阅读说明：本技术 一种使君子配方颗粒特征图谱的构建及检测方法 (Construction and detection method of rangooncreeper fruit formula particle characteristic spectrum ) 是由 周厚成 胡昌江 辜新月 陈玉梅 宋媛 叶志萍 周维 于 2020-12-31 设计创作，主要内容包括：本发明公开了一种使君子配方颗粒特征图谱的构建及检测方法,属于中药分析及质量鉴别、控制技术领域。在本技术方案中,基于高效液相色谱仪建立特征图谱,并进行特征图谱的检测,能整体控制使君子配方颗粒中的特征成分,弥补了现有质量控制技术的不足,所得特征图谱中的特征峰信息量丰富,能有效保证使君子配方颗粒的整体质量的稳定性,使使君子配方颗粒的质量控制技术更为完善、科学；且本方法操作简单,具有精密度高、稳定性好、重复性好及准确度高等优势,为中药材质量鉴别提供有效依据。(The invention discloses a construction and detection method of a rangooncreeper fruit formula particle characteristic spectrum, and belongs to the technical field of traditional Chinese medicine analysis, quality identification and control. In the technical scheme, the characteristic spectrum is established based on a high performance liquid chromatograph, the detection of the characteristic spectrum is carried out, the characteristic components in the rangooncreeper fruit formula particles can be integrally controlled, the defects of the existing quality control technology are overcome, the information content of characteristic peaks in the obtained characteristic spectrum is rich, the stability of the overall quality of the rangooncreeper fruit formula particles can be effectively guaranteed, and the quality control technology of the rangooncreeper fruit formula particles is more perfect and scientific; the method is simple to operate, has the advantages of high precision, good stability, good repeatability, high accuracy and the like, and provides an effective basis for quality identification of the traditional Chinese medicinal materials.)

1. A construction method of a rangooncreeper fruit formula particle characteristic spectrum is characterized by comprising the following steps:

A. preparation of control solutions: dissolving ellagic acid control substance with 80% methanol to obtain solution containing 20 μ g per 1 mL;

B. preparation of a test solution: taking the product, grinding, weighing 1.0g, placing into a conical flask with a plug, adding 10mL of methanol, performing ultrasonic treatment for 30min, cooling, shaking up, filtering, and taking the subsequent filtrate to obtain the final product;

C. establishing: respectively injecting the obtained reference substance solution and the obtained test solution into a high performance liquid chromatograph, measuring, and establishing an HPLC characteristic spectrum;

the chromatographic conditions in the high performance liquid chromatograph meet:

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 is 5 mu m;

column temperature of the chromatographic column: 30 ℃;

mobile phase: methanol is taken as a mobile phase A, and 0.2% phosphoric acid aqueous solution is taken as a mobile phase B;

flow rate of mobile phase: 1.0 mL/min;

sample introduction amount: 10 mu L of the solution;

detection wavelength: 360 nm;

the number of theoretical plates is not less than 5000 calculated according to ellagic acid peak;

the mobile phase is eluted according to the gradient program:

0-18 min, wherein the mobile phase A is 23 → 41%, and the mobile phase B is 77 → 59%;

18-23 min, wherein the mobile phase A is 41 → 43%, and the mobile phase B is 59 → 57%;

23-35 min, wherein the mobile phase A is 43 → 52%, and the mobile phase B is 57 → 48%;

35-42 min, wherein the mobile phase A is 52 → 55%, and the mobile phase B is 48 → 45%;

42-60 min, 55 → 90% of mobile phase A and 45 → 10% of mobile phase B.

2. The method for constructing the rangooncreeper formula particle feature map of claim 1, wherein in the ultrasonic treatment process in the step B, the power is 600W, and the frequency is 40 kHz.

3. The method for constructing the characteristic spectrum of the rangooncreeper formula particle according to claim 2, wherein the HPLC characteristic spectrum of the rangooncreeper formula particle comprises six characteristic peaks, wherein the peak 2 is ellagic acid, the peak 2 is a reference peak S peak, and the relative retention time of each characteristic peak is as follows: peak 1 was 0.56, peak 3 was 1.12, peak 4 was 1.21, peak 5 was 1.42 and peak 6 was 1.63, the relative retention time was within ± 10% of the specified value.

