UPLC identification method for rhizoma dryopteris crassirhizomae, rhizoma osmundae, cycas fern rhizome and cyrtomium fortunei and formula granules

Abstract

The invention discloses a UPLC (Ultra Performance Liquid Chromatography) identification method for medicinal materials and formula granules of rhizoma dryopteris crassirhizomae, rhizoma osmundae, sago fern rhizome and cyrtomium fortunei, which comprises the following steps: determining specified values of relative retention time of the medicinal materials and the formula granules of rhizoma dryopteris crassirhizomae, rhizoma osmundae, sago fern rhizome and cyrtomium fortunei through a high performance liquid chromatography; the relative retention time of the corresponding test solution is within + / -10% of the corresponding specified value of the rhizoma dryopteris crassirhizomae, rhizoma osmundae, cynoglossum cyrtomium rhizome and cyrtomium fortunei medicinal materials and formula granules. The method can identify rhizoma dryopteris crassirhizomae, rhizoma osmundae, cycas fern rhizome and cyrtomium fortunei medicinal materials and formula granules according to the existence of characteristic peaks.

Description

Technical Field

[0001] The invention relates to the technical field of identification of medicinal materials and formula granules, and specifically proposes a UPLC identification method for the medicinal materials and formula granules of Cyperus rotundus, Cyperus rotundus, Cyperus rotundus and Cyperus rotundus. Background Art

[0002] Dryopteris crassipes Dryopteris crassirhizoma Nakai The dried rhizome and petiole residues of Osmanthus fragrans Osmunda japonica Thunb The dried rhizome and petiole residues of . ; Cycad fern is a plant of the Pteridaceae family Cycad fern Brainea insignis(Hook.)J.Sm. Rhizome of the plant Cyperus rotundus; Cyperus rotundus is a plant of the Dryopteris family Cyrtomium fortunei J. Sm. The four medicinal materials are similar in appearance and are mostly referred to as "Guanzhong" in the market, making it difficult to distinguish them.

[0003] Traditional Chinese medicine granules are made by extracting single herbal pieces using water as the solvent, and then using physical methods such as solid-liquid separation, concentration, drying, and granulation to produce the granules. These granules retain multiple active ingredients from the medicinal materials.

[0004] In the prior art, the identification research of Rhizoma Cyrthospermi, Rhizoma Ostreae, Rhizoma Cycas and Rhizoma Scutellariae mainly adopts thin layer identification and characteristic spectrum identification method, such as CN110426487A adopts thin layer identification method, uses 4 kinds of thin layer chromatography conditions to inspect 18 spots in Rhizoma Cyrthospermi medicinal materials, but this method does not distinguish between various Rhizoma Cyrthospermi. CN119936285A distinguishes Rhizoma Cyrthospermi from other Rhizoma Cyrthospermi by measuring the content of flavonoid acid AB. This method can be used for distinguishing Rhizoma Cyrthospermi from confused products, but there are multiple confused product contents of 0, and it is impossible to distinguish between confused products. CN119470671A distinguishes Rhizoma Cyrthospermi from Rhizoma Ostreae and Rhizoma Cycas by establishing characteristic spectrum. This method does not distinguish Rhizoma Cyrthospermi, and can only be used for Rhizoma Cyrthospermi to distinguish from other Rhizoma Cyrthospermi, and it is impossible to distinguish Rhizoma Ostreae, Rhizoma Cycas and Rhizoma Scutellariae. Summary of the Invention

[0005] In view of this, the present invention proposes a method for simultaneously identifying the medicinal materials and formula granules of Cyperus rotundus, Cycas revoluta, and Cyperus rotundus, which effectively identifies the medicinal materials and formula granules of Cyperus rotundus, Cyperus rotundus, and Cyperus rotundus through high performance liquid chromatography.

