A method for constructing HPLC characteristic spectrum of granules of scutellaria baicalensis

Abstract

The present invention provides a method for constructing an HPLC characteristic spectrum of a chicken bone grass formula particle, including: A) dissolving a test sample raw material with a solvent, extracting, and obtaining a test liquid; B) determining the test liquid by high performance liquid chromatography to obtain an HPLC characteristic spectrum of a chicken bone grass formula particle; the chromatographic conditions of the high performance liquid chromatography are: a chromatographic column is a C 18 column; mobile phase A is a 0.1% formic acid aqueous solution, and mobile phase B is acetonitrile, and gradient elution. The present invention adopts high performance liquid chromatography, selects acetonitrile 0.1% formic acid solution as the mobile phase for gradient elution, and uses gallic acid, abrinine, erythrine, isoschaftoside, schaftoside, and vicinin 2 as reference substances to establish an HPLC characteristic spectrum of a chicken bone grass formula particle, with good repeatability and precision, stable and reliable method, and can control the quality of the chicken bone grass formula particle.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a method for constructing an HPLC characteristic spectrum of tricholoma foetida formula particles. Background Art

[0002] Herba Cercidiphyllum is a plant of the genus Abrus in the Leguminosae family. It has a long history of medicinal use in my country. The whole plant is often used as medicine. It has the effects of removing dampness and jaundice, clearing away heat and detoxifying, soothing the liver and relieving pain. It is often used clinically to treat damp-heat jaundice, rib discomfort, stomach pain, mastitis, fatty liver disease, hepatitis and internal injuries. In addition, Herba Cercidiphyllum also has multiple pharmacological effects such as anti-tumor, antioxidant, antibacterial, antiviral, anti-inflammatory and analgesic, promoting wound healing, and immunomodulation. The wide range of pharmacological effects of Herba Cercidiphyllum, coupled with the characteristics of being cheap and easy to cultivate, make it have extremely high economic value and social benefits, and have huge development potential and broad market prospects. According to literature review, there are few studies on the relevant quality standards of Herba Cercidiphyllum formula granules.

[0003] At present, there is a lack of an effective means to control its quality. The present invention aims to construct a high-performance liquid phase characteristic spectrum method for the formula granules of Gastrodia elata, which comprehensively reflects the quality level of the formula granules of Gastrodia elata and its related preparations, and provides guidance for the quality control of the medicinal materials, decoction pieces and their related preparations of Gastrodia elata formula granules. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for constructing an HPLC characteristic spectrum of tricholoma polyphylla formula granules. The HPLC characteristic spectrum method of tricholoma polyphylla formula granules constructed by the present invention is stable and reliable, and can control the quality of tricholoma polyphylla formula granules.

[0005] The present invention provides a method for constructing an HPLC characteristic spectrum of tricholoma serrata formula particles, comprising:

[0006] A) Dissolve the test sample raw material with a solvent, extract, and obtain a test solution;

[0007] B) the liquid to be tested is measured by high performance liquid chromatography to obtain HPLC characteristic spectrum of the granules of the herbaceous foetida formula;

[0008] 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 a 0.1% formic acid aqueous solution, the mobile phase B is acetonitrile, and the elution is gradient.

[0009] Preferably, the method further comprises preparing a reference solution: taking gallic acid, abrinine, erythrine, isoschaftoside, schaftoside, and vecinin-2 respectively, dissolving them in methanol to obtain a reference solution;

[0010] The reference solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of the HPLC characteristic spectrum of the herbaceous foetida formula granules are qualitatively identified according to the chromatogram of the reference.

[0011] Preferably, the concentrations of the reference solution are as follows: 50 μg / mL of gallic acid, 50 μg / mL of abrinine, 60 μg / mL of erythrine, 55 μg / mL of isoschaftoside, 50 μg / mL of schaftoside, and 30 μg / mL of vecinin-2.

[0012] Preferably, the gradient elution is specifically:

[0013] 0-4min, phase A: 98-90%, phase B: 2-10%;

[0014] 4-16 min, phase A: 90%-90%, phase B: 10%-10%;

[0015] 16-17 min, phase A: 90%-86%, phase B: 10%-14%;

[0016] 17-20min, phase A: 86%-85%, phase B: 14%-15%;

[0017] 20-21 min, phase A: 85%-85%, phase B: 15%-15%;

[0018] 21-22 min, phase A: 85%-83%, phase B: 15%-17%;

[0019] 22~50min, phase A: 83%~83%, phase B: 17%~17%.

[0020] Preferably, the chromatographic column is InertSustain C18 250×4.6mm5μm, phenomenexC18250×4.6mm5μm, Agilent 5TC-C18(2)250×4.6mm5μm; the column temperature is 35°C.

[0021] Preferably, the flow rate of the mobile phase is 1.0 mL / min; the detection wavelength is 280 nm; and the injection volume is 10 μL.