4. A method for detecting the characteristic spectrum of rangooncreeper formula granules according to any one of claims 1 to 3, comprising the following steps:

s1, taking the product, grinding, weighing 1.0g, placing the product in a conical flask with a plug, adding 10mL of methanol, carrying out ultrasonic treatment for 30min, cooling, shaking up, filtering, and taking a subsequent filtrate to obtain the product;

s2, detection: injecting 10 μ L of the test solution obtained in step S1 into a high performance liquid chromatograph, measuring, comparing the high performance liquid chromatogram with the HPLC characteristic spectrum, and detecting the quality and authenticity of the fructus quisqualis formula granule;

the chromatographic conditions in the high performance liquid chromatograph meet:

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 is 5 mu m;

column temperature of the chromatographic column: 30 ℃;

mobile phase: methanol is taken as a mobile phase A, and 0.2% phosphoric acid aqueous solution is taken as a mobile phase B;

flow rate of mobile phase: 1.0 mL/min;

sample introduction amount: 10 mu L of the solution;

detection wavelength: 360 nm;

the number of theoretical plates is not less than 5000 calculated according to ellagic acid peak;

the mobile phase is eluted according to the gradient program:

0-18 min, wherein the mobile phase A is 23 → 41%, and the mobile phase B is 77 → 59%;

18-23 min, wherein the mobile phase A is 41 → 43%, and the mobile phase B is 59 → 57%;

23-35 min, wherein the mobile phase A is 43 → 52%, and the mobile phase B is 57 → 48%;

35-42 min, wherein the mobile phase A is 52 → 55%, and the mobile phase B is 48 → 45%;

42-60 min, 55 → 90% of mobile phase A and 45 → 10% of mobile phase B.

5. The method for detecting the rangooncreeper formula particle feature map of claim 4, wherein in the ultrasonic treatment process of the step S1, the power is 600W, and the frequency is 40 kHz.

Technical Field

The invention relates to a method for constructing a characteristic spectrum of a traditional Chinese medicine, in particular to a method for constructing and detecting a characteristic spectrum of rangooncreeper fruit formula granules, and belongs to the technical field of analysis, quality identification and control of traditional Chinese medicines.

Background

The fructus Quisqualis is dry mature fruit of Quisqualis indica L. of Combretaceae, is produced mainly in tropical and subtropical regions, has sweet taste and warm property, can astringe deficiency heat and check diarrhea and dysentery, and has effects of killing parasite and benefiting spleen and stomach.

The rangooncreeper fruit formula particle is prepared by carrying out water extraction, concentration, drying, granulation and the like on a single traditional Chinese medicine rangooncreeper fruit decoction piece, and compared with medicinal materials and decoction pieces, the rangooncreeper fruit formula particle loses the inherent form and has corresponding changes of corresponding active ingredients, so that the inherent quality is difficult to be comprehensively reflected only by quantitative analysis of content-indicating ingredients of the medicinal materials or the decoction pieces, and the current qualitative detection technology for the rangooncreeper fruit formula particle is almost not available.

The characteristic spectrum is a quality evaluation method for reflecting the integral characteristics of chemical components of the traditional Chinese medicine, and can effectively detect and control the authenticity, the consistency and the stability of the quality of the traditional Chinese medicine and the preparation thereof. Therefore, it is necessary to establish an HPLC characteristic spectrum method for rapidly identifying the quisqualis indica formula granules, which provides a basis for effectively controlling and relatively comprehensively evaluating the quality of the quisqualis indica formula granules.

In the prior art, a UPLC-MS/MS method simultaneously determines the content of 9 components in quisqualis medicinal materials, such as Wenxuilian and the like, and discloses that: establishing ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry (UPLC-MS/MS) and simultaneously determining the content of 9 components (trigonelline, vanillic acid, ferulic acid, syringic acid, catechin, ellagic acid, malic acid, gallic acid and methyl gallate) in the quisqualis indica medicinal material. The method comprises the following steps: gradient elution with a Welch Xtimate C18 column (100 mm. times.2.1 mm, 1.8 μm) using 0.1% aqueous formic acid (A) -acetonitrile (B) as the mobile phase at a flow rate of 0.25mL min-1, column temperature 45 ℃; the mass spectrum adopts an ESI ion source and a multi-reaction monitoring mode. Detecting the content of 9 components in 34 batches of rangooncreeper fruit of different producing areas (Fujian, Sichuan, Guangxi and Guangdong), and screening the quality control markers of the rangooncreeper fruit by combining chemometrics analysis.

