosa Ledeb in different solvents;

FIG. 9 is a chromatogram of the agrimony formula granules obtained by different extraction methods;

FIG. 10 is a chromatogram of Agrimonia pilosa formula granules taken at different extraction times;

FIG. 11 shows the chromatogram peak identification of the agrimony formula granule;

FIG. 12 is a chromatogram of a granule formulation of Agrimonia pilosa Ledeb using different instruments;

FIG. 13 is a chromatogram of agrimony formula granules using different chromatographic columns;

FIG. 14 is a graph showing the characteristic spectrum of the granule prepared from 3 batches of Agrimonia pilosa Ledeb;

FIG. 15 is a control profile of Agrimonia pilosa Ledeb formula granules; peak 1: green tea acid; peak 3: chlorogenic acid (S1); peak 4: cryptochlorogenic acid; peak 8: quercetin (S2).

Detailed Description

1. Laboratory instruments and materials

High performance liquid chromatograph: waters model 2695-e model 2998 high performance liquid chromatograph, Agilent model 1260 high performance liquid chromatograph, Shimadzu model LC-20AT high performance liquid chromatograph;

an electronic balance: ME204E/02, MS205DU, XP26 (Mettler-Tollido instruments, Inc.);

an ultra-pure water machine: cell type 1810A (Shanghai Mohler scientific instruments, Inc.);

an ultrasonic cleaner: model KQ5200DB (600W, 40 KHz; ultrasonic instruments, Inc. of Kunshan);

a chromatographic column: agilent ZORBAX Eclipse XDB-C185 μm 4.6X 250mm, Waters Xbridge C185 μm 4.6X 250mm, Kromasil C185 μm 4.6X 250 mm.

Quercetin (Chinese food and drug assay researcher, batch No.: 111538-201606, content is calculated by 90.6%),

chlorogenic acid (China institute for food and drug assay, batch No. 110753-201415, content in 96.2%),

chlorogenic acid (Dopperfeld Biotechnology Co., Ltd., lot number: 17062003, content 98%),

cryptochlorogenic acid (Dopperfeld Biotechnology Co., Ltd., lot number: 17061401, content 98%),

herba et Gemma Agrimoniae control drug (China institute for testing and testing food and drug, lot number: 120966-201307),

acetonitrile, methanol (SIGMA, chromatographically pure); the water is ultrapure water, and other reagents are analytically pure.

Agrimony formula particle GR-XHC-04, agrimony formula particle GR-XHC-01, GR-XHC-02, GR-XHC-03.

2. Method for preparing high-efficiency liquid effect detection

Preparing a reference solution, namely taking 1.0g of a hairyvein agrimony reference medicinal material, precisely weighing, placing the hairyvein agrimony reference medicinal material in a conical flask with a plug, precisely adding 25mL of 50% methanol, sealing the plug, weighing, carrying out ultrasonic treatment (the power is 600W, the frequency is 40kHz) for 30 minutes, cooling, weighing again, complementing the weight loss by using 50% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the reference solution; taking appropriate amount of quercetin, chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid as reference substances, precisely weighing, and adding 50% methanol to obtain solution containing 30 μ g per 1 ml.

Preparing test solution by accurately weighing 0.5g of the product, placing in a conical flask with a plug, accurately adding 25ml of 50% methanol, weighing, ultrasonically treating (power 600W, frequency 40kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking, filtering, and collecting the subsequent filtrate.

Chromatographic conditions are as follows: a chromatographic column: octadecylsilane chemically bonded silica is used as a filler, the column length is 250mm, the inner diameter is 4.6mm, and the particle size of the silica is 5 microns; a detector: a diode array detector, wherein the detection wavelength is 324nm, and the number of theoretical plates is not less than 5000 according to the calculation of a quercitrin peak; column temperature: 30 ℃; flow rate: 1.0 mL/min; mobile phase: taking methanol-acetonitrile as a mobile phase A and 0.1% formic acid solution as a mobile phase B, and performing gradient elution; the gradient elution procedure was:

the mobile phase A rises from 8% (v/v) to 10% (v/v) within 0-5 min;

5-20min, the mobile phase A rises from 10% (v/v) to 16% (v/v);

20-25min, keeping the mobile phase A at 16% (v/v);

the mobile phase A rises from 16% (v/v) to 23% (v/v) within 25-30 min;

30-50min, the mobile phase A rises from 23% (v/v) to 30% (v/v);

the mobile phase A is increased from 30% (v/v) to 32% (v/v) within 50-55 min;

the mobile phase A rises from 32% (v/v) to 55% (v/v) within 55-70 min.