[0022] Preferably, the similarity of the HPLC characteristic spectrum of the herbaceous granules is evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and a HPLC standard characteristic spectrum of the herbaceous granules composed of 7 characteristic peaks is obtained, wherein peak 1 is gallic acid, peak 2 is abrinine, peak 3 is erythrine, peak 4 is vecinine-2, peak 5 is schaftoside, and peak 6 is isoschaftoside.

[0023] Preferably, in the standard characteristic spectrum, taking Vitzenin-2 as the reference peak S peak, the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within ±8% of the specified value, and the specified values ​​are: 0.308 (peak 1), 0.459 (peak 2), 0.601 (peak 3), 1.000 (peak 4, S), 1.092 (peak 5), 1.132 (peak 6), 1.245 (peak 7).

[0024] Preferably, in step A), the solvent is water; the extraction is heating reflux extraction or ultrasonic extraction; the heating reflux extraction time is 20 to 40 minutes; the ultrasonic power is 600W, the frequency is 40kHz; and the ultrasonic time is 30 minutes.

[0025] Preferably, in step A), the ratio of the mass g of the test sample raw material to the volume mL of the solvent is (0.2-0.3): (20-30);

[0026] The test sample raw material is chicken bone grass formula granules.

[0027] Compared with the prior art, the present invention provides a method for constructing an HPLC characteristic spectrum of granules of scutellaria baicalensis, 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 characteristic spectrum of granules of scutellaria baicalensis; the chromatographic conditions of the high performance liquid chromatography method are: a chromatographic column is a C18 column; mobile phase A is a 0.1% formic acid aqueous solution, and mobile phase B is acetonitrile, and gradient elution. The present invention adopts high performance liquid chromatography, selects acetonitrile-0.1% formic acid solution as the mobile phase for gradient elution, and uses gallic acid, abrinine, erythrine, isoschaftoside, schaftoside, and vecinin-2 as reference substances to establish an HPLC characteristic spectrum of granules of scutellaria baicalensis, which has good repeatability and precision, and the method is stable and reliable, and the quality of granules of scutellaria baicalensis can be controlled. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the ultraviolet absorption spectrum of gallic acid;

[0029] Figure 2 Abrusine UV absorption spectrum;

[0030] Figure 3 UV absorption spectrum of erythrine;

[0031] Figure 4 Ultraviolet absorption spectrum of schaftoside;

[0032] Figure 5 UV absorption spectrum of isoschaftoside;

[0033] Figure 6 UV absorption spectrum of Vetsenin-2;

[0034] Figure 7 3D chromatogram of the chicken bone grass formula granules;

[0035] Figure 8 Comparison chart of each wavelength of the chicken bone grass formula granules;

[0036] Fig. 9 This is the result diagram of the chromatographic column temperature investigation;

[0037] Fig.10 This is the flow velocity investigation result diagram;

[0038] Fig.11 This is the delay test result diagram;

[0039] Fig.12 This is the result diagram of the investigation of the extraction solvent;

[0040] Fig.13 This is the result diagram of the investigation of extraction methods;

[0041] Fig.14 This is the result diagram of the extraction time investigation;

[0042] Fig.15 This is the result of the investigation on the amount of solvent;

[0043] Fig.16 Identification of chromatographic peaks of characteristic spectrum of granules of Gastrodia elata formula;

[0044] Fig.17 Figures showing survey results from different instruments;

[0045] Fig.18 Investigation on the durability of chromatographic columns containing granules of scutellaria baicalensis formula;

[0046] Fig.19 Characteristics of the granules of the chicken bone grass formula;

[0047] Fig. 20 This is the comparative characteristic spectrum of the Herba Codonopsis pilosulae formula granules;

[0048] Fig.21 It is a comparison diagram of different flow phases;

[0049] Fig. 22 The gradient diagrams of different mobile phases. DETAILED DESCRIPTION

[0050] The present invention provides a method for constructing an HPLC characteristic spectrum of a herbaceous granule, and 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.

[0051] The present invention provides a method for constructing an HPLC characteristic spectrum of tricholoma serrata formula particles, comprising:

[0052] A) Dissolve the test sample raw material with a solvent, extract, and obtain a test solution;

[0053] B) the liquid to be tested is measured by high performance liquid chromatography to obtain HPLC characteristic spectrum of the granules of the herbaceous foetida formula;

[0054] 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 a 0.1% formic acid aqueous solution, the mobile phase B is acetonitrile, and the elution is gradient.

[0055] The raw materials of the test sample of the present invention include but are not limited to the granules of scutellaria baicalensis formula.

[0056] The present invention provides a method for constructing an HPLC characteristic spectrum of the herbaceous granules. First, the herbaceous granules are raw materials, dissolved with a solvent, and extracted to obtain a test solution. The solvent is preferably water.

