Construction method for measuring HPLC (High Performance Liquid Chromatography) content of nandina

Abstract

The invention provides a construction method for determining the HPLC (High Performance Liquid Chromatography) content of nandina domestica, which comprises the following steps: A) dissolving a test raw material by adopting a solvent, and extracting to obtain a to-be-detected solution; b) determining the to-be-detected liquid by adopting a high performance liquid chromatography to obtain an HPLC spectrum of the nandina domestica; chromatographic conditions of the high performance liquid chromatography are as follows: a chromatographic column is a C18 column; a mobile phase A is an acetonitrile solution, a mobile phase B is a 0.1% phosphoric acid aqueous solution, and gradient elution is carried out; the gradient elution is specifically as follows: 0-20 min, the phase A is 10%-11%, and the phase B is 90%-89%; 20-25 min, the phase A accounts for 11%-18%, and the phase B accounts for 89%-82%; 25-40 min, the phase A accounts for 18%-40%, and the phase B accounts for 82%-60%. According to the present invention, the high performance liquid chromatography is adopted, the acetonitrile-0. 1% phosphoric acid solution is adopted as the mobile phase to perform gradient elution, and the chlorogenic acid reference substance, the neochlorogenic acid reference substance and the cryptochlorogenic acid reference substance are adopted as the reference substances to establish the HPLC content determination method of the nandina domestica so as to provide more scientific technical means for controlling the medicinal quality of the nandina domestica.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a construction method for determining the content of pyracantha glabra by HPLC. Background Art

[0002] The name of Nandina domestica was first recorded in the "Wood Section" of Volume 6 of "Compendium of Materia Medica Supplement", which records Nandina domestica: "Nandina domestica is a species of poplar tree. People now plant it in their yards. It produces red seeds in winter for people to play with. It is not the Nandina domestica." "The seeds are called red loquat seeds. They can be used to treat star lice. Mash them with mercury and rub them on the affected area. They can also be soaked in wine to treat rheumatism."

[0003] At present, there is no relevant standard for the content determination of Radix Gastrodiae and its decoction pieces. The relevant literature mainly studies the flavonoids and alkaloid components. There is a lack of qualitative and quantitative research literature on Radix Gastrodiae formula granules and related preparations. Through our research, we found that after Radix Gastrodiae was decocted, the flavonoids and alkaloid components were low. It is difficult to evaluate the quality of Radix Gastrodiae, its decoction pieces, standard decoctions, extracts and formula granules and other related preparations by using the content limit as a quality control indicator. Modern research shows that phenolic acid components such as chlorogenic acid have antibacterial, antiviral, white blood cell increase, liver protection and choleretic, anti-tumor, blood pressure lowering, blood lipid lowering, free radical scavenging and central nervous system excitation effects.

[0004] Therefore, the present invention aims to construct a high-performance liquid phase content determination method for Ganoderma lucidum, which can comprehensively reflect the quality level of Ganoderma lucidum and its related preparations, and provide guidance for the quality control of Ganoderma lucidum medicinal materials, decoction pieces and their related preparations. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a method for constructing an HPLC content determination method for Fructus Caryophylli, and the method of the present invention is accurate and reliable in detecting chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid.

[0006] The present invention discloses the construction and application of a high-performance liquid phase content determination detection method for Fructus gynaecogenis. The method establishes a content determination method with the total amount of chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid as an indicator, fully displays the chemical composition characteristics of Fructus gynaecogenis, and comprehensively reflects the quality information of Fructus gynaecogenis, thereby being able to comprehensively and effectively control the quality of Fructus gynaecogenis medicinal materials, decoction pieces and related preparations thereof.

[0007] A method for constructing an HPLC content determination method of Fructus Sieb.

[0008] A) Dissolve the test sample raw material with a solvent, extract, and obtain a test solution;

[0009] B) the test solution is measured by high performance liquid chromatography to obtain an HPLC spectrum of Fructus pyrifera;

[0010] The chromatographic conditions of the high performance liquid chromatography are: the chromatographic column is a C18 column; preferably C18(2)100A250×4.6mm, 5μm; C18 250×4.6mm, 5μm; ZORBAX Eclipse Plus C18 250×4.6mm, 5μm chromatographic columns.

[0011] The mobile phase A of the present invention is acetonitrile solution, the mobile phase B is 0.1% phosphoric acid aqueous solution, and the gradient elution is performed;

[0012] The gradient elution is specifically:

[0013] 0-20min, phase A: 10%→11%, phase B: 90→89%;

[0014] 20-25 min, phase A: 11%→18%, phase B: 89%→82%;

[0015] 25-40min, phase A: 18%→40%, phase B: 82%→60%.

[0016] The test sample raw material of the present invention is one or more of the following: Radix Gastrodiae, Radix Gastrodiae decoction pieces, Radix Gastrodiae standard decoction or Radix Gastrodiae formula granules.