[0006] The technical solution of the present invention is achieved as follows: The present invention provides a UPLC identification method for the medicinal materials and formula granules of Cyperus rotundus, Cyperus rotundus, and Cyperus rotundus, comprising the following steps: (1) Prepare a reference solution containing protocatechuic acid, protocatechuic aldehyde, neochlorogenic acid, trans-coumaric acid-4-glucoside, caffeic acid-4-O-glucoside, chlorogenic acid, and cryptochlorogenic acid; (2) Prepare the test solution: Take the sample to be identified, add 30% methanol solution, ultrasonicate and filter; (3) Perform UPLC analysis: Mobile phase A: methanol, B: 0.1% phosphoric acid; Gradient elution program: At 0-12 min, the volume ratio of the mobile phase A in the mobile phase is 5-6%, and the volume ratio of the mobile phase B in the mobile phase is 95-94%; At 12-21.6 min, the volume ratio of the mobile phase A in the mobile phase is 6-8%, and the volume ratio of the mobile phase B in the mobile phase is 94-92%; At 21.6-30 min, the volume ratio of the mobile phase A in the mobile phase is 8-12%, and the volume ratio of the mobile phase B in the mobile phase is 92-88%; At 30-48 min, the volume ratio of the mobile phase A in the mobile phase is 12-20%, and the volume ratio of the mobile phase B in the mobile phase is 88-80%; Column: C18, particle size 1.7 μm; Detection wavelength: 300nm; (4) Identification: Rhizoma Cynanchum: Based on the retention time of the chlorogenic acid peak, there is a characteristic peak with a relative retention time of 1.24±10%; Osmanthus fragrans: Based on the retention time of the chlorogenic acid peak, there is a characteristic peak at a relative retention time of 1.55±10%, and there is no characteristic peak at a relative retention time of 1.24±10%; Cycad fern Cyperus rotundus: Based on the retention time of the chlorogenic acid peak, there are characteristic peaks at relative retention times of 1.37±10% and 1.55±10%, and no characteristic peaks at relative retention times of 1.04±10% and 1.24±10%. Xiao Guanzhong: Based on the retention time of the protocatechuic acid peak, there is a characteristic peak with a relative retention time of 2.73±10%, and there are no chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid related peaks.

[0007] In some embodiments, the chromatography column has a size of 150 mm×2.1 mm.

[0008] In some embodiments, the flow rate of the mobile phase through the chromatography column is 0.25 ml / min.

[0009] In some embodiments, the column temperature of the chromatography column is 25-30°C.

[0010] In some embodiments, in the preparation of the test solution, 0.2-1.0 g of medicinal material powder is taken, 5-20 ml of 30% methanol is added, and ultrasonic power is 250 W, frequency is 35 kHz, and treatment is carried out for 20 minutes.

[0011] In some embodiments, in the preparation of the test solution, 0.1-0.5 g of the formula particles are taken, 5-20 ml of 30% methanol is added, and the ultrasonic power is 250 W and the frequency is 35 kHz for 10 minutes.

[0012] In some embodiments, the preparation method of the reference solution includes taking an appropriate amount of protocatechuic acid, protocatechuic aldehyde, neochlorogenic acid, trans-coumaric acid-4-glucoside, caffeic acid-4-O-glucoside, chlorogenic acid, and cryptochlorogenic acid reference substances, accurately weighing them, and adding 30% methanol to prepare a mixed solution containing 10 μg each of protocatechuic acid, protocatechuic aldehyde, neochlorogenic acid, trans-coumaric acid-4-glucoside, caffeic acid-4-O-glucoside, chlorogenic acid, and cryptochlorogenic acid per 1 ml.

[0013] The present invention has the following beneficial effects compared to the prior art: This technical solution achieves the simultaneous and accurate identification of four easily confused medicinal materials, namely, Rhizoma Cyperi, Rhizoma Bauhiniae, Rhizoma Cycas, and Rhizoma Scutellariae, and their formula granules for the first time by establishing a UPLC characteristic spectrum identification system based on a combination of seven phenolic acid markers, a precise four-segment gradient elution program, and two-dimensional judgment criteria. This breaks through the limitations of existing technologies that can only distinguish a single type or rely on a single component. Through high-performance liquid chromatography, Rhizoma Cyperi, Rhizoma Bauhiniae, Rhizoma Cycas, and Rhizoma Scutellariae and their formula granules can be simultaneously identified. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0015] Figure 1 Characteristic spectra of reference substances for protocatechuic acid, protocatechuic aldehyde, neochlorogenic acid, trans-coumaric acid-4-glucoside, caffeic acid-4-O-glucoside, chlorogenic acid, and cryptochlorogenic acid; Figure 2 This is the characteristic spectrum of the Mianma Guanzhong formula granules in Example 1 of the present invention; Figure 3 This is the characteristic spectrum of the Ziqi Guanzhong formula granules in Example 2 of the present invention; Figure 4This is the characteristic spectrum of the Cycas revoluta granules in Example 3 of the present invention; Figure 5 This is the characteristic spectrum of the Xiaoguanzhong formula granules in Example 4 of the present invention; Figure 6 This is the characteristic spectrum of the medicinal material of Mianma Guanzhong in Example 5 of the present invention; Figure 7 This is the characteristic spectrum of the medicinal material of Ziqi Guanzhong in Example 6 of the present invention; Figure 8 This is the characteristic spectrum of the medicinal material Cycad Pteris Cyperus in Example 7 of the present invention; Figure 9 This is the characteristic spectrum of the medicinal material Rhizoma Cyperi in Example 8 of the present invention. DETAILED DESCRIPTION