[0057] Specifically, the contents of the chicken bone grass formula granules are dissolved with a solvent, extracted, cooled, shaken well, and filtered to obtain the product.

[0058] The extraction of the present invention is heating reflux extraction or ultrasonic extraction; the time of the heating water flow extraction is preferably 15 to 45 minutes; more preferably 20 to 40 minutes; most preferably 30 minutes.

[0059] The power of the ultrasound is 600W, the frequency is 40kHz, and the ultrasound time is 30min.

[0060] Among them, the ratio of the mass g of the raw material of the Acanthopanax formula granules to the volume mL of the solvent is preferably (0.2-0.3): (20-30); more preferably 0.5:25.

[0061] 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.

[0062] The present invention also comprises preparing a reference solution: taking gallic acid, abrinine, erythrine, isoschaftoside, schaftoside and vecinin-2 respectively, dissolving them in methanol to obtain a reference solution.

[0063] Among them, gallic acid was 50μg / mL, abrinine was 50μg / mL, erythrine was 60μg / mL, isoschaftoside was 55μg / mL, schaftoside was 50μg / mL, and vecinin-2 was 30μg / mL.

[0064] The reference solution is measured by high performance liquid chromatography to obtain the chromatograms of the reference; and the components of the HPLC characteristic spectrum of the herbaceous spatholobi formula granules are qualitatively measured according to the chromatograms of the reference.

[0065] The mobile phase A of the present invention is 0.1% formic acid aqueous solution, the mobile phase B is acetonitrile, and the elution is performed by gradient.

[0066] The gradient elution of the present invention is preferably specifically:

[0067] 0-4min, phase A: 98-90%, phase B: 2-10%;

[0068] 4-16 min, phase A: 90%-90%, phase B: 10%-10%;

[0069] 16-17 min, phase A: 90%-86%, phase B: 10%-14%;

[0070] 17-20min, phase A: 86%-85%, phase B: 14%-15%;

[0071] 20-21 min, phase A: 85%-85%, phase B: 15%-15%;

[0072] 21-22 min, phase A: 85%-83%, phase B: 15%-17%;

[0073] 22~50min, phase A: 83%~83%, phase B: 17%~17%.

[0074] The present invention has good baseline separation under the above elution gradient, good separation of each peak and a stable baseline.

[0075] InertSustain C18 250×4.6mm5μm, phenomenonexC18 250×4.6mm 5μm, Agilent5TC-C18(2)250×4.6mm5μm.

[0076] The present invention adopts a chromatographic column to perform durability detection, and has good durability.

[0077] The column temperature of the present invention is preferably 35° C. At the above chromatographic column temperature, the chromatographic peak shape of the present invention is good and the separation degree is moderate.

[0078] The mobile phase flow rate is preferably 1.0 mL / min.

[0079] The present invention found that the chromatographic peaks were better separated at a flow rate of 1.0 mL / min, and the separation degree was moderate, which was the most preferred solution.

[0080] The column temperature of the present invention is preferably 35°C.

[0081] The detection wavelength of the present invention is preferably 280 nm.

[0082] The inventors found that at 280 nm, the chromatographic peak has a larger amount of information, the chromatogram baseline is more stable, and the peak area of ​​each peak is larger.

[0083] The injection volume of the present invention is 10 to 15 μL, preferably 10 μL.

[0084] The beneficial effects of the present invention are that, under a liquid chromatography condition, the substance group of the herbaceous spatholobi formula granules is controlled by a characteristic spectrum, and the characteristic spectrum is located by gallic acid, abrinine, erythrine, isoschaftoside, schaftoside, and vecinin-2; the cost of detection can be greatly reduced, and qualitative detection can be achieved.

[0085] The similarity of the HPLC characteristic spectra of the Herba Corydalis Glucosides was evaluated by the Chinese medicine chromatographic fingerprint similarity evaluation system, and the HPLC standard characteristic spectrum of the Herba Corydalis Glucosides was obtained, which consisted of 7 characteristic peaks, among which peak 1 was gallic acid, peak 2 was abrinine, peak 3 was erythrine, peak 4 was vecinine-2, peak 5 was schaftoside, and peak 6 was isoschaftoside.

[0086] In the standard characteristic spectrum, taking Vitzenin-2 as the reference peak S peak, the relative retention time of each characteristic peak and the S peak was calculated, and the relative retention time was within ±8% of the specified value, and the specified values ​​were: 0.308 (peak 1), 0.459 (peak 2), 0.601 (peak 3), 1.000 (peak 4, S), 1.092 (peak 5), 1.132 (peak 6), and 1.245 (peak 7).

[0087] Quality judgment standard: Take a sample of the herba syringae formula granules and operate according to the same method as above to obtain the characteristic spectrum of the herba syringae formula granules. Use the 2012 edition of the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" of the National Pharmacopoeia Committee to analyze the standard characteristic spectrum of the herba syringae formula granules and the sample characteristic spectrum, and the similarity is greater than 0.90.