[0017] The invention provides a method for constructing HPLC content determination of guanyin seeds. Firstly, a test sample raw material is dissolved and extracted by a solvent to obtain a test solution.

[0018] Specifically, the test sample raw material is dissolved in a solvent, extracted, cooled, shaken, and filtered to obtain the product. The present invention does not limit the specific methods and operations of cooling, shaking, and filtering, which are well known to those skilled in the art.

[0019] The extraction of the present invention is heating reflux extraction or ultrasonic extraction, preferably ultrasonic treatment. The power of the ultrasonic treatment is 600W, the frequency is 40kHz, and the ultrasonic treatment time is 15 to 60 minutes, preferably 30 minutes.

[0020] According to the present invention, the ratio of the mass g of the test sample raw material to the volume mL of the solvent is 0.1-1:25-100;

[0021] The solvent of the present invention is 50% to 70% methanol; specifically, it can be 50% methanol, 60% methanol, 70% methanol; preferably, it is 50% methanol.

[0022] In some specific embodiments of the present invention, the ratio of the mass g of the test sample raw material to the volume mL of the solvent is 0.1 to 1:50;

[0023] In some specific embodiments of the present invention,

[0024] Medicinal material test solution: Take 1.0 g of powder of Gynecology daphne, accurately weigh it, put it in a stoppered conical flask, accurately add 50 ml of 50% methanol, weigh it, ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0025] Decoction piece test solution: Take 1.0g of Tianzhuzi decoction piece powder, weigh it accurately, put it in a stoppered conical flask, add 50ml of 50% methanol accurately, weigh it, treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0026] Standard decoction test solution: Take an appropriate amount of Tianzhuzi standard decoction, take about 0.1g, accurately weigh, put it in a stoppered conical flask, accurately add 50ml of 50% methanol, stopper it, weigh the weight, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, and take the filtrate to obtain.

[0027] Formula granules for test solution: Take an appropriate amount of Tianzhuzi formula granules, grind them into powder, take about 0.1g, accurately weigh, put it in a stoppered conical flask, accurately add 50ml of 50% methanol, stopper it, weigh it, ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0028] The above raw materials of the present invention can all be subjected to quality control and qualitative and quantitative detection by the method of the present invention.

[0029] The present invention also includes preparing a reference solution: taking a chlorogenic acid reference substance, a neochlorogenic acid reference substance and a cryptochlorogenic acid reference substance respectively, dissolving them in 50% methanol to obtain a reference solution;

[0030] The reference solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of the dahliae fructus are qualitatively identified according to the chromatogram of the reference.

[0031] The concentrations of the reference solutions of the present invention are preferably as follows: chlorogenic acid reference substance is 2.6763696 μg / ml to 267.6369600 μg / ml, neochlorogenic acid reference substance is 1.622296 μg / ml to 162.229600 μg / ml, and cryptochlorogenic acid reference substance is 1.03082 μg / ml to 103.08200 μg / ml.

[0032] Specifically, the linear regression equation of neochlorogenic acid is y=13.2522X-3.7026, and the correlation coefficient R 2 =1.0000, indicating that the linear relationship between the concentration of chlorogenic acid and the peak area is good in the concentration range of 1.622296μg / ml to 162.229600μg / ml; the linear regression equation of chlorogenic acid is y=15.4919X-7.2945, and the correlation coefficient R 2 =1.0000, indicating that the linear relationship between chlorogenic acid concentration and peak area is good in the concentration range of 2.6763696μg / ml to 267.6369600μg / ml; the linear regression equation of cryptochlorogenic acid is y=12.242X-2.0249, and the correlation coefficient R 2 =1.0000, indicating that the linear relationship between the concentration of chlorogenic acid and the peak area is good in the concentration range of 1.03082μg / ml to 103.08200μg / ml.

[0033] The test solution is measured by high performance liquid chromatography to obtain the HPLC characteristic spectrum of Fructus Dictamni.

[0034] The HPLC chromatographic conditions are: the chromatographic column is a C18 column, the size is 250×4.6mm 5μm, the column temperature is 25-35° C., preferably 25° C. At the above chromatographic column temperature, the chromatographic peaks of the present invention are symmetrical, the separation is good, and the peaks are more complete.

[0035] The mobile phase A of the invention is acetonitrile solution, the mobile phase B is 0.1% phosphoric acid aqueous solution, and the gradient elution is performed.

[0036] Specifically, the gradient elution is specifically:

[0037] 0-20min, phase A: 10%→11%, phase B: 90→89%;

[0038] 20-25 min, phase A: 11%→18%, phase B: 89%→82%;

[0039] 25-40min, phase A: 18%→40%, phase B: 82%→60%.

[0040] The theoretical plate number calculated based on chlorogenic acid should not be less than 5000.

[0041] The present invention has good baseline separation under the above elution gradient, good separation of each peak and stable baseline.