[0016] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the present invention belong. If the definitions set forth in this section are contrary to or otherwise inconsistent with definitions set forth in the patents, patent applications, published patent applications, and other publications incorporated herein by reference, the definitions listed in this section take precedence over the definitions incorporated herein by reference.

[0018] Unless otherwise specified, the methods used in the following examples are conventional methods. The materials, reagents, and instruments used are conventional materials, reagents, and instruments in the art, unless otherwise specified, and can be obtained commercially by those skilled in the art.

[0019] When an amount, concentration or other value or parameter is expressed as a range, a preferred range or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper range limit or preferred value with any lower range limit or preferred value, regardless of whether the range is disclosed alone. For example, when a range "1 to 5" is disclosed, the described range should be interpreted as including the ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within the range. In the present specification and claims, range definitions may be combined and / or interchanged, and if not otherwise stated, such ranges include all subranges contained therein.

[0020] Example 1 This example provides an identification method for Mianma Guanzhong formula granules: Standard solution configuration: Take appropriate amounts of protocatechuic acid, protocatechuic aldehyde, neochlorogenic acid, trans-coumaric acid-4-glucoside, caffeic acid-4-O-glucoside, chlorogenic acid, and cryptochlorogenic acid reference substances, accurately weigh them, and add 30% methanol to prepare a mixed solution containing 10 μg of each component per 1 ml, which is used as the reference solution.

[0021] Configuration of the sample to be tested (Mianma Guanzhong Formula Granules): Take 0.5g of Mianma Guanzhong granules, grind them into powder, weigh them accurately, put them into a stoppered conical flask, accurately add 20ml of 30% methanol, stopper it tightly, and ultrasonically treat it for 10 minutes (power 250W, frequency 35kHz), cool it, shake it well, filter it, and take the filtrate as the test solution.

[0022] UPLC chromatographic conditions: Mobile phase: Phase A is methanol, phase B is 0.1% phosphoric acid; Gradient elution program: 0-12min: the volume ratio of phase A increases linearly from 5% to 6%, and the volume ratio of phase B decreases linearly from 95% to 94%; 12-21.6 min: The volume ratio of phase A increases linearly from 6% to 8%, and the volume ratio of phase B decreases linearly from 94% to 92%; 21.6-30min: The volume ratio of phase A increases linearly from 8% to 12%, and the volume ratio of phase B decreases linearly from 92% to 88%; 30-48min: The volume ratio of phase A increases linearly from 12% to 20%, and the volume ratio of phase B decreases linearly from 88% to 80%; Chromatographic column: octadecylsilane bonded silica gel (150 mm × 2.1 mm, 1.7 μm); Flow rate: 0.25ml / min; Column temperature: 25℃-30℃; Detection wavelength: 300nm; Injection volume: 2 μL; Theoretical plate number: not less than 10,000 calculated based on the chlorogenic acid peak.

[0023] Identification results: Reference material spectrum Figure 1 As shown, peaks 1-7 are protocatechuic acid, protocatechuic aldehyde, neochlorogenic acid, trans-coumaric acid-4-glucoside, caffeic acid-4-O-glucoside, chlorogenic acid, and cryptochlorogenic acid, respectively.