[0088] The method provided by the present invention can effectively monitor the quality of different batches of tricholoma foetida formula granules to stabilize the quality. The method has the characteristics of high precision and good reproducibility, and is conducive to comprehensive monitoring of product quality.

[0089] The characteristic spectrum of the herbaceous spatholobi formula granules established by the present invention uses uracil, adenine and hypoxanthine as references, focuses on the order of each characteristic peak and the correlation with medicinal materials and intermediate products, can comprehensively evaluate the overall quality features of the product, and the method is scientific and reliable.

[0090] The invention provides a method for constructing an HPLC characteristic spectrum of a herbaceous herb formula granule, comprising: A) dissolving a test sample raw material with a solvent, extracting, and obtaining a test liquid; B) determining the test liquid with a high performance liquid chromatography method, and obtaining an HPLC characteristic spectrum of the herbaceous herb formula granule; the chromatographic conditions of the high performance liquid chromatography method are: a chromatographic column is a C18 column; mobile phase A is a 0.1% formic acid aqueous solution, and mobile phase B is acetonitrile, and gradient elution. The invention adopts high performance liquid chromatography, selects acetonitrile-0.1% formic acid solution as a mobile phase for gradient elution, and uses gallic acid, abrinine, erythrine, isoschaftoside, schaftoside, and vecinin-2 as reference substances to establish an HPLC characteristic spectrum of the herbaceous herb formula granule, which has good repeatability and precision, and the method is stable and reliable, and the quality of the herbaceous herb formula granule can be controlled.

[0091] To further illustrate the present invention, a method for constructing an HPLC characteristic spectrum of a herba scutellariae formula granules provided by the present invention is described in detail below in conjunction with examples.

[0092] High performance liquid chromatograph: Waters 2695-e2998 high performance liquid chromatograph, Agilent 1260 high performance liquid chromatograph, Shimadzu LC-20AD high performance liquid chromatograph;

[0093] Electronic balance: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instrument Co., Ltd.);

[0094] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0095] Ultrasonic cleaner: KQ5200DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0096] Chromatographic columns: InertSustainC18 250×4.6mm 5μm, phenomenex C18 250×4.6mm5μm, Agilent 5-TC C18(2)5μm 4.6×250mm;

[0097] Acetonitrile and formic acid were of chromatographic grade, water was ultrapure water, and the other reagents were of analytical grade;

[0098] Gallic acid (China Food and Drug Inspection Institute, batch number: 110831-201611, content is 95.6%);

[0099] Abrin (China Food and Drug Inspection Institute, batch number: 111808-201302, content 96.6%);

[0100] Erythrina alkaloids (Sichuan Weikeqi Biotechnology Co., Ltd., batch number: wkq18012302, content ≥92%);

[0101] Weicaining-2 (Sichuan Weiqi Biotechnology Co., Ltd., batch number: wkq18012302, content ≥98%);

[0102] Schaftoside (Beijing Century Aoke Biotechnology Co., Ltd., batch number: 180320, content ≥98%);

[0103] Isoschaftoside (Beijing Century Aoke Biotechnology Co., Ltd., batch number: 170813, content ≥ 98%);

[0104] Aconitum carmichaelii control medicinal material (Beijing Century Aoke Biotechnology Co., Ltd., batch number: 171110).

[0105] Aconitum formula granules (SY1810001, SY1810002, SY1810003, 1802001).

[0106] Example 1 Chromatographic Condition Screening

[0107] 1.2 Chromatographic conditions

[0108] Octadecylsilane bonded silica gel was used as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); 0.1% formic acid was used as mobile phase A, acetonitrile was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 1.0 mL per minute; the column temperature was 35°C; the detection wavelength was 280 nm. The theoretical plate number calculated based on the Vescinin-2 peak should be no less than 6000.

[0109]

[0110]

[0111] 1.2.1 Wavelength selection

[0112] Based on the experimental conditions proposed above, the diode array detector was used to perform full-band scanning of the test solution, and the chromatograms of the test solution at wavelengths of 250nm, 270nm, 280nm, 300nm, and 330nm were extracted respectively. Figure 1-Figure 8 .in Figure 1 is the ultraviolet absorption spectrum of gallic acid;

[0113] Figure 2 Abrusine UV absorption spectrum; Figure 3 UV absorption spectrum of erythrine; Figure 4 Ultraviolet absorption spectrum of schaftoside; Figure 5 UV absorption spectrum of isoschaftoside; Figure 6 UV absorption spectrum of Vetsenin-2; Figure 7 3D chromatogram of the chicken bone grass formula granules; Figure 8 Comparison chart of each wavelength of the chicken bone grass formula granules. The results show that when the detection wavelength is 280nm, the chromatographic peak information is larger, the chromatogram baseline is more stable, and the peak area of ​​each peak is larger, so the detection wavelength is determined to be 280nm.