[0042] The flow rate of the mobile phase of the present invention is 0.8-1.2 mL / min, and more preferably 1.0 mL / min.

[0043] The present invention found that the chromatographic peaks were well separated and the separation degree was moderate under the above flow rate, which is the most preferred solution.

[0044] The injection volume is 1 to 5 μL.

[0045] The detection wavelength is 325 nm. The inventors found that at 325 nm, the chromatographic peak information volume is larger, the chromatogram baseline is more stable, and the peak area of ​​each peak is larger.

[0046] The invention provides a quality evaluation method for a dianthus fragrans raw material and a preparation thereof, wherein the method described in any one of the above technical schemes is used to perform detection and analyze the detection result.

[0047] (1) The present invention establishes a method for determining the content of Ziziphus truncatus, and uses the total amount of chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid as an indicator, which can control the intrinsic quality of Ziziphus truncatus and its related preparations from an overall and macroscopic perspective, thereby ensuring the efficacy of the drug and allowing the medicinal material and its related preparations to be subjected to more formal quality control.

[0048] (2) The method of the present invention has good stability, high precision, good reproducibility, is convenient and easy to master.

[0049] The invention provides a method for constructing HPLC content determination of jasmine, comprising: A) dissolving a test sample raw material with a solvent, extracting, and obtaining a test solution; B) determining the test solution with a high performance liquid chromatography method, and obtaining an HPLC spectrum of jasmine; the chromatographic conditions of the high performance liquid chromatography method are: a chromatographic column is a C18 column; a mobile phase A is an acetonitrile solution, a mobile phase B is a 0.1% phosphoric acid aqueous solution, and gradient elution is performed; the gradient elution is specifically as follows: 0-20 min, phase A: 10%→11%, phase B: 90→89%; 20-25 min, phase A: 11%→18%, phase B: 89%→82%; 25-40 min, phase A: 18%→40%, phase B: 82%→60%. The present invention adopts high performance liquid chromatography, selects acetonitrile-0.1% phosphoric acid solution as mobile phase for gradient elution, takes chlorogenic acid reference substance, neochlorogenic acid reference substance and cryptochlorogenic acid reference substance as reference substances, establishes an HPLC content determination method of geranium seeds, and provides more scientific technical means for controlling the medicinal quality of geranium seeds. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Mobile phase selection chart;

[0051] Figure 2 UV absorption spectrum of new chlorogenic acid-reference substance;

[0052] Figure 3 Neochlorogenic acid-UV absorption spectrum of the test sample;

[0053] Figure 4Chlorogenic acid-reference substance UV absorption spectrum;

[0054] Figure 5 Chlorogenic acid-UV absorption spectrum of the test sample;

[0055] Figure 6 Ultraviolet absorption spectrum of cryptochlorogenic acid-reference substance;

[0056] Figure 7 Ultraviolet absorption spectrum of cryptochlorogenic acid-test sample;

[0057] Figure 8 Flow rate survey results;

[0058] Fig. 9 Column temperature investigation results;

[0059] Fig.10 Investigation on the specificity of Indian basil;

[0060] Fig.11 New chlorogenic acid standard curve;

[0061] Fig.12 Standard curve of chlorogenic acid;

[0062] Fig.13 Standard curve of cryptochlorogenic acid;

[0063] Fig.14 Chromatographic column inspection;

[0064] Fig.15 It is the chromatogram of Comparative Example 1;

[0065] Fig.16 It is the chromatogram of Comparative Example 2;

[0066] Fig.17 It is the chromatogram of Comparative Example 3. DETAILED DESCRIPTION

[0067] The present invention provides a method for constructing the HPLC content determination of Fructus Sieb. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they all belong to the scope of protection of the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0068] To further illustrate the present invention, a method for constructing an HPLC content determination method of Fructus Dictamni provided by the present invention is described in detail below in conjunction with examples.

[0069] Experimental instruments and materials

[0070] High performance liquid chromatograph: Agilent 1260 high performance liquid chromatograph;

[0071] Electronic balance: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instrument Co., Ltd.);

[0072] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0073] Ultrasonic cleaner: KQ600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0074] Chromatographic column: C18(2)100A250×4.6mm, 5μm; C18 250×4.6mm, 5μm; ZORBAX Eclipse Plus C18 250×4.6mm, 5μm

[0075] Chlorogenic acid (China Food and Drug Inspection Institute, batch number: 110753-202119, purity: 96.3%);

[0076] Neochlorogenic acid (Chengdu Desite Biotechnology Co., Ltd., batch number: DSTDX001504, purity: 98.80%),

[0077] Chlorogenic acid (Chengdu Desit Biotechnology Co., Ltd., batch number: DST220104-035, purity: 99.30%),

[0078] Methanol, acetonitrile and phosphoric acid were of chromatographic grade; water was ultrapure water, and other reagents were of analytical grade.