[0024] The characteristic spectrum of the test sample is as follows Figure 2 As shown, 12 characteristic peaks are present, of which 7 peaks correspond to the retention times of the 7 components of the reference solution. Taking the chlorogenic acid peak as the reference peak (S peak), the relative retention time values ​​calculated are: 0.16 (peak 1), 0.78 (peak 7), 1.04 (peak 9), 1.24 (peak 11), and 1.37 (peak 12).

[0025] It can be seen that the relative retention time of peak 11 falls within the range of ±10% of the specified value of 1.24, and it is determined to be Mianma Guanzhong formula granules.

[0026] Example 2 Identification of Ziqi Guanzhong Formula Granules Standard solution configuration: Same as Example 1.

[0027] Configuration of the sample to be tested (Ziqi Guanzhong Formula Granules): Take 0.1g of Ziqi Guanzhong granules, grind them into powder, weigh them accurately, put them into a stoppered conical flask, accurately add 5ml of 30% methanol, stopper it tightly, and treat it ultrasonically for 10 minutes (power 250W, frequency 35kHz), cool it, shake it well, filter it, and take the filtrate as the test solution.

[0028] UPLC chromatographic conditions: Same as Example 1.

[0029] Identification results: The characteristic spectrum of the test sample is as follows Figure 3 As shown, there are 12 characteristic peaks, 7 of which correspond to the retention times of the 7 components of the reference solution. Taking the chlorogenic acid peak as the reference peak (S peak), the relative retention time values ​​were calculated as follows: 0.16 (peak 1), 0.78 (peak 7), 1.04 (peak 9), 1.37 (peak 11), and 1.55 (peak 12).

[0030] The relative retention time 1.55 of peak 12 of the test sample falls within the specified value range of 1.55±10%, and there is no characteristic peak within the relative retention time range of S peak 1.24±10%, so it is determined to be Ziqi Guanzhong formula granules.

[0031] Example 3 Identification of Cycad Fern and Guanzhong Formula Granules Standard solution configuration: Same as Example 1.

[0032] Configuration of the sample to be tested (Cycad fern granules): Take 0.2g of Cycad Fern and Cyperus rotundus granules, grind them into powder, weigh them accurately, put them into a stoppered conical flask, accurately add 10ml of 30% methanol, stopper it tightly, and ultrasonically treat it for 10 minutes (power 250W, frequency 35kHz), cool it, shake it well, filter it, and take the filtrate as the test solution.

[0033] UPLC chromatographic conditions: Same as Example 1.

[0034] Identification results: The characteristic spectrum of the test sample is as follows Figure 4 As shown, it exhibits 10 characteristic peaks, 7 of which correspond to the retention times of the 7 components of the reference solution. Using the chlorogenic acid peak as the reference peak (S peak), the relative retention time values ​​were calculated to be: 0.16 (peak 1), 1.37 (peak 9), and 1.55 (peak 10).

[0035] The relative retention times of peaks 9 and 10 of the test sample fell within the ±10% range of the specified values ​​of 1.37 and 1.55, and there were no characteristic peaks within the relative retention time range of 1.04±10% and 1.24±10% of the S peak, and it was determined to be the Cycad Fern and Cyperus rotundus formula granules.

[0036] Example 4 Identification of Xiaoguanzhong Formula Granules Standard solution configuration: Same as Example 1.

[0037] Configuration of the sample to be tested (Xiaoguanzhong formula granules): Take 0.1g of Xiaoguanzhong granules, grind them into powder, weigh them accurately, put them into a stoppered conical flask, accurately add 5ml of 30% methanol, stopper it tightly, and treat it ultrasonically for 10 minutes (power 250W, frequency 35kHz), cool it, shake it well, filter it, and take the filtrate as the test solution.

[0038] UPLC chromatographic conditions: Same as Example 1.

[0039] Identification results The characteristic spectrum of the test sample is as follows Figure 5As shown, there are four characteristic peaks, two of which correspond to the retention times of the protocatechuic acid and protocatechualdehyde reference peaks. Using the protocatechuic acid peak as the reference peak (S peak), the relative retention time values ​​were calculated to be 0.56 (peak 1) and 2.73 (peak 4).

[0040] The relative retention time of peak 4 of the test sample fell within the specified value range of 2.73±10%, and there was no characteristic peak corresponding to the neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid reference substances, so it was determined to be Xiaoguanzhong formula granules.

[0041] Example 5 Identification of the medicinal material of Mianma Guanzhong Standard solution configuration: Same as Example 1.