[0114] 1.2.2 Column temperature investigation

[0115] Based on the experimental conditions proposed above, the column temperatures of 25℃, 30℃ and 35℃ were investigated respectively. Fig. 9 , Table 1. Fig. 9 This is the result diagram of the chromatographic column temperature investigation;

[0116] Table 1 Column temperature investigation - characteristic peak relative retention time ratio

[0117]

[0118] The results showed that when the column temperature was 25℃, 30℃, and 35℃, the relative retention time RSD of each characteristic peak was 0.00%-4.64%. When the column temperature was 35℃, the chromatogram peak shape was better and the separation was moderate. Therefore, the column temperature was determined to be 35℃.

[0119] 1.2.3 Flow rate investigation

[0120] Based on the experimental conditions proposed above, the flow rates of 0.8ml / min, 1ml / min, and 1.2ml / min were investigated. Fig.10 , Table 2; Fig.10 This is the flow rate investigation result diagram.

[0121] Table 2 Flow rate investigation - characteristic peak relative retention time ratio

[0122]

[0123] The results showed that when the flow rates were 0.8mL / min, 1.0mL / min, and 1.2mL / min, the relative retention time RSD of each characteristic peak was 0.00%-16.39%. When the flow rate was 1.0mL / min, the chromatogram peak shape was good and the separation was moderate. Therefore, the flow rate was determined to be 1.0mL / min.

[0124] 1.2.4 Delay test

[0125] Based on the above experimental conditions, the chromatogram acquisition time was extended to 100 min. Fig.11 , Fig.11 This is a graph showing the delay test results.

[0126] The results show that the chromatogram peaks have been completely collected when the chromatogram acquisition time reaches 50 minutes. Therefore, the chromatogram acquisition time is determined to be 50 minutes.

[0127] 1.3 Preparation of reference solution

[0128] Take about 0.5 g of Glechoma longituba reference medicinal material, accurately weigh it, accurately add 50 ml of water, weigh it, ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with water, shake it well, filter it, and take the filtrate as the reference solution of the reference medicinal material.

[0129] Take appropriate amounts of gallic acid reference substance, abrinine reference substance, erythrine reference substance, isoschaftoside reference substance, schaftoside reference substance, and vicinin-2 reference substance, weigh them accurately, and add methanol to prepare mixed solutions containing 50 μg of gallic acid, 50 μg of abrinine, 60 μg of erythrine, 55 μg of isoschaftoside, 50 μg of schaftoside, and 30 μg of vicinin-2 per 1 ml as reference substance solutions.

[0130] 1.4 Preparation of test solution

[0131] 1.4.1 Investigation of extraction solvent

[0132] Take about 0.25g of this product (batch number: 1802001), weigh it accurately, put it in a stoppered conical flask, and test the extraction solvents of the test sample as water, methanol, 80% methanol, and 50% methanol solution. Add 25mL of each extraction solvent, stopper it, weigh it, and treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with the extraction solvent, shake it well, filter it, and take the filtrate to obtain it. Fig.12 , Fig.12 This is a diagram showing the results of the extraction solvent investigation.

[0133] The results showed that when the extraction solvent was water, the chromatogram obtained had a large amount of chromatographic peak information, good peak shape and good separation, so water was selected as the extraction solvent for the characteristic spectrum of the herbaceous granules.

[0134] 1.4.2 Investigation of extraction methods

[0135] Take about 0.25g of this product (batch number: 1802001), weigh it accurately, put it in a stoppered conical flask, add 25mL of water accurately, stopper it, weigh it, and examine the extraction method of the test product, which is reflux extraction and ultrasonic extraction, respectively. The extraction time is 30 minutes, let it cool, weigh it again, make up the lost weight with water, shake it well, filter it, and take the filtrate. Fig.13 , Fig.13 This is a graph showing the results of the extraction method investigation.

[0136] The results showed that there was no significant difference in the peak shape and peak area of ​​the chromatograms obtained when the test samples were subjected to ultrasonic extraction and reflux extraction respectively. Therefore, the simple ultrasonic method was selected as the extraction method for the preparation of the test sample solution of the Herba Codonopsis pilosulae formula granules.

[0137] 1.4.3 Extraction time investigation

[0138] Take about 0.25g of this product (batch number: 1802001), weigh three portions accurately, place in a stoppered conical flask, add 25mL of water accurately, stopper, weigh, and ultrasonically treat (power 600W, frequency 40kHz), and observe the extraction time of the test product for 10 minutes, 30 minutes, and 50 minutes respectively, let cool, weigh again, make up the lost weight with water, shake well, filter, and take the filtrate to obtain. Fig.14 . Fig.14 This is the result of the extraction time investigation. The results show that when the extraction time is 30 minutes, the extraction is sufficient and the peak shape of the obtained chromatogram is good. Therefore, the extraction time of the test sample is determined to be 30 minutes.

