Method for determining content of ferulic acid in caulis sargentodoxae standard decoction and method for constructing specific chromatogram of ferulic acid in caulis sargentodoxae standard decoction

Abstract

The invention relates to the technical field of pharmaceutical analysis, in particular to a method for determining the content of ferulic acid in a caulis sargentodoxae standard decoction and a method for constructing a specific chromatogram of the ferulic acid in the caulis sargentodoxae standard decoction. High performance liquid chromatography is adopted for determination, and the conditions of the high performance liquid chromatography are as follows: octadecylsilane chemically bonded silica is taken as a filling agent of a chromatographic column; acetonitrile is used as a mobile phase A, a phosphoric acid aqueous solution with the volume percentage of 0.1-0.3% is used as a mobile phase B, and gradient elution is carried out. The method is high in specificity, good in repeatability and durability and high in stability and accuracy; the constructed specific chromatogram and the detection method can rapidly and comprehensively realize the purpose of combined control of multiple components in the standard decoction of the stephania lancea, realize quality control of the stephania lancea from medicinal materials to preparations, and are beneficial to guarantee of effectiveness of clinical medication.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for determining the content of ferulic acid in a standard decoction of Rhizoma Cibotii and a method for constructing a characteristic spectrum. Background Art

[0002] The original plant of Jasminum lanceolaria Roxburgh is the dried vine stem of Jasminum lanceolaria Roxburgh, a plant of the Oleaceae family. The whole plant can be used as medicine, and the name of the medicinal material is Jasminum lanceolaria Roxburgh (broken bone wind). The medicinal part of Jasminum lanceolaria Roxburgh medicinal materials listed in the Hunan Provincial Chinese Medicinal Materials Standard and the Chongqing Municipal Chinese Medicinal Materials Quality Standard is the dried vine stem, and the medicinal part of Jasminum lanceolaria Roxburgh medicinal materials listed in the Guangxi Chinese Medicinal Materials Standard is the dried whole plant. It is widely distributed in the hillsides, shrubs, valleys and dense forests in Anhui, Taiwan, Fujian, Jiangxi, Hubei, Hunan, Hainan, Guangdong, Guangxi, Guizhou, Sichuan, Yunnan and other places, with an altitude of less than 2,200 meters. It has the effects of dispelling wind and dampness, cooling blood and detoxifying, and is often used for rheumatic pain, traumatic injuries, unknown poisonous sores, and gynecological inflammation; it is also used by the people to treat fever, rheumatic pain, anti-inflammatory, eye pain, etc.

[0003] The document "HPLC Determination of Ferulic Acid Content in Qingxiangteng" discloses: A method for determining the ferulic acid content in Qingxiangteng medicinal material was established using high performance liquid chromatography, using a VP-ODS-C18 chromatographic column (4.6mm×150mm, 5μm); and acetonitrile-0.085% phosphoric acid aqueous solution (17:83) as the mobile phase.

[0004] The research object of the above literature is the medicinal material of Qingxiangteng. What is different from it is that the standard decoction of Qingxiangteng is a freeze-dried powder made from Qingxiangteng slices through water extraction, concentration, drying, etc. Compared with the medicinal materials and slices, it has lost its inherent morphology, and the corresponding active ingredients have also changed accordingly. At present, only the Hunan Provincial Traditional Chinese Medicine Standard (2009 Edition), Chongqing Traditional Chinese Medicine Quality Standard, and Guangxi Traditional Chinese Medicine Standard have carried out quality control on the microscopic and thin layer identification of Qingxiangteng medicinal materials, which is difficult to fully reflect its intrinsic quality. At present, there are very few literature reports on the high-performance liquid phase qualitative and quantitative detection technology of Qingxiangteng. Therefore, it is necessary to establish a HPLC content determination and characteristic spectrum method for the standard decoction of Qingxiangteng to provide a basis for effective control and more comprehensive evaluation of the quality of the standard decoction of Qingxiangteng. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a method for determining the content of ferulic acid in a standard decoction of Rhizoma Cibotii and a method for constructing a characteristic spectrum, which provides a guarantee for the quality and consistency evaluation of the standard decoction of Rhizoma Cibotii.