The determination method comprises precisely sucking 10 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, and determining.

3. Inspection of chromatographic conditions

3.1 wavelength investigation

On the basis of the experimental conditions, a diode array detector is utilized to respectively carry out full-wave-band scanning on the quercetin, the chlorogenic acid, the neochlorogenic acid, the cryptochlorogenic acid and the test solution, and chromatograms of the test solution at wavelengths of 250nm, 272nm, 286nm, 300nm, 324nm and 360nm are respectively extracted. As shown in fig. 1-5.

The result shows that the information content of the chromatographic peak is larger when the detection wavelength is 324nm, the base line of the chromatogram is more stable, and therefore, the detection wavelength is determined to be 324 nm.

3.2 column temperature investigation

Based on the experimental conditions set forth above, the column temperatures were examined at 20 ℃, 30 ℃ and 40 ℃. As shown in fig. 6 and table 1.

TABLE 1 column temperature investigation-ratio of characteristic peaks to retention time

The result shows that when the column temperature is 30 ℃, the chromatogram has symmetrical peak shapes, good separation degrees, peak relative retention time less than 8 percent, and good durability. Therefore, the column temperature is determined to be 30 ℃ for subsequent investigation.

3.3 investigation of flow Rate

Based on the experimental conditions set forth above, the flow rates were examined at 0.8mL/min, 1mL/min, and 1.2mL/min, respectively. As shown in fig. 7 and table 2.

Table 2 flow rate investigation-characteristic peak relative retention time

The result shows that when the flow rates are respectively 0.8ml/min, 1.0ml/min and 1.2ml/min, the relative retention time RSD of each characteristic peak is 0.06-12.58%, and when the flow rate is 1.0ml/min, the chromatogram peak shape is good and the separation degree is moderate. Therefore, the flow rate was determined to be 1.0 ml/min.

4. Investigation of preparation of test solution

4.1 examination of extraction solvent

The results of using methanol, 50% methanol, 70% methanol, water and 50% ethanol as extraction solvents respectively show that the chromatographic peak information obtained by using 50% methanol as the extraction solvent is large and the separation degree is good, so that the method selects 50% methanol as the extraction solvent and is consistent with the content measurement. As shown in fig. 8.

4.2 examination of extraction methods

Compared with the reflux extraction and the ultrasonic extraction, the chromatographic peak information is approximately the same, and finally, the ultrasonic extraction with simple operation is selected for extraction. As shown in fig. 9.

4.3 investigation of extraction time

Ultrasonic extraction was compared for 20min, 30min, 40min, and finally ultrasonic extraction was selected for 30 min. As shown in fig. 10.

4.4 method for preparing final definite sample

Taking 0.5g of the product granules (batch number: GR-XHC-04), grinding, accurately weighing, placing in a conical flask with a plug, accurately adding 25ml of 50% methanol, weighing, carrying out ultrasonic treatment for 30 minutes, cooling to room temperature, weighing again, complementing the weight loss by 50% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the product.

5. Methodology investigation

5.1 chromatographic peak assignment

Preparation of a test solution: preparing the test solution of the agrimony formula granules according to the experimental conditions.

Preparation of reference solutions

Preparation of control solutions: taking appropriate amount of quercetin, chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid as reference substances, precisely weighing, and adding 50% methanol to obtain solution containing 30 μ g per 1 ml.

Preparation of reference drug solution: precisely weighing herba et Gemma Agrimoniae control material about 1.0g, placing into conical flask with plug, precisely adding 50% methanol 25ml, sealing, weighing, ultrasonic treating (power 600W, frequency 40kHz) for 30min, cooling, weighing again, supplementing lost weight with 50% methanol, shaking, filtering, and collecting filtrate.

Preparation of negative control solution: negative control solution of agrimony-lacking formula granules is prepared according to the experimental conditions set forth above.

And (5) positioning the peak of the characteristic spectrum of the agrimony formula granules. As shown in fig. 11.

5.2 precision test

Taking a test solution of herba et Gemma Agrimoniae formula granule (batch number: GR-XHC-04), continuously injecting sample for 6 times according to a formulated experimental method, 10 mul each time, and calculating relative retention time and relative peak area of each characteristic peak. As shown in tables 3 and 4.

TABLE 1 precision investigation-Retention time

TABLE 2 precision investigation-Peak area

The results show that the instrument is accurate.

5.3 repeatability test

6 parts of hairyvein agrimony formula particles (batch number: GR-XHC-04) are precisely weighed, and are prepared and measured according to a proposed experimental method. As shown in table 5.