[0139] 1.4.4 Examination of solvent dosage

[0140] Take about 0.25g of this product (batch number: 1802001), weigh three portions accurately, place in a stoppered conical flask, add water accurately, stopper, weigh, and ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes. Test the amount of the test solvent at 10mL, 25mL, and 50mL respectively, cool, weigh again, make up the lost weight with water, shake well, filter, and take the filtrate. Fig.15 . Fig.15 This is a graph showing the results of investigating solvent usage.

[0141] The results showed that when the solvent dosage was 25 mL, the chromatographic peak area was moderate and the peak shape was good. Therefore, the solvent dosage of the test sample was determined to be 25 mL.

[0142] 1.4.5 Finalize the test sample preparation method

[0143] Take an appropriate amount of this product, grind it into powder, take about 0.25g, weigh it accurately, put it in a stoppered conical flask, accurately add 25mL of water, stopper it, 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 water, shake it well, filter it, and take the filtrate to obtain it.

[0144] Example 2 Methodological Investigation

[0145] 2.5.1 Chromatographic peak identification

[0146] Preparation of test solution: According to the experimental conditions proposed above, prepare the test solution of Acanthopanax formula granules.

[0147] Preparation of reference solution: Take about 0.5g of Herba Corydalis as a reference medicinal material, weigh it accurately, add 50ml of water 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 water, shake it well, filter it, and take the filtrate as the reference medicinal material reference solution. Separately take appropriate amount of gallic acid reference substance, abrinine reference substance, erythrine reference substance, isoschaftoside reference substance, schaftoside reference substance, and vecinin-2 reference substance, weigh them accurately, add methanol to prepare solutions containing 50μg of gallic acid, 50μg of abrinine, 60μg of erythrine, 55μg of isoschaftoside, 50μg of schaftoside, and 30μg of vecinin-2 per ml, as reference substance reference solution.

[0148] Preparation of negative control solution: According to the experimental conditions proposed above, prepare the negative control solution of Acanthopanax formula granules.

[0149] Locate the characteristic spectrum peaks of the granules of the herbaceous sage. Fig.16 . Fig.16 Chromatographic peak identification of characteristic spectrum of granules of radix scutellariae formula: The results show that peak 1 is gallic acid; peak 2 is abrinine; peak 3 is erythrine; peak 4 is vecinin-2; peak 5 is schaftoside; peak 6 is isoschaftoside. In the following methodological investigation, 7 peaks in the sample were investigated.

[0150] 2.5.2 Precision test

[0151] Take the test solution of the Herba Corydalis Formula Granules (batch number: 1802001), inject 6 times continuously according to the proposed experimental method, 10 μl each time, and calculate the retention time and peak area of ​​each peak. See Table 3 and Table 4.

[0152] Table 3 Precision characteristic peak retention time investigation

[0153]

[0154] Table 4 Precision investigation - characteristic peak area

[0155]

[0156] The results showed that the RSD values ​​of the retention time of each peak were 0.16%-0.49%, and the RSD of the peak area was 0.08%-2.75%, indicating that the instrument had good precision.

[0157] 2.5.3 Repeatability study

[0158] 6 portions of Herba Codonopsis pilosulae formula granules (batch number: 1802001) were accurately weighed and prepared and tested according to the proposed experimental method. See Table 5 and Table 6.

[0159] Table 5 Repeatability study - characteristic peak relative retention time ratio

[0160]

[0161] Table 6 Repeatability study - characteristic peak relative peak area ratio

[0162]

[0163]

[0164] The results showed that the RSD of the relative retention time of each characteristic peak was between 0.00% and 0.29%, and the RSD of the relative peak area of ​​each characteristic peak was between 0.00% and 5.78%. The method had good repeatability.

[0165] 2.5.4 Intermediate precision study

[0166] 2.5.4.1 Investigation with different instruments

[0167] Based on the experimental conditions proposed above, three portions of Herba Codonopsis pilosulae granules (batch number: 1802001) were accurately weighed to prepare test solutions, which were then measured on Agilent 1260, Waters e2695-2998, and Shimadzu LC-20AD high performance liquid chromatographs (the chromatographic columns were Agilent 5TC-C18 (2) 250×4.6mm5μm). Fig.17 , Table 7, Table 8. Fig.17 Figures showing survey results from different instruments;

[0168] Table 7 Different instruments investigated - relative retention time ratio of characteristic peaks

[0169]

[0170] Table 8 Different instruments investigated - characteristic peak relative peak area ratio

[0171]

[0172] The results showed that when the three instruments were used to detect the test samples, the RSD of the relative retention time of each characteristic peak was between 0.00% and 2.70%; the RSD of the relative peak area of ​​each characteristic peak was between 0.00% and 20.04%, and the method had good durability.