[0006] The first aspect of the present invention provides a method for determining the content of ferulic acid in a standard decoction of Rhizoma Cibotii by high performance liquid chromatography, wherein the conditions of the high performance liquid chromatography include:

[0007] The chromatographic column is filled with octadecylsilane bonded silica gel;

[0008] Acetonitrile was used as mobile phase A, 0.1% by volume phosphoric acid aqueous solution was used as mobile phase B, and gradient elution was performed;

[0009] The conditions of the gradient elution are:

[0010]

[0011] The percentage sign in the table means volume percentage.

[0012] According to some embodiments of the present invention, the mobile phase B is a 0.1% by volume phosphoric acid aqueous solution.

[0013] According to some embodiments of the present invention, the chromatographic column is ACQUITY UPLC HSS T3 C18.

[0014] According to some embodiments of the present invention, the specifications of the chromatographic column are 2.1 mm×100 mm, 1.8 μm.

[0015] According to some embodiments of the present invention, the conditions of the high performance liquid chromatography method further include: the flow rate of the mobile phase is 0.28 to 0.32 mL / min, preferably 0.3 mL / min.

[0016] According to some embodiments of the present invention, the conditions of the high performance liquid chromatography method further include: the column temperature of the chromatographic column is 28-32°C, preferably 30°C.

[0017] According to some embodiments of the present invention, the conditions of the high performance liquid chromatography method further include: an injection volume of 1 μL.

[0018] According to some embodiments of the present invention, the conditions of the high performance liquid chromatography method further include: a detection wavelength of 320 nm.

[0019] According to some embodiments of the present invention, the detector of the high performance liquid chromatography is a diode array detector.

[0020] According to some embodiments of the present invention, the theoretical plate number of the high performance liquid chromatography, calculated based on the ferulic acid peak, is not less than 2000.

[0021] According to some embodiments of the present invention, the content determination method further comprises the preparation of a test solution; the preparation step comprises: mixing the standard decoction of Rhizoma Coptidis with a solvent, and ultrasonicating to obtain the solution.

[0022] According to some embodiments of the present invention, the mass concentration of the test solution is 3-6 mg / mL.

[0023] Furthermore, the solvent of the test solution is water or a mixed solution of alcohol and water.

[0024] Furthermore, the solvent is a 50-70% by volume methanol aqueous solution, a 50-70% by volume ethanol aqueous solution or water; preferably, the solvent is a 50-70% by volume ethanol aqueous solution; more preferably, the solvent is a 50% by volume ethanol aqueous solution.

[0025] According to some embodiments of the present invention, the ultrasonic conditions of the present invention include at least one of the following i-iii:

[0026] i. Power: 400~600W;

[0027] ii. Frequency: 30~50kHz;

[0028] iii. Time: 20 to 40 minutes.

[0029] According to some embodiments of the present invention, the content determination method further comprises the preparation of a reference solution, and the preparation step comprises: mixing the reference solution with a solvent.

[0030] According to some embodiments of the present invention, the reference substance is ferulic acid.

[0031] According to some embodiments of the present invention, the mass concentration of the reference solution is 10-50 μg / mL.

[0032] Furthermore, the solvent of the reference solution is water or a mixed solution of alcohol and water.

[0033] Furthermore, the solvent is a 50-70% by volume methanol aqueous solution, a 50-70% by volume ethanol aqueous solution or water; preferably, the solvent is a 50-60% by volume methanol aqueous solution; more preferably, the solvent is a 50% by volume methanol aqueous solution.

[0034] The second aspect of the present invention provides a method for constructing a characteristic spectrum of a standard decoction of Rhizoma Coptidis, which is obtained by respectively measuring a reference solution, a reference medicinal material solution and a test solution by high performance liquid chromatography and calibrating the common peaks; the high performance liquid chromatography adopts the above-mentioned conditions.

[0035] According to some embodiments of the present invention, the characteristic spectrum includes six characteristic peaks, among which, taking peak 3 ferulic acid as the reference peak, the relative retention time of the remaining characteristic peaks and the reference peak is calculated, and the relative retention time should be within ±10% of the specified value, and the specified value is: peak 1: 0.77, peak 2: 0.89, peak 4: 1.33, peak 5: 1.39, peak 6: 1.95.