[0079] Tianzhuzi formula granules: TZZ-KL-01, TZZ-KL-02, TZZ-KL-03;

[0080] Tianzhuzi medicinal materials: TZZ-YC-01, TZZ-YC-02, TZZ-YC-03, TZZ-YC-04, TZZ-YC-05, TZZ-YC-06, TZZ-YC-07, TZZ-YC-08, TZZ-YC-09, TZZ-YC-10, TZ Z-YC-11, TZZ-YC-12, TZZ-YC-13, TZZ-YC-14, TZZ-YC-15, TZZ-YC-16, TZZ-YC-17, TZZ-YC-18, TZZ-YC-19, TZZ-YC-20, TZZ-YC-21;

[0081] Tianzhuzi decoction pieces: TZZ-YP-01, TZZ-YP-02, TZZ-YP-03, TZZ-YP-04, TZZ-YP-05, TZZ-YP-06, TZZ-YP-07, TZZ-YP-08, TZZ-YP-09, TZZ-YP-10, TZ Z-YP-11, TZZ-YP-12, TZZ-YP-13, TZZ-YP-14, TZZ-YP-15, TZZ-YP-16, TZZ-YP-17, TZZ-YP-18, TZZ-YP-19, TZZ-YP-20, TZZ-YP-21;

[0082] Tianzhuzi standard decoction: TZZ-BT-01, TZZ-BT-02, TZZ-BT-03, TZZ-BT-04, TZZ-BT-05, TZZ-BT-06, TZZ-BT-07, TZZ-BT-08, TZZ-BT-09, TZZ-BT-10, T ZZ-BT-11, TZZ-BT-12, TZZ-BT-13, TZZ-BT-14, TZZ-BT-15, TZZ-BT-16, TZZ-BT-17, TZZ-BT-18, TZZ-BT-19, TZZ-BT-20, TZZ-BT-21.

[0083] Example chromatographic conditions investigation

[0084] 2.2.1 Chromatographic conditions and system suitability test

[0085] Octadecylsilane bonded silica gel was used as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 1.0 ml per minute; the column temperature was 25°C; the detection wavelength was 325 nm. The theoretical plate number calculated based on the chlorogenic acid peak should be no less than 5000.

[0086]

[0087] 2.2.2 Preparation of reference solution

[0088] Take appropriate amounts of chlorogenic acid reference substance, neochlorogenic acid reference substance, and cryptochlorogenic acid reference substance, weigh them accurately, and add 50% methanol to prepare a mixed solution containing 50 μg of neochlorogenic acid, 30 μg of chlorogenic acid, and 20 μg of cryptochlorogenic acid per 1 ml.

[0089] 2.2.3 Preparation of test solution

[0090] Medicinal material test solution: Take about 1.0 g of Gypsophila japonica powder (passed through No. 3 sieve), weigh it accurately, put it in a stoppered conical flask, accurately add 50 ml of 50% methanol, weigh it, treat it with ultrasound (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0091] Decoction piece test solution: Take about 1.0g of Tianzhuzi decoction piece powder (passed through No. 3 sieve), weigh it accurately, put it in a stoppered conical flask, accurately add 50ml of 50% methanol, weigh it, treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0092] Standard decoction test solution: Take an appropriate amount of Tianzhuzi standard decoction, take about 0.1g, accurately weigh, put it in a stoppered conical flask, accurately add 50ml of 50% methanol, stopper it, weigh the weight, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, and take the filtrate to obtain.

[0093] Formula granules for test solution: Take an appropriate amount of Tianzhuzi formula granules, grind them into powder, take about 0.1g, accurately weigh, put it in a stoppered conical flask, accurately add 50ml of 50% methanol, stopper it, weigh it, ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0094] 2.2.4 Determination method

[0095] Accurately pipette 5 μl of reference solution and test solution respectively, inject into liquid chromatograph, and measure to obtain the result.

[0096] 2.3 Establishment of content determination method

[0097] 2.3.1 Chromatographic conditions and system suitability test

[0098] 2.3.1.1 Mobile phase selection

[0099] Based on the experimental conditions proposed above, acetonitrile-0.1% phosphoric acid was selected for investigation. Figure 1 . Figure 1 Mobile phase selection diagram; the results show that when acetonitrile-0.1% phosphoric acid is used as the mobile phase, the peak shape and separation effect of the target peak are good, so acetonitrile-0.1% phosphoric acid solution is selected as the mobile phase for subsequent investigation.

[0100] 2.3.1.2 Wavelength selection

[0101] Based on the experimental conditions proposed above, the diode array detector was used to perform full-band scanning on the neochlorogenic acid reference solution, chlorogenic acid reference solution, cryptochlorogenic acid reference solution and the test solution. Figure 2-7 . Figure 2 UV absorption spectrum of new chlorogenic acid-reference substance; Figure 3 Neochlorogenic acid-UV absorption spectrum of the test sample; Figure 4 Chlorogenic acid-reference substance UV absorption spectrum; Figure 5 Chlorogenic acid-UV absorption spectrum of the test sample; Figure 6 Ultraviolet absorption spectrum of cryptochlorogenic acid-reference substance; Figure 7 Ultraviolet absorption spectrum of chlorogenic acid-test sample.