[0173] 2.5.4.2 Investigation by different personnel and time

[0174] Based on the experimental conditions proposed above, different personnel (A, B) accurately weighed one portion of the Herba Corydalis Formula Granules (batch number: 1802001) at different times (T1, T2) to prepare the test sample for determination. See Table 9 and Table 10.

[0175] Table 9 Different personnel and time investigation - characteristic peak relative retention time ratio

[0176]

[0177] Table 10: Different personnel and time surveys - characteristic peak relative peak area ratios

[0178]

[0179] The results showed that under different sample preparation personnel and different sample preparation time conditions, the RSD of the relative retention time of each characteristic peak was between 0.00% and 0.07%; the RSD of the relative peak area of ​​each characteristic peak was between 0.00% and 0.90%, and the method had good precision.

[0180] 2.5.5 Durability test

[0181] 2.5.5.1 Study on the durability of chromatographic columns Based on the experimental conditions proposed above, the chromatographic columns InertSustain C18 250×4.6mm 5μm, phenomenex C18 250×4.6mm 5μm, and Agilent 5TC-C18 (2) 250×4.6mm 5μm were studied. Fig.18 , Table 11, Table 12. Fig.18 Investigation on the durability of chromatographic columns containing granules formulated with Acanthopanax gracilis.

[0182] Table 11 Column durability investigation - relative retention time of characteristic peaks

[0183]

[0184]

[0185] Table 12 Column durability investigation - characteristic peak relative peak area ratio

[0186]

[0187] The results showed that when the samples were detected using the three chromatographic columns, the RSD of the characteristic peak relative retention time was between 0.00% and 3.21%, and the RSD of the peak relative peak area was between 0.00% and 24.61%, indicating that the method had good durability.

[0188] 2.5.5.2 Stability

[0189] Based on the above experimental conditions, the same test solution was taken and measured at 0h, 2h, 4h, 9h, 17h, and 24h. See Table 13 and Table 14.

[0190] Table 13 Stability study - characteristic peak retention time ratio

[0191]

[0192] Table 14 Stability study - characteristic peak area ratio

[0193]

[0194]

[0195] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.00% and 0.46%, and the RSD of the characteristic peak area was between 0.00% and 6.11%, indicating that the method had good stability.

[0196] In summary, the RSD of the relative retention time of each characteristic peak meets the requirements in the above investigations, and the method is good. The above 7 characteristic peaks were included in the subsequent investigation.

[0197] Example 3

[0198] 3.5.6 Determination of characteristic peaks and establishment of reference spectrum

[0199] 3.5.6.1 Verification results of three batches of scutellaria baicalensis formula granules

[0200] The proposed method was used to measure the characteristic spectra of three batches of samples of this product, and the relative retention time and relative peak area were calculated. Fig.19 , Tables 15-16. Fig.19 Characteristic spectrum of the granules of Acanthopanax salsa.

[0201] Table 15 Relative retention time of three batches of granules of scutellaria baicalensis formula

[0202]

[0203] Table 16 Relative peak areas of three batches of granules of radix scutellariae

[0204]

[0205] According to the principle of stable relative retention time and the fact that each batch of samples can be detected and the peaks are relatively high, a total of 7 peaks with good repeatability were selected as characteristic peaks. The RSD of the relative retention time of the 7 characteristic peaks of the 3 batches of chicken bone grass formula granules was 0.00% to 0.02%, and the RSD of the relative retention time of the 7 characteristic peaks during the methodological investigation was 0.00% to 16.39%, indicating that the method has good repeatability.

[0206] 3.5.6.2 Establishment of relative retention time limit values

[0207] The summary of the methodology investigation items and verification results is shown in Table 17:

[0208] Table 2 Summary of RSD% results of each item of the methodology Standard - relative retention time

[0209]

[0210] It can be seen from the above table that different chromatographic columns have a great influence on the retention time. In order to increase the reproducibility and applicability of the method, the relative retention time of each peak is temporarily set at ±8%.

[0211] Final regulations: The characteristic chromatogram of the test product should show 7 characteristic peaks, among which the peak corresponding to the reference substance of Vecinin-2 is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±8% of the specified value. The specified values ​​are: 0.308 (peak 1), 0.459 (peak 2), 0.601 (peak 3), 1.000 (peak 4, S), 1.092 (peak 5), 1.132 (peak 6), 1.245 (peak 7).

[0212] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize three batches of granules of Herba Glechomae var. erythrorhizoma, and a comparison spectrum of the characteristic spectrum of Herba Glechomae var. erythrorhizoma granules was established. Fig. 20 . Fig. 20 This is the comparative characteristic spectrum of the chicken bone grass formula granules.