[0042] Configuration of the sample to be tested (Cyperus rotundus medicinal material): Take 0.2g of the powder of the medicinal material of Mianma Guanzhong, accurately weigh it, place it in a stoppered conical flask, accurately add 5ml of 30% methanol, seal it tightly, and ultrasonically treat it for 20 minutes (power 250W, frequency 35kHz), cool it, shake it well, filter it, and take the filtrate as the test solution.

[0043] UPLC chromatographic conditions: Same as Example 1.

[0044] Identification results: The characteristic spectrum of the test sample is as follows Figure 6 As shown, it exhibits 11 characteristic peaks, 7 of which correspond to the retention times of the 7 components of the reference solution. Using the chlorogenic acid peak as the reference peak (S peak), the relative retention time values ​​calculated are: 0.78 (peak 6), 1.04 (peak 8), 1.24 (peak 10), and 1.37 (peak 11).

[0045] The relative retention time of peak 10 of the test sample fell within the specified value range of 1.24±10%, and was determined to be the medicinal material Rhizoma Cyrthospermi.

[0046] Example 6 Identification of the medicinal material Ziqi Guanzhong Standard solution configuration: Same as Example 1.

[0047] Configuration of the sample to be tested (Ziqi Guanzhong medicinal material): Take 0.2 g of the powder of Ziqi Guanzhong medicinal material, accurately weigh it, place it in a stoppered conical flask, accurately add 5 ml of 30% methanol, stopper it tightly, and ultrasonically treat it for 20 minutes (power 250 W, frequency 35 kHz). Cool it, shake it well, filter it, and take the filtrate as the test solution.

[0048] UPLC chromatographic conditions Same as Example 1.

[0049] Identification results: The characteristic spectrum of the test sample is as follows Figure 7 As shown, 11 characteristic peaks are present, of which 7 peaks correspond to the retention times of the 7 components of the reference solution. Using the chlorogenic acid peak as the reference peak (S peak), the relative retention time values ​​calculated are: 0.78 (peak 6), 1.04 (peak 8), 1.37 (peak 10), and 1.55 (peak 11).

[0050] The relative retention time of peak 11 of the test sample falls within the specified value of 1.55±10%, and there is no characteristic peak within the relative retention time of S peak of 1.24±10%, so it is determined to be the medicinal material Ziqi Guanzhong.

[0051] Example 7 Identification of the medicinal material Cycad fern Standard solution configuration: Same as Example 1.

[0052] Configuration of the sample to be tested (Cycad fern and Cycas cycad) Take 0.2 g of Cycad Pteris Cyperus powder, accurately weigh it, place it in a stoppered conical flask, accurately add 5 ml of 30% methanol, stopper it tightly, and ultrasonically treat it for 20 minutes (power 250 W, frequency 35 kHz), cool it, shake it well, filter it, and take the filtrate as the test solution.

[0053] UPLC chromatographic conditions: Same as Example 1.

[0054] Identification results: The characteristic spectrum of the test sample is as follows Figure 8 As shown, it exhibits eight characteristic peaks, six of which correspond to the retention times of protocatechuic acid, protocatechuic aldehyde, neochlorogenic acid, trans-coumaric acid-4-glucoside, caffeic acid-4-O-glucoside, and chlorogenic acid reference peaks. Using the chlorogenic acid peak as the reference peak (S peak), the relative retention time values ​​were calculated to be 1.37 (peak 7) and 1.55 (peak 8).

[0055] The relative retention times of peaks 7 and 8 of the test sample fell within the ±10% range of the specified values ​​of 1.37 and 1.55, and there were no characteristic peaks within the relative retention time range of 1.04±10% and 1.24±10% of the S peak, and it was determined to be the medicinal material Cycad fern.

[0056] Example 8 Identification of Xiao Guanzhong Medicinal Material Standard solution configuration: Same as Example 1.

[0057] Configuration of the sample to be tested (Xiao Guanzhong medicinal material): Take 0.2 g of Xiao Guanzhong medicinal material powder, accurately weigh it, place it in a stoppered conical flask, accurately add 5 ml of 30% methanol, seal it tightly, and ultrasonically treat it for 20 minutes (power 250 W, frequency 35 kHz), cool it, shake it well, filter it, and take the filtrate as the test solution.