Disclosure of Invention

The invention aims to solve the problems in the prior art and provides a construction and detection method of a rangooncreeper fruit formula particle characteristic spectrum. In the technical scheme, the characteristic spectrum is established based on a high performance liquid chromatograph, the detection of the characteristic spectrum is carried out, the characteristic components in the rangooncreeper fruit formula particles can be integrally controlled, the defects of the existing quality control technology are overcome, the information content of characteristic peaks in the obtained characteristic spectrum is rich, the stability of the overall quality of the rangooncreeper fruit formula particles can be effectively guaranteed, and the quality control technology of the rangooncreeper fruit formula particles is more perfect and scientific; the method is simple to operate, has the advantages of high precision, good stability, good repeatability, high accuracy and the like, and provides an effective basis for quality identification of the traditional Chinese medicinal materials.

In order to achieve the technical purpose, the following technical scheme is proposed:

a construction method of a rangooncreeper fruit formula particle characteristic spectrum comprises the following steps:

A. preparation of control solutions: dissolving ellagic acid control substance with 80% methanol to obtain solution containing 20 μ g per 1 mL;

B. preparation of a test solution: taking the product, grinding, weighing 1.0g, placing into a conical flask with a plug, adding 10mL of methanol, performing ultrasonic treatment (power 600W and frequency 40kHz) for 30min, cooling, shaking up, filtering, and taking the subsequent filtrate to obtain the final product;

C. establishing: respectively injecting the obtained reference substance solution and the obtained test solution into a high performance liquid chromatograph, measuring, and establishing an HPLC characteristic spectrum;

the chromatographic conditions in the high performance liquid chromatograph meet:

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 is 5 mu m;

column temperature of the chromatographic column: 30 ℃;

mobile phase: methanol is taken as a mobile phase A, and 0.2% phosphoric acid aqueous solution is taken as a mobile phase B;

flow rate of mobile phase: 1.0 mL/min;

sample introduction amount: 10 mu L of the solution;

detection wavelength: 360 nm;

the number of theoretical plates is not less than 5000 calculated according to ellagic acid peak;

the mobile phase is eluted according to the gradient program:

further, the HPLC characteristic spectrum of the monarch drug combination granule includes six characteristic peaks, wherein the peak 2 is ellagic acid, the peak 2 is a reference peak S peak, and the relative retention time of each characteristic peak is as follows: peak 1 was 0.56, peak 3 was 1.12, peak 4 was 1.21, peak 5 was 1.42 and peak 6 was 1.63, the relative retention time was within ± 10% of the specified value.

A detection method of a rangooncreeper fruit formula particle characteristic spectrum comprises the following steps:

s1, taking the product, grinding, weighing 1.0g, placing the product in a conical flask with a plug, adding 10mL of methanol, carrying out ultrasonic treatment (power 600W and frequency 40kHz) for 30min, cooling, shaking up, filtering, and taking a subsequent filtrate to obtain the product;

s2, detection: injecting 10 μ L of the test solution obtained in step S1 into a high performance liquid chromatograph, measuring, comparing the high performance liquid chromatogram with the HPLC characteristic spectrum, and detecting the quality and authenticity of the fructus quisqualis formula granule;

the chromatographic conditions in the high performance liquid chromatograph meet:

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 is 5 mu m;

column temperature of the chromatographic column: 30 ℃;

mobile phase: methanol is taken as a mobile phase A, and 0.2% phosphoric acid aqueous solution is taken as a mobile phase B;

flow rate of mobile phase: 1.0 mL/min;

sample introduction amount: 10 mu L of the solution;

detection wavelength: 360 nm;

the number of theoretical plates is not less than 5000 calculated according to ellagic acid peak;

the mobile phase is eluted according to the gradient program:

by adopting the technical scheme, the beneficial technical effects brought are as follows:

1) the invention provides a construction and detection method of a rangooncreeper fruit formula particle characteristic spectrum, six common peaks such as ellagic acid and the like are determined, chromatographic peaks in the constructed HPLC characteristic spectrum are well separated, characteristic spectrum information is rich, chromatographic peak shapes are good, main chemical component information of rangooncreeper fruit formula particles can be comprehensively reflected, and the detection method has good stability and repeatability and is convenient for large-scale popularization and application;

2) the HPLC characteristic spectrum of the rangooncreeper fruit formula particle constructed by the invention fills the blank of quality control of the rangooncreeper fruit formula particle in the prior art, and provides an effective research basis for the subsequent research of the rangooncreeper fruit formula particle;

3) the invention establishes the characteristic spectrum based on the high performance liquid chromatograph, and detects the characteristic spectrum, can integrally control the characteristic components in the rangooncreeper fruit formula granules, can effectively ensure the stability of the overall quality of the rangooncreeper fruit formula granules, and enables the quality control technology of the rangooncreeper fruit formula granules to be more perfect and scientific; the method is simple to operate, has the advantages of high precision, good stability, good repeatability, high accuracy and the like, and provides an effective basis for quality identification of the traditional Chinese medicinal materials.