TABLE 3 repeatability test-relative retention time ratio

The result shows that the method has good repeatability.

5.4 intermediate precision investigation

5.41 investigation of different instruments

Based on the experimental conditions, accurately weighing two parts of herba et Gemma Agrimoniae formula granule (batch number: GR-XHC-04) respectively, preparing test solution, and measuring on Agilent 1260, Shimadzu LC-20AT and Waters2695-e2998 type high performance liquid chromatograph respectively. As shown in fig. 12, table 6 and table 7.

TABLE 4 Instrument durability test-relative Retention time ratio

TABLE 5 Instrument durability test-relative Peak area ratio

The results show that the RSD of each characteristic peak relative retention time is less than 4% when the test sample is detected by the 3 instruments.

5.42 different personnel and time review

Based on the experimental conditions, two parts of agrimony formula granules (batch number: GR-XHC-04) are precisely weighed by different persons (A, B) at different time (T1, T2) respectively to prepare a test sample for determination. As shown in tables 8 and 9.

TABLE 6 ratio of person and time review-relative retention time

TABLE 7 personnel and time survey-relative Peak area ratios

The result shows that different people can determine the same sample at different time, and the method has better stability.

5.5 durability test

5.51 durability test of column

On the basis of the experimental conditions set forth above, the chromatographic columns Agilent ZORBAX XDB-C18, Waters Xbridge C18, and Kromasil C18 were examined, respectively. As shown in fig. 13, table 10 and table 11.

TABLE 8 column durability test-relative retention time ratio

TABLE 9 column durability test-relative peak area ratio

The results show that the RSD of the characteristic peak relative retention time is 0.66-8.50% and the RSD of the characteristic peak relative peak area is 2.02-20.28% when the sample is detected by the 3 chromatographic columns.

5.52 stability test

Based on the experimental conditions, the same test solution is taken and respectively measured at 0h, 3h, 6h, 9h, 12h and 24 h. As shown in table 12.

Table 10 stability investigation-retention time

The results show that the RSD of the retention time of the corresponding characteristic peak is between 0.27 and 1.45 percent, and the sample solution is stable within 24 hours.

5.53 methodology summary

When peak 8 was selected as the S peak, the RSD of each stage versus retention time is shown in table 27. RSD for each phase versus retention time when peak 3 was selected as the S peak. As shown in tables 13 and 14.

TABLE 11 RSD values when Peak 8 is the S peak

TABLE 12 RSD values when Peak 3 is the S peak

In summary, when peak 3 is selected as the S peak from peaks 2 to 6, the durability satisfies the requirement of + -5%; peaks 7-10 the durability satisfied ± 5% when peak 8 was selected as the S peak, so peak 3 was selected as the S1 and peak 8 was selected as the S2 peak.

In summary, the RSD of each characteristic peak relative retention time meets the requirements in the above studies, and the method is good. The 10 characteristic peaks are included in the subsequent examination.

The technical solutions for achieving the objects of the present invention are further illustrated by the following specific examples, and it should be noted that the technical solutions claimed in the present invention include, but are not limited to, the following examples.

Example 1

Verification result of 3 batches of agrimony formula granules

And (3) measuring the characteristic spectrum of the 3 batches of samples of the product by a drawn-up method, and calculating the relative retention time and the relative peak area. As shown in fig. 14, table 15 and table 16.

TABLE 133 Agrimonia pilosa formula relative Retention time

Relative peak area of granule of Table 143 batches of Agrimonia pilosa

According to the principle that the relative retention time is stable, samples of each batch can be detected, and the peaks are relatively high, 10 peaks with good repeatability are selected as characteristic peaks. The results show that when peak 3 and peak 8 are regarded as S peaks, the relative retention time RSD of the characteristic peak of 3 batches of agrimony formula particles is less than 1%.

Example 2

Making of specified value limit of relative retention time

The methodology for each survey item and the validation results are summarized in table 17:

TABLE 15 methodology results RSD% summary criteria for each project-relative retention time

From the above table, it is understood that the column temperature, flow rate, and relative retention time of different instruments on peak 1 have the greatest effect, so the flow rate is set to 1ml/min, and the specified value of relative retention time of each peak is temporarily set to 5% in order to increase the reproducibility and applicability of the method.