[0058] UPLC chromatographic conditions: Same as Example 1.

[0059] Identification results: The characteristic spectrum of the test sample is as follows Figure 9 As shown, it exhibits three characteristic peaks, two of which correspond to the retention times of the protocatechuic acid and protocatechualdehyde reference peaks. Using the protocatechuic acid peak as the reference peak (S peak), the calculated relative retention time is 2.73 (peak 3).

[0060] The relative retention time of peak 3 of the test sample fell within the specified value range of 2.73±10%, and there was no characteristic peak corresponding to the neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid reference substances, so it was determined to be the medicinal material Xiao Guanzhong.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A UPLC identification method for the medicinal materials and formula granules of Cyperus rotundus, Cyperus rotundus, Cyperus rotundus, and Cyperus rotundus, characterized in that: The steps include: (1) Prepare a reference solution containing protocatechuic acid, protocatechuic aldehyde, neochlorogenic acid, trans-coumaric acid-4-glucoside, caffeic acid-4-O-glucoside, chlorogenic acid, and cryptochlorogenic acid; (2) Prepare the test solution: Take the sample to be identified, add 30% methanol solution, ultrasonicate and filter; (3) Perform UPLC analysis: Mobile phase A: methanol, B: 0.1% phosphoric acid; Gradient elution program: At 0-12 min, the volume ratio of the mobile phase A in the mobile phase is 5-6%, and the volume ratio of the mobile phase B in the mobile phase is 95-94%; At 12-21.6 min, the volume ratio of the mobile phase A in the mobile phase is 6-8%, and the volume ratio of the mobile phase B in the mobile phase is 94-92%; At 21.6-30 min, the volume ratio of the mobile phase A in the mobile phase is 8-12%, and the volume ratio of the mobile phase B in the mobile phase is 92-88%; At 30-48 min, the volume ratio of the mobile phase A in the mobile phase is 12-20%, and the volume ratio of the mobile phase B in the mobile phase is 88-80%; Column: C18, particle size 1.7 μm; Detection wavelength: 300nm; (4) Identification: Rhizoma Cynanchum: Based on the retention time of the chlorogenic acid peak, there is a characteristic peak with a relative retention time of 1.24±10%; Osmanthus fragrans: Based on the retention time of the chlorogenic acid peak, there is a characteristic peak at a relative retention time of 1.55±10%, and there is no characteristic peak at a relative retention time of 1.24±10%; Cycad fern Cyperus rotundus: Based on the retention time of the chlorogenic acid peak, there are characteristic peaks at relative retention times of 1.37±10% and 1.55±10%, and no characteristic peaks at relative retention times of 1.04±10% and 1.24±10%. Xiao Guanzhong: Based on the retention time of the protocatechuic acid peak, there is a characteristic peak with a relative retention time of 2.73±10%, and there are no chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid related peaks.

2. The method according to claim 1, wherein The size of the chromatographic column is 150 mm×2.1 mm.

3. The method according to claim 1, wherein The flow rate of the mobile phase through the column was 0.25 ml / min.

4. The method according to claim 1, wherein The column temperature was 25-30°C.

5. The method according to claim 1, wherein In the preparation of the test solution, 0.2-1.0 g of medicinal material powder was added to 5-20 ml of 30% methanol, and ultrasonic power was 250 W and frequency was 35 kHz for 20 minutes.

6. The method according to claim 1, wherein In the preparation of the test solution, take 0.1-0.5g of the formula granules, add 5-20ml of 30% methanol, and treat with ultrasound power of 250W and frequency of 35kHz for 10 minutes.

7. The method according to claim 1, wherein The preparation method of the reference solution includes taking an appropriate amount of protocatechuic acid, protocatechuic aldehyde, neochlorogenic acid, trans-coumaric acid-4-glucoside, caffeic acid-4-O-glucoside, chlorogenic acid, and cryptochlorogenic acid reference substances, accurately weighing them, and adding 30% methanol to prepare a mixed solution containing 10 μg each of protocatechuic acid, protocatechuic aldehyde, neochlorogenic acid, trans-coumaric acid-4-glucoside, caffeic acid-4-O-glucoside, chlorogenic acid, and cryptochlorogenic acid per 1 ml.

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