Drawings

FIG. 1 is a chromatogram of the results of the extraction mode examination in example 3;

FIG. 2 is a chromatogram of the examination result of the extraction solvent in example 3;

FIG. 3 is a chromatogram of the extraction time examination result in example 3;

FIG. 4 is a chromatogram of the material ratio test in example 3;

FIG. 5 is a graph of the UV absorption spectrum of the ellagic acid control in the wavelength investigation of example 4;

FIG. 6 is a 3D chromatogram of quisqualis indica formula granules from wavelength investigation of example 4;

FIG. 7 is a chromatogram of different wavelengths of quisqualis indica formula granules in the wavelength investigation of example 4;

FIG. 8 is a chromatogram of column temperature examination results in example 4;

FIG. 9 is a chromatogram of the flow rate investigation result in example 4;

FIG. 10 is a chromatogram peak assignment result chart of the characteristic spectrum of the rangooncreeper fruit formula particle in example 5;

FIG. 11 is a graph of the feature spectrum of three batches of rangooncreeper fruit formula granules in example 5;

FIG. 12 is a comparative profile of the rangooncreeper formula granules in example 5 (wherein, Peak 2 (S): ellagic acid).

Detailed Description

In the following, the technical solutions in the embodiments of the present invention are clearly and completely described, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the following examples, reference is made to an apparatus comprising:

high performance liquid chromatograph: shimadzu LC-20AD high performance liquid chromatograph,

a chromatographic column: agilent 5 TC-C18250X 4.6mm, Kromasil 100-5-C184.6X 250mm, Phenomenex Luna5 mu m C18(2)100A 4.6X 250mm,

an electronic balance: ME204E/02, MS205D Mm, XP26 (Mettler-Tollido instruments, Inc.),

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

an ultrasonic cleaner: model KQ600DB (600W, 40 KHz; ultrasonic instruments, Inc. of Kunshan);

in the following examples, the reagents involved included:

methanol (chromatographically pure), phosphoric acid (chromatographically pure), water (ultrapure water) in the mobile phase A, and other reagents are analytically pure;

in the following examples, the test articles referred to include:

the rangooncreeper fruit formula granule batch number is as follows: SJZ1, SJZ2, SJZ 3;

in the following examples, reference controls include:

ellagic acid (China institute for food and drug assay, batch No.: 111959-201602, content is 97.8%).

Example 1

A construction method of a rangooncreeper fruit formula particle characteristic spectrum comprises the following steps:

A. preparation of control solutions: dissolving ellagic acid control substance with 80% methanol to obtain solution containing 20 μ g per 1 mL;

B. preparation of a test solution: taking the product, grinding, weighing 1.0g, placing into a conical flask with a plug, adding 10mL of methanol, performing ultrasonic treatment (power 600W and frequency 40kHz) for 30min, cooling, shaking up, filtering, and taking the subsequent filtrate to obtain the final product;

C. establishing: respectively injecting the obtained reference substance solution and the obtained test solution into a high performance liquid chromatograph, measuring, and establishing an HPLC characteristic spectrum;

the chromatographic conditions in the high performance liquid chromatograph meet:

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 is 5 mu m;

column temperature of the chromatographic column: 30 ℃;

mobile phase: methanol is taken as a mobile phase A, and 0.2% phosphoric acid aqueous solution is taken as a mobile phase B;

flow rate of mobile phase: 1.0 mL/min;

sample introduction amount: 10 mu L of the solution;

detection wavelength: 360 nm;

the number of theoretical plates is not less than 5000 calculated according to ellagic acid peak;

the mobile phase was subjected to a gradient elution procedure, as specified in table 1 below:

TABLE 1 gradient elution

Example 2

Based on example 1, this example provides a method for detecting a characteristic spectrum of rangooncreeper fruit formula granules, which includes the following steps:

s1, taking the product, grinding, weighing 1.0g, placing the product in a conical flask with a plug, adding 10mL of methanol, carrying out ultrasonic treatment (power 600W and frequency 40kHz) for 30min, cooling, shaking up, filtering, and taking a subsequent filtrate to obtain the product;

s2, detection: injecting 10 μ L of the test solution obtained in step S1 into a high performance liquid chromatograph, measuring, comparing the high performance liquid chromatogram with the HPLC characteristic spectrum, and detecting the quality and authenticity of the fructus quisqualis formula granule;

the chromatographic conditions in the high performance liquid chromatograph meet:

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 is 5 mu m;

column temperature of the chromatographic column: 30 ℃;

mobile phase: methanol is taken as a mobile phase A, and 0.2% phosphoric acid aqueous solution is taken as a mobile phase B;

flow rate of mobile phase: 1.0 mL/min;

sample introduction amount: 10 mu L of the solution;

detection wavelength: 360 nm;

the number of theoretical plates is not less than 5000 calculated according to ellagic acid peak;

the mobile phase is eluted according to the gradient program:

the mobile phase was subjected to a gradient elution procedure, as specified in table 2 below:

TABLE 2 gradient elution

Example 3

Based on examples 1-2, this example will further describe the technical scheme by considering the extraction method, the kind of extraction solvent, the extraction time, and the material ratio in the preparation of the sample solution.

First, examination of extraction method

Two samples (lot SJZ1) were ground into fine powder and weighed to 1.0g, and placed in conical flasks with stoppers, 10mL of methanol was added, followed by heating and refluxing (conventional process conditions) for 30min, ultrasonic treatment (power 600W, frequency 40kHz) for 30min, cooling, shaking, and filtering to obtain a filtrate, and the results are shown in FIG. 1.

The results show that: when the ultrasonic extraction and reflux extraction are carried out on the test solution, the extraction effect has no great difference, so that the ultrasonic extraction method which is simple and convenient is selected for the test solution extraction method.

Second, investigation of extraction solvent

Five samples (batch No. SJZ1) were taken, ground, weighed to 1.0g, placed in a conical flask with a stopper, respectively added with 10mL of methanol, 10mL of 50% methanol, 10mL of water, 10mL of ethanol and 10mL of 50% ethanol, treated with ultrasound (power 600W, frequency 40kHz) for 30min, cooled, shaken, filtered, and the subsequent filtrate was taken, and the results are shown in FIG. 2.

The results show that: when the extraction solvent is methanol, the chromatographic peak information amount is large, and the separation degree is good, so the extraction solvent of the test sample in the technical scheme is selected to be methanol.

Third, investigation of extraction time

Three parts of a test sample (batch No. SJZ1) are taken, ground, weighed to be 1.0g, placed in a conical flask with a plug, respectively added with 10mL of methanol, weighed, respectively treated by ultrasonic treatment (power 600W, frequency 40kHz) for 20min, 30min and 40min, cooled, shaken evenly and filtered, and a subsequent filtrate is taken, and the obtained result is shown in figure 3.

The results show that: when the extraction time is 20min, the peak separation degree and the like are good, but in order to ensure sufficient extraction and measurement efficiency of the target component in the solution, the extraction time of the sample solution is selected to be 30 min.

Fourth, survey of material ratio

Three parts of a test sample (batch No. SJZ1) are taken, ground, weighed to be 0.5g, 1.0g and 1.5g respectively, placed in a conical flask with a plug, 10mL of methanol is added respectively, the weight is weighed, ultrasonic treatment (power 600W, frequency 40kHz) is carried out for 30min respectively, the mixture is cooled, shaken evenly and filtered, and a subsequent filtrate is taken, and the obtained result is shown in figure 4.

The results show that: when the sample amount is 1.0g, the ratio of the chromatographic peak height is suitable, and therefore, the sample amount of the sample is determined to be 1.0 g.

In summary, in the preparation of the test solution, the specific scheme is defined as follows: taking the product, grinding, taking 1.0g, placing into a conical flask with a plug, adding 10mL of methanol, performing ultrasonic treatment (power 600W, frequency 40KHz) for 30min, cooling, shaking, filtering, and taking the subsequent filtrate to obtain the final product.

Example 4

Based on examples 1-3, this example considers the chromatographic conditions (wavelength, column temperature, flow rate) during construction to further illustrate the present technical solution.