[0213] Comparative Example 1 Comparison of different flow phases

[0214] Octadecylsilane bonded silica gel was used as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); 0.1% formic acid or 0.1% phosphoric acid was used as mobile phase A, acetonitrile was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 1.0 mL per minute; the column temperature was 35°C; the detection wavelength was 280 nm. The theoretical plate number calculated based on the Vescinin-2 peak should be no less than 6000.

[0215]

[0216]

[0217] The comparison results of 0.1% formic acid and 0.1 phosphoric acid are shown in the figure. Fig.21 .Depend on Fig.21 It can be seen that the peak shape and separation degree of each chromatographic peak when 0.1% formic acid is used as the mobile phase are better than those with 0.1% phosphoric acid.

[0218] Comparative Example 2 Different mobile phase gradients

[0219] Octadecylsilane bonded silica gel was used as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); 0.1% formic acid was used as mobile phase A, acetonitrile was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 1.0 mL per minute; the column temperature was 35°C; the detection wavelength was 280 nm. The theoretical plate number calculated based on the Vescinin-2 peak should be no less than 6000.

[0220]

[0221] See Figure 22, Fig. 22 It can be seen that at 21-22 minutes, the organic phase ratio changes from 85 to 80; at 22-50 minutes, the organic phase ratio changes from 80 to 80. In this mobile phase ratio, compared with the better method, the organic phase ratio decreases, resulting in a poor separation of all peaks from 25 minutes to 30 minutes. Therefore, the proposed chromatographic conditions are the best chromatographic conditions.

[0222] 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 characteristic spectrum of a herbaceous granule, comprising: A) dissolving the test sample raw material with a solvent, extracting, and obtaining a test solution; the solvent is water; The extraction is heating reflux extraction or ultrasonic extraction; B) using high performance liquid chromatography to determine the test solution to obtain an HPLC characteristic spectrum of the herbaceous granules; and further comprising preparing a reference solution: taking gallic acid, abrinine, erythrine, isoschaftoside, schaftoside, and vecinin-2 respectively, dissolving them in 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 HPLC characteristic spectrum of the herbaceous stalk formula granules are qualitatively analyzed according to the chromatogram of the reference; 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 a 0.1% formic acid aqueous solution, the mobile phase B is acetonitrile, and the gradient elution is performed; The gradient elution is specifically: 0-4min, phase A: 98-90%, phase B: 2-10%; 4-16 min, phase A: 90%-90%, phase B: 10%-10%; 16-17 min, phase A: 90%-86%, phase B: 10%-14%; 17-20 min, phase A: 86%-85%, phase B: 14%-15%; 20-21 min, phase A: 85%-85%, phase B: 15%-15%; 21-22 min, phase A: 85%-83%, phase B: 15%-17%; 22~50min, phase A: 83%~83%, phase B: 17%~17%.

2. The method according to claim 1, characterized in that: The concentrations of the reference solution are specifically as follows: 50 μg / mL of gallic acid, 50 μg / mL of abrinine, 60 μg / mL of erythrine, 55 μg / mL of isoschaftoside, 50 μg / mL of schaftoside, and 30 μg / mL of vecinin-2.

3. The method according to claim 1, characterized in that The chromatographic columns are InertSustain C18 250×4.6mm5μm, phenomenexC18 250×4.6mm 5μm, Agilent 5 TC-C18 (2) 250×4.6mm5μm; the column temperature is 35°C.

4. The method according to claim 1, characterized in that: The flow rate of the mobile phase was 1.0 mL / min; the detection wavelength was 280 nm; and the injection volume was 10 μL.

5. The method according to claim 1, characterized in that The similarity of the HPLC characteristic spectra of the Herba Corydalis Glucosides was evaluated by the Chinese medicine chromatographic fingerprint similarity evaluation system, and the HPLC standard characteristic spectrum of the Herba Corydalis Glucosides was obtained, which consisted of 7 characteristic peaks, among which peak 1 was gallic acid, peak 2 was abrinine, peak 3 was erythrine, peak 4 was vecinine-2, peak 5 was schaftoside, and peak 6 was isoschaftoside.

6. The method according to claim 5, characterized in that In the standard characteristic spectrum, taking Vitzenin-2 as the reference peak S peak, the relative retention time of each characteristic peak and the S peak was calculated, and the relative retention time was within ±8% of the specified value, and the specified values ​​were: 0.308 (peak 1), 0.459 (peak 2), 0.601 (peak 3), 1.000 (peak 4, S), 1.092 (peak 5), 1.132 (peak 6), and 1.245 (peak 7).

7. The method according to claim 1, characterized in that Step A) The heating reflux extraction time is 20-40 min; the ultrasonic power is 600 W, the frequency is 40 kHz; and the ultrasonic time is 30 min.

8. The method according to claim 7, characterized in that Step A) The ratio of the mass g of the test sample raw material to the volume mL of the solvent is (0.2~0.3): (20~30); The test sample raw material is chicken bone grass formula granules.

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