Finally, the following steps are provided: the test sample should have 10 characteristic peaks, wherein peak 1 should have the same retention time as the corresponding reference peak, wherein the peak corresponding to the chlorogenic acid reference is the S1 peak, and the relative retention time of peaks 1-6 and the S1 peak is calculated and should be within + -5% of the specified value. The specified values are: 0.882 (peak 2), 1.000 (peak 3, S1), 1.099 (peak 4), 1.444 (peak 5), 1.483 (peak 6); the peak corresponding to the quercitrin reference was the S2 peak, and the relative retention times of peaks 7-10 and S2 were calculated to be within ± 5% of the specified values between the relative retentions, which were 0.972 (peak 7), 1.000 (peak 8, S2), 1.023 (peak 9), and 1.457 (peak 10).

Synthesizing 3 batches of agrimony formula particles by adopting a traditional Chinese medicine chromatography fingerprint similarity evaluation system (2012 edition), and establishing a control map of the characteristic map of the agrimony formula particles. As shown in fig. 15.

The above-mentioned embodiments are further described in detail for the purpose of illustrating the invention, the technical solutions and the advantages, it should be understood that the above-mentioned embodiments are only exemplary of the invention, and are not intended to limit the invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the invention should be included in the protection scope of the invention.

Claims

1. A method for identifying a characteristic spectrum of hairyvein agrimonia herb formula particles is characterized by comprising the following steps:

(2) and preparing a reference substance solution: precisely weighing herba et Gemma Agrimoniae control medicinal material 1.0g, placing in conical flask with plug, precisely adding extraction solvent, sealing plug, weighing, extracting for 20-40min, cooling, weighing again, supplementing loss weight with extraction solvent, shaking, filtering, and collecting filtrate; precisely weighing appropriate amount of quercetin, chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid as reference substances, and adding 50% methanol to obtain solution;

(3) the chromatographic conditions were as follows: a chromatographic column: octadecylsilane chemically bonded silica is used as a filler, the column length is 250mm, the inner diameter is 4.6mm, and the particle size of the silica is 5 microns; a detector: a diode array detector, wherein the detection wavelength is 324nm, and the number of theoretical plates is not less than 5000 according to the calculation of a quercitrin peak; column temperature: 30 ℃; flow rate: 1.0 mL/min; mobile phase: taking methanol-acetonitrile as a mobile phase A and 0.1% formic acid solution as a mobile phase B, and performing gradient elution; the gradient elution procedure was:

the mobile phase A rises from 8% (v/v) to 10% (v/v) within 0-5 min;

5-20min, the mobile phase A rises from 10% (v/v) to 16% (v/v);

20-25min, keeping the mobile phase A at 16% (v/v);

the mobile phase A rises from 16% (v/v) to 23% (v/v) within 25-30 min;

30-50min, the mobile phase A rises from 23% (v/v) to 30% (v/v);

the mobile phase A is increased from 30% (v/v) to 32% (v/v) within 50-55 min;

the time is 55-70min, and the mobile phase A is increased from 32% (v/v) to 55% (v/v);

2. The method for identifying the characteristic spectrum of the agrimony formula granules according to claim 1, wherein the method comprises the following steps: in the step (1), the sample extraction solvent is methanol or 50% methanol or 70% methanol or water or 50% ethanol.

3. The method for identifying the characteristic spectrum of the agrimony formula granules according to claim 1, wherein the method comprises the following steps: in the step (1), the extraction mode is reflux extraction or ultrasonic extraction.

4. The method for identifying the characteristic spectrum of the agrimony formula granules according to claim 1, wherein the method comprises the following steps: in the step (1), the extraction time is 20min or 30min or 40 min.

5. The method for identifying the characteristic spectrum of the agrimony formula granules according to claim 1, wherein the method comprises the following steps: in the step (2), the reference substance extraction solvent is 50% methanol.

6. The method for identifying the characteristic spectrum of the agrimony formula granules according to claim 1, wherein the method comprises the following steps: in the step (2), a proper amount of quercetin, chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid reference substances are precisely weighed, and 50% methanol is added to prepare a solution containing 30 micrograms per 1mL, so as to obtain the product.

7. The method for identifying the characteristic spectrum of the agrimony formula granules according to claim 1, wherein the method comprises the following steps: the characteristic map should present 10 characteristic peaks, wherein peak 1 should have the same retention time as the corresponding reference peak, wherein the peak corresponding to the chlorogenic acid reference is the S1 peak, the relative retention time of peaks 1-6 and S1 peak should be calculated, the relative retention time should be within ± 5% of the specified value, and the specified value of each peak is: peak 20.882, peak 31.000, peak 41.099, peak 51.444, peak 61.483; the peak corresponding to the quercitrin reference was the S2 peak, and the relative retention times of peaks 7-10 and S2 were calculated to be within ± 5% of the specified value between the relative retention, which was peak 70.972, peak 81.000, peak 91.023, and peak 101.457.