First, wavelength investigation

Scanning ellagic acid reference substance solution and test solution with diode array detector respectively, extracting chromatogram of the test solution at wavelengths of 230nm, 250nm, 270nm, 290nm, 310nm, 330nm, and 360nm, determining the rest chromatogram conditions in the same way as in example 1, and obtaining results shown in FIGS. 5-7.

The results show that: when the detection wavelength is 360nm, the amount of information on the chromatographic peak is large, and therefore, the detection wavelength is determined to be 360 nm.

Second, column temperature investigation

The column temperatures were set to 25 ℃, 30 ℃ and 35 ℃ respectively, and the chromatographic conditions were the same as in example 1, and the results obtained were as shown in table 3 below and fig. 8.

TABLE 3 column temperature investigation-ratio of characteristic peaks relative retention time

The results show that: when the column temperature is 25 ℃, 30 ℃ and 35 ℃, the chromatogram peaks are symmetrical, and the separation degree meets the requirement, so the column temperature is limited to 30 ℃, and the subsequent investigation is convenient.

Third, investigation of flow velocity

The flow rates were set to 0.8mL/min, 1.0mL/min and 1.2mL/min, respectively, and the other chromatographic conditions were the same as in example 1, and the results were obtained as shown in Table 4 below and FIG. 9.

TABLE 4 flow Rate investigation-ratio of characteristic Peak to Retention time

The result shows that when the flow rates are 0.8mL/min and 1.0mL/min respectively, the relative retention time chromatogram of each characteristic peak has symmetrical peak shapes, and the separation degree meets the requirement, so the flow rate under the chromatographic condition is determined to be 1.0 mL/min.

Example 5

The embodiment provides a method for constructing a characteristic map of rangooncreeper fruit formula granules by taking the rangooncreeper fruit formula granules as a test sample, and the method comprises the following steps:

A. preparation of control solutions: dissolving ellagic acid control substance with 80% methanol to obtain solution containing 20 μ g per 1 mL;

B. preparation of a test solution: taking the product, grinding, weighing 1.0g, placing into a conical flask with a plug, adding 10mL of methanol, performing ultrasonic treatment (power 600W and frequency 40kHz) for 30min, cooling, shaking up, filtering, and taking the subsequent filtrate to obtain the final product;

C. establishing: respectively injecting the obtained reference substance solution and the obtained test solution into a high performance liquid chromatograph, measuring, and establishing an HPLC characteristic spectrum;

the chromatographic conditions in the high performance liquid chromatograph meet:

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 is 5 mu m;

column temperature of the chromatographic column: 30 ℃;

mobile phase: methanol is taken as a mobile phase A, and 0.2% phosphoric acid aqueous solution is taken as a mobile phase B;

flow rate of mobile phase: 1.0 mL/min;

sample introduction amount: 10 mu L of the solution;

detection wavelength: 360 nm;

the number of theoretical plates is not less than 5000 calculated according to ellagic acid peak;

the mobile phase was subjected to a gradient elution procedure, as specified in table 5 below:

TABLE 5 gradient elution

In addition, based on the above method for constructing the HPLC profile of the quisqualis indica formula granule, the following methodology investigation is also performed in this embodiment, which specifically includes:

identification of chromatographic peak

1. Preparation of control solutions: dissolving ellagic acid control substance with 80% methanol to obtain solution containing 20 μ g per 1 mL;

2. preparation of a test solution: taking the product, grinding, weighing 1.0g, placing into a conical flask with a plug, adding 10mL of methanol, performing ultrasonic treatment (power 600W and frequency 40kHz) for 30min, cooling, shaking up, filtering, and taking the subsequent filtrate to obtain the final product;

3. preparation of negative control solution: taking 10mL of methanol, placing the methanol in a conical flask with a plug, carrying out ultrasonic treatment (power 600W and frequency 40kHz) for 30min, cooling, shaking up, filtering, and taking a subsequent filtrate to obtain the product;

identifying peaks of a characteristic diagram of the rangooncreeper fruit formula granules, and obtaining a result as shown in figure 10, wherein the peak 2 is ellagic acid, and six peaks in a sample diagram are examined in subsequent methodology examination.

Second, precision test

Taking the test solution of the quisqualis indica formula particles, continuously injecting samples for six times according to the construction method of the HPLC characteristic spectrum of the quisqualis indica formula particles, wherein each time is 10 mu L, calculating the retention time and the peak area, and obtaining the results as shown in the following tables 6-7.