[0102] By comparing the maximum absorption wavelengths of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid in the spectral diagram and referring to the chromatogram, it was found that the index peak had greater absorption when the detection wavelength was 325nm, and the baseline of the chromatogram was more stable, so the detection wavelength was determined to be 325nm.

[0103] 2.3.1.3 Flow rate investigation

[0104] According to the above proposed test conditions, the flow rates were 0.8ml / min, 1.0ml / min, and 1.2ml / min. Figure 8 , Table 1. Figure 8 Flow rate survey results.

[0105] Table 1 Analysis results of different flow rates

[0106]

[0107] The results showed that when the flow rates were 0.8ml / min, 1.0ml / min and 1.2ml / min, the chromatogram peak shape and separation effect met the requirements, and the proposed flow rate was 1.0ml / min.

[0108] 2.3.1.4 Column temperature investigation

[0109] Based on the above proposed experimental conditions, the column temperatures of 25℃, 30℃ and 35℃ were investigated. Fig. 9 , Table 2. Fig. 9 Column temperature investigation results.

[0110] Table 2 Analysis results of different column temperatures

[0111]

[0112]

[0113] The results show that when the column temperature is 25℃, 30℃, and 35℃, the chromatogram peak shape and separation effect meet the requirements. The column temperature is temporarily set at 25℃.

[0114] The chromatographic conditions and system usability test for the content determination of Tianzhuzi formula granules are tentatively set as follows: octadecylsilane bonded silica gel is used as the filler (column length is 250mm, inner diameter is 4.6mm, particle size is 5μm); acetonitrile is used as mobile phase A, 0.1% phosphoric acid solution is used as mobile phase B, and gradient elution is performed according to the provisions in the following table; the flow rate is 1.0ml per minute; the column temperature is 25℃; the detection wavelength is 325nm. The theoretical plate number calculated based on the chlorogenic acid peak should be no less than 5000.

[0115]

[0116] 2.3.2 Preparation of test solution

[0117] 2.3.2.1 Investigation of extraction methods

[0118] Take an appropriate amount of Tianzhuzi formula granules (batch number: TZZ-KL-01), grind to about 0.1g, accurately weigh, place in a stoppered conical bottle, accurately add 50ml of 50% methanol, weigh the weight, reflux, ultrasonic treatment (power 600W, frequency 40kHz) for 30 minutes, cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, and take the filtrate. Accurately take 5μl of the test solution, inject into the liquid chromatograph, and calculate the total amount of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid under different extraction methods. The results are shown in Table 3.

[0119] Table 3 Investigation of extraction methods

[0120]

[0121] The results showed that the extraction method had little effect on the contents of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid, so the extraction method chosen was ultrasound, which was relatively simple to operate.

[0122] 2.3.2.2 Investigation of extraction solvent

[0123] Take an appropriate amount of Tianzhuzi formula granules (batch number: TZZ-KL-01), grind to about 0.1g, weigh, place in a stoppered conical flask, accurately add 50ml of water, 10% methanol, 50% methanol, 70% methanol, methanol, 50% ethanol, weigh, ultrasonic treatment (power 600W, frequency 40kHz) for 30 minutes, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, and take the filtrate. Accurately take 5μl of the test solution, inject it into the liquid chromatograph, and calculate the total amount of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid under different extraction solvents. The results are shown in Table 4.

[0124] Table 4 Extraction solvent investigation results

[0125]

[0126]

[0127] From the above, it can be seen that the extraction solvents of 50% methanol, 70% methanol and 50% ethanol have little effect on the contents of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid, so the extraction solvent is tentatively set to 50% methanol.

[0128] 2.3.2.3 Investigation of the amount of extraction solvent added

[0129] Take an appropriate amount of Tianzhuzi formula granules (batch number: TZZ-KL-01), grind to about 0.1g, accurately weigh, place in a stoppered conical bottle, accurately add 50% methanol 25ml, 50ml and 100ml, weigh, ultrasonic treatment (power 600W, frequency 40kHz) for 30 minutes, let cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, and take the filtrate. Accurately take 1μl of the test solution, inject into the liquid chromatograph, and calculate the total amount of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid under different extraction solvents. The results are shown in Table 5.

[0130] Table 5 Investigation of the amount of extraction solvent added

[0131]

[0132] The results showed that when the amount of solvent added was 25ml, 50ml and 100ml, the content difference was not large. The amount of sample added with a peak area close to that of the reference substance was taken, so the amount of solvent added was determined to be 50ml.