TABLE 6 precision investigation-Retention time

TABLE 7 precision investigation-Peak area

The results show that: the retention time and RSD of the peak area of each characteristic peak are 0.03-0.17%, and the RSD of the relative peak area of each characteristic peak is 0.06-1.31%, so that the instrument has good precision.

Third, repeatability inspection

Six portions of the rangooncreeper fruit formula particles are taken, the preparation and the measurement of the test solution are carried out according to the construction method of the HPLC characteristic map provided by the rangooncreeper fruit formula particles, and the obtained results are shown in the following tables 8-9.

TABLE 8 repeated investigation-relative retention time

TABLE 9 repeatability test-relative peak area

The results show that: the RSD of each characteristic peak relative retention time is 0.05-0.25%, and the RSD of each characteristic peak relative peak area is 1.58-1.87%, indicating that the method has good repeatability.

Fourth, intermediate precision investigation (different persons and time)

Based on the above-mentioned construction method of the HPLC profile of the quisqualis indica formula granules, two portions of the quisqualis indica formula granules are weighed respectively under different personnel (A, B) and different time (T1, T2) conditions to prepare test solution, and the results are shown in the following tables 10-11.

TABLE 10 personnel and time review-relative Retention time

TABLE 11 personnel and time survey-relative Peak area

The results show that: different persons can measure the same sample at different times, and the method has better stability.

Fifth, durability investigation (durability of chromatographic column)

Based on the construction method of the HPLC characteristic map of the rangooncreeper formula particles, three chromatographic columns of Phenomenex 5 mu m C18(2)100A 4.6X 250mm, Kromasil 100-5-C184.6X 250mm and Agilent 5 TC-C18250X 4.6mm are respectively examined, and the obtained results are shown in the following tables 12-13.

TABLE 12 column durability examination-relative retention time

TABLE 13 column durability test-relative peak area

The results show that: the three chromatographic columns are used for detecting the rangooncreeper fruit formula particle sample, the RSD of the relative retention time of the characteristic peak is 0.10-1.32%, and the RSD of the relative peak area of the characteristic peak is 1.48-3.65%. The method is proved to have good durability of the chromatographic column.

Sixthly, stability investigation

Based on the construction method of the HPLC characteristic map of the rangooncreeper fruit formula granules, the same test solution of the rangooncreeper fruit formula granules is respectively measured at 0h, 4h, 8h, 12h, 18h and 24h, and the obtained results are shown in the following tables 14-15.

Table 14 stability study-retention time

TABLE 15 stability survey-Peak area

The result shows that the RSD of the retention time of the corresponding characteristic peak is 0.18-1.67%, and the sample solution is stable within 24 hours.

In summary, the following steps: the RSD of each characteristic peak relative retention time meets the requirements in the above researches, the method is good, and the six characteristic peaks are included in the subsequent researches.

Seventhly, verification of characteristic spectrum of formula particles

By adopting the construction method of the HPLC characteristic map of the rangooncreeper fruit formula granules, the characteristic maps of three batches of samples are measured, the relative retention time and the relative peak area are calculated, and the verification results are shown in the following tables 16-17 and fig. 11.

TABLE 16 relative retention times of three quisqualis indica formula granules

TABLE 17 relative peak area of three Combretaceae formula granules

According to the principle that the relative retention time is stable, samples of each batch can be detected, and the peak is relatively high, six peaks with good repeatability are selected as characteristic peaks. The results show that: when the peak 2 is taken as an S peak, the relative retention time RSD of the characteristic peak of the three batches of rangooncreeper fruit formula granules is less than 2.0 percent.

Therefore, it is finally specified that: six characteristic peaks are presented in the chromatogram of the test sample, wherein the peak corresponding to the ellagic acid control is an S peak, and the relative retention time of the S peak of each characteristic peak is calculated and is within + -10% of the specified value. The specified values are: 0.56 (peak 1), 1.12 (peak 3), 1.21 (peak 4), 1.42 (peak 5), 1.63 (peak 6).

In addition, a traditional Chinese medicine chromatography fingerprint similarity evaluation system (2012 edition) is adopted to synthesize three batches of rangooncreeper fruit formula granules, a comparison characteristic spectrum of the rangooncreeper fruit formula granules is established, and the quality of the rangooncreeper fruit formula granules can be controlled relatively accurately and integrally as shown in figure 12 (wherein, peak 2 (S): ellagic acid).