[0133] 2.3.2.4 Extraction time investigation

[0134] Take an appropriate amount of Tianzhuzi formula granules (batch number: TZZ-KL-01), grind to about 0.1g, accurately weigh, place in a stoppered conical bottle, accurately add 50ml of 50% methanol, weigh the weight, ultrasonically treat (power 600W, frequency 40kHz) for 15 minutes, 30 minutes and 60 minutes, cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, and take the filtrate. Accurately take 5μl of the test solution, inject into the liquid chromatograph, and calculate the total amount of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid under different extraction solvents. The results are shown in Table 6.

[0135] Table 6 Extraction time investigation

[0136]

[0137]

[0138] The results showed that the content difference was not significant when the extraction time was 15 minutes, 30 minutes and 60 minutes, so 30 minutes of extraction was sufficient. Therefore, the extraction time of the test sample was determined to be 30 minutes.

[0139] In summary, the preparation method of the test sample for the determination of the content of Gandhizi is tentatively as follows: take an appropriate amount of Gandhizi formula granules, grind them into powder, take about 0.1g, accurately weigh it, put it in a stoppered conical flask, accurately add 50ml of 50% methanol, stopper it, weigh it, ultrasonically treat it (power 600W, frequency 40KHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0140] 2.3.2.5 Determination of the content of baicalensis

[0141] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 1.0 ml per minute; the column temperature was 25°C; the detection wavelength was 325 nm. The theoretical plate number calculated based on the chlorogenic acid peak should be no less than 5000.

[0142]

[0143] Preparation of reference solution: Take appropriate amounts of chlorogenic acid reference, neochlorogenic acid reference and cryptochlorogenic acid reference, weigh them accurately, add 50% methanol to prepare a mixed solution containing 50 μg of neochlorogenic acid, 30 μg of chlorogenic acid and 20 μg of cryptochlorogenic acid per 1 ml.

[0144] Preparation of test solution: Take an appropriate amount of Tianzhuzi formula granules, grind them into powder, take about 0.1g, weigh it accurately, put it in a stoppered conical flask, accurately add 50ml of 50% methanol, stopper it tightly, weigh it, treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0145] Determination method: Accurately pipette 5μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0146] 2.3.3 Methodological investigation

[0147] 2.3.3.1 Specificity Investigation

[0148] Preparation of reference solution: Take appropriate amount of neochlorogenic acid reference substance, chlorogenic acid reference substance, and cryptochlorogenic acid reference substance, accurately weigh them, add 50% methanol to make a solution containing 50 μg of each per 1 ml, as the reference solution.

[0149] Preparation of test solution: Take an appropriate amount of Tianzhuzi formula granules, grind them into powder, take about 0.1g, weigh it accurately, put it in a stoppered conical flask, accurately add 50ml of 50% methanol, stopper it tightly, weigh it, ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0150] Preparation of negative control solution: According to the above proposed method for preparing the test solution, prepare a negative control solution lacking dahliae. Fig.10 . Fig.10 Investigation on the specificity of Indian basil; Fig.10 It can be seen that the negative solution has no interference with the determination of the peak to be tested, indicating that the method has good specificity.

[0151] 2.3.3.2 Precision investigation

[0152] The mixed solution of neochlorogenic acid reference substance, chlorogenic acid reference substance and cryptochlorogenic acid reference substance was sampled continuously for 6 times, the peak area was recorded, and the RSD value was calculated. The results are shown in Table 7.

[0153] Table 7 Precision investigation results

[0154]

[0155]

[0156] From the above, it can be seen that in the precision investigation, the peak area RSD value of neochlorogenic acid is 1.0%, the peak area RSD value of chlorogenic acid is 0.1%, and the peak area RSD value of cryptochlorogenic acid is 1.6%, indicating that the instrument has good precision.

[0157] 2.3.3.3 Linear Relationship

[0158] The mixed mother liquor reference solution of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid was accurately aspirated to prepare reference solutions of different concentrations (for content determination), 5 μl of each was aspirated, and injected into the liquid chromatograph to obtain the peak area. The response curve was drawn with the reference concentration (X, μg / ml) as the abscissa and the peak area (Y) as the ordinate. The results are shown in Tables 8, 9, and 10. Fig.11 , 12 、13. Fig.11 New chlorogenic acid standard curve; Fig.12 Standard curve of chlorogenic acid; Fig.13 Standard curve of cryptochlorogenic acid.

[0159] Table 8 New chlorogenic acid standard curve analysis results

[0160]

[0161] Table 9 Chlorogenic acid standard curve analysis results

[0162]

[0163] Table 10 Analysis results of standard curve of cryptochlorogenic acid

[0164]

[0165] The results showed that the linear regression equation of neochlorogenic acid was y=13.2522X-3.7026, and the correlation coefficient R 2 =1.0000, indicating that the linear relationship between the concentration of chlorogenic acid and the peak area is good in the concentration range of 1.622296μg / ml to 162.229600μg / ml; the linear regression equation of chlorogenic acid is y=15.4919X-7.2945, and the correlation coefficient R 2 =1.0000, indicating that the linear relationship between chlorogenic acid concentration and peak area is good in the concentration range of 2.6763696μg / ml to 267.6369600μg / ml; the linear regression equation of cryptochlorogenic acid is y=12.242X-2.0249, and the correlation coefficient R 2 =1.0000, indicating that the linear relationship between the concentration of chlorogenic acid and the peak area is good in the concentration range of 1.03082μg / ml to 103.08200μg / ml.

[0166] 2.3.3.4 Repeatability

[0167] Take an appropriate amount of Tianzhuzi formula granules (batch number: TZZ-KL-01), grind it into powder, about 0.1g, accurately weigh 6 portions, and prepare the test solution by the same operator according to the determined method. Accurately pipette 5μl of the test solution respectively, inject it into the liquid chromatograph, and calculate the total amount of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid in the 6 samples. The results are shown in Table 11.

[0168] Table 11 Repeatability test results

[0169]

[0170]

[0171] The results showed that the RSD value of the total content was 0.7%, indicating that the method had good repeatability.

[0172] 2.3.3.5 Intermediate precision

[0173] Based on the experimental conditions proposed above, different personnel carried out the determination on different instruments at different times. An appropriate amount of Tianzhuzi granules (batch number: TZZ-KL-01) was accurately weighed and ground into powder. About 0.1 g was taken to prepare the test sample for determination. The results of the contents of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid were calculated, and the results are shown in Table 12.

[0174] Table 12 Intermediate precision inspection results

[0175]

[0176] The results showed that the RSD value of the total content was 0.8%, and the intermediate precision of this method was good.

[0177] 2.3.3.6 Sample recovery rate

[0178] Take the test sample with known content (batch number: TZZ-KL-01, chlorogenic acid content of 10.7 mg / g, neochlorogenic acid content of 4.6 mg / g, cryptochlorogenic acid content of 5.2 mg / g), grind it into powder, about 0.05 g, a total of 6 portions, accurately weighed, and accurately added a certain amount of neochlorogenic acid reference substance (4.566536 μg / ml), chlorogenic acid reference substance (10.306026 μg / ml), and cryptochlorogenic acid reference substance (5.256942 μg / ml) respectively. Prepare and determine the test solution according to the proposed method, calculate the recovery rate, and the results are shown in Table 13. The calculation formula is as follows:

[0179]

[0180] Table 13 Sample recovery test results

[0181]

[0182]

[0183] The results showed that the average recovery rate of neochlorogenic acid was 99.3% with an RSD of 1.3%, the average recovery rate of chlorogenic acid was 101.9% with an RSD of 1.5%, and the average recovery rate of cryptochlorogenic acid was 101.6% with an RSD of 1.3%. This method has good accuracy.

[0184] 2.3.3.7 Durability test

[0185] 2.3.3.7.1 Column durability study

[0186] Based on the above experimental conditions, C18(2)100A250×4.6mm, 5μm; C18 250×4.6mm, 5μm; ZORBAX Eclipse Plus C18 250×4.6mm, 5μm chromatographic columns were used for investigation. 5μl of the test solution was accurately drawn and injected into the liquid chromatograph to calculate the total amount of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid in the sample. The results are shown in Table 14. Fig.14 . Fig.14 Chromatographic column inspection.

[0187] Table 14 Results of the investigation on the durability of the chromatographic column

[0188]

[0189] The results showed that the analytical chromatographic indicators of different chromatographic columns were good.

[0190] 2.3.3.8 Stability test

[0191] According to the experimental conditions proposed above, a test solution was prepared, and the peak area of ​​uridine was measured at 0h, 2h, 4h, 8h, 16h and 24h respectively. The results are shown in Table 15.

[0192] Table 15 Stability test results

[0193]

[0194]

[0195] The results showed that under the experimental conditions, the RSD value of the neochlorogenic acid content was 0.6%, the RSD value of the chlorogenic acid content was 0.6%, and the RSD value of the cryptochlorogenic acid content was 1.3%. The test solution had good stability within 24 hours.

[0196] 2.3.4 Sample content determination verification

[0197] According to the proposed method, 21 batches of medicinal materials, 21 batches of decoction pieces, 21 batches of standard decoctions and 3 batches of formula granule samples of Gastrodia elata were tested, and the results are shown in Tables 16-20.

[0198] Table 16 Results of content determination of 21 batches of Fructus Sieb.

[0199]

[0200] Table 17 Content determination results of 21 batches of Fructus Sieb.

[0201]

[0202]

[0203] Table 18 Content determination results of 21 batches of standard decoction of Fructus dasyphyllae

[0204]

[0205] Table 19 Test results of content of 3 batches of Tianzhuzi formula granules

[0206]

[0207] From the above, it can be seen that the method for determining the content of Ganoderma lucidum can effectively detect Ganoderma lucidum medicinal materials, decoction pieces, standard decoctions, and formula granule samples, making the method stable and feasible.

[0208] Comparative Example 1

[0209] The pretreatment was the same as in the previous example. Octadecylsilane bonded silica gel was used as the filler (column length was 250 mm, inner diameter was 4.6 mm, particle size was 5 μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 1.0 ml per minute; the column temperature was 25° C.; and the detection wavelength was 325 nm.

[0210]

[0211] The results are as follows Fig.15 , Fig.15 Be the chromatogram of comparative example 1; By Fig.15 It can be seen that the chromatographic peaks of neochlorogenic acid and cryptochlorogenic acid are poorly separated from the adjacent impurity peaks, and the gradient ratio was changed to continue optimization.

[0212] Comparative Example 2

[0213] The pretreatment was the same as in the previous example. Octadecylsilane bonded silica gel was used as the filler (column length was 250 mm, inner diameter was 4.6 mm, particle size was 5 μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 1.0 ml per minute; the column temperature was 25°C; the detection wavelength was 325 nm

[0214]

[0215] The results are as follows Fig.16 , Fig.16 Be the chromatogram of comparative example 2; By Fig.16 It can be seen that the chromatographic peaks of neochlorogenic acid and cryptochlorogenic acid are poorly separated from the adjacent impurity peaks, and the gradient ratio was changed to continue optimization.

[0216] Comparative Example 3

[0217] The pretreatment was the same as in the previous example. The results of different mobile phases were compared. Octadecylsilane bonded silica gel was used as the filler (column length was 250 mm, inner diameter was 4.6 mm, particle size was 5 μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid, 0.1% formic acid, and 0.1% glacial acetic acid solution were used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 1.0 ml per minute; the column temperature was 25°C; and the detection wavelength was 325 nm.

[0218]

[0219] The results are as follows Fig.17 , Fig.17 Be the chromatogram of comparative example 3; By Fig.17 It can be seen that when 0.1% phosphoric acid is used as mobile phase B, the three chromatographic peaks of the sample are better separated, and the proposed mobile phase B is 0.1% phosphoric acid.

[0220] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing an HPLC content determination method of Fructus Sieb. A) Dissolve the test sample raw material with a solvent, extract, and obtain a test solution; B) the test solution is measured by high performance liquid chromatography to obtain an HPLC spectrum of Fructus pyrifera; The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is an acetonitrile solution, the mobile phase B is a 0.1% phosphoric acid aqueous solution, and the gradient elution is performed; The gradient elution is specifically: 0-20min, phase A: 10%→11%, phase B: 90→89%; 20-25 min, phase A: 11%→18%, phase B: 89%→82%; 25-40min, phase A: 18%→40%, phase B: 82%→60%.

2. The method according to claim 1, characterized in that The invention also includes preparing a reference solution: taking a chlorogenic acid reference substance, a neochlorogenic acid reference substance and a cryptochlorogenic acid reference substance respectively, dissolving them in 50% methanol to obtain a reference solution; The reference solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of the dahliae fructus are qualitatively identified according to the chromatogram of the reference.

3. The method according to claim 2, characterized in that The concentrations of the reference solutions are specifically as follows: 2.6763696 μg / ml to 267.6369600 μg / ml for the chlorogenic acid reference substance, 1.622296 μg / ml to 162.229600 μg / ml for the neochlorogenic acid reference substance, and 1.03082 μg / ml to 103.08200 μg / ml for the cryptochlorogenic acid reference substance.

4. The method according to claim 1, characterized in that: The chromatographic column is C 18 250×4.6mm 5μm; column temperature is 25℃~35℃; the theoretical plate number calculated based on chlorogenic acid should be no less than 5000.

5. The method according to claim 4, characterized in that The flow rate of the mobile phase is 0.8-1.2 mL / min; the injection volume is 1-5 μL.

6. The method according to claim 4, characterized in that The detection wavelength is 325 nm.

7. The method according to claim 5, characterized in that The column temperature was 25° C. and the mobile phase flow rate was 1.0 mL / min.

8. The method according to claim 1, characterized in that In step A), the solvent is 50% to 70% methanol; the extraction is ultrasonic extraction; the ultrasonic power is 600W, the frequency is 40kHz; and the ultrasonic time is 15 to 60 minutes.

9. The method according to claim 8, characterized in that The ratio of the mass g of the test material to the volume mL of the solvent is 0.1-1:25-100; The test sample raw material is one or more of the following: guava seeds medicinal materials, guava seeds decoction pieces, guava seeds standard decoction or guava seeds formula granules.

10. A method for evaluating the quality of Fructus Gynurae ginseng raw materials and preparations thereof, comprising: performing detection by the method described in any one of claims 1 to 9 and analyzing the detection results.

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