[0036] According to some embodiments of the present invention, the step of preparing the control medicinal material solution includes: mixing the control medicinal material with a solvent, and ultrasonicating.

[0037] According to some embodiments of the present invention, the mass concentration of the control medicinal material solution is 3-6 mg / mL.

[0038] According to some embodiments of the present invention, the solvent of the present invention is water or a mixed solution of alcohol and water;

[0039] According to some preferred embodiments of the present invention, the solvent is a 50-70% by volume methanol aqueous solution, a 50-70% by volume ethanol aqueous solution or water. Preferably, the solvent is a 50-70% by volume ethanol aqueous solution; more preferably, the solvent is a 50% by volume ethanol aqueous solution.

[0040] According to some embodiments of the present invention, the ultrasonic condition includes at least one of the following i-iii:

[0041] i. Power: 400~600W;

[0042] ii. Frequency: 30~50kHz;

[0043] iii. Time: 20 to 40 minutes.

[0044] The third aspect of the present invention provides the application of the above-mentioned content determination method or construction method in detecting the quality of the standard decoction of Qingxiangteng.

[0045] Beneficial effects:

[0046] The present invention provides a characteristic spectrum and content determination method of a standard decoction of Rhizoma Cibotii, determines six common peaks such as ferulic acid, and the chromatographic peaks in the constructed HPLC characteristic spectrum are well separated, the characteristic spectrum information is rich, the chromatographic peak shape is good, and the main chemical component information of the standard decoction of Rhizoma Cibotii can be fully reflected. The detection method has good stability and repeatability, and is easy to promote and apply.

[0047] The present invention constructs the HPLC characteristic spectrum of the standard decoction of Caulis Agrimoniae based on a high performance liquid chromatograph, and detects the characteristic spectrum, which can comprehensively control the characteristic components in the standard decoction of Caulis Agrimoniae, effectively ensure the stability of the overall quality of the standard decoction of Caulis Agrimoniae, and make the quality control technology of the standard decoction of Caulis Agrimoniae more complete and scientific; and the method is simple to operate, and has the advantages of high precision, good stability, good repeatability and high accuracy, etc., and provides an effective basis for the quality identification of Chinese medicinal materials.

[0048] definition:

[0049] The fragrant vine described in the present invention refers to the dried vine stems of the fragrant vine.

[0050] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0052] Figure 1 The chromatogram (210 nm) of the optimal absorption wavelength in Example 1;

[0053] Figure 2 The chromatogram (254 nm) of the optimal absorption wavelength in Example 1;

[0054] Figure 3 The chromatogram (320 nm) of the optimal absorption wavelength in Example 1;

[0055] Figure 4 The chromatogram is the gradient elution result in Example 1;

[0056] Figure 5 The chromatogram of the column temperature investigation result in Example 1;

[0057] Figure 6 The chromatogram is the flow rate investigation result in Example 1;

[0058] Figure 7 The chromatogram is the double retention time result in Example 1;

[0059] Figure 8 This is the chromatogram of the results of the extraction method investigation in Example 1;

[0060] Fig. 9 This is the chromatogram of the extraction time investigation result in Example 1;

[0061] Fig.10 The chromatogram is the result of the investigation of the amount of solvent used in Example 1;

[0062] Fig.11 This is the chromatogram of the solvent extraction results in Example 1;

[0063] Fig.12 The chromatogram is a graph showing the specificity of the characteristic spectrum in Example 1;

[0064] Fig.13 The characteristic spectrum of the ferulic acid reference substance in Example 1;

[0065] Fig.14 The characteristic spectrum of the reference medicinal material of the genus Rhizoma Coptidis in Example 1;

[0066] Fig.15 It is an overlay of characteristic spectra of 18 batches of standard decoction of Qingxiangteng in Example 1;

[0067] Fig.16 The reference characteristic spectrum of the standard decoction of Qingxiangteng in Example 1;

[0068] Fig.17 The standard curve of the ferulic acid reference substance in Example 2;

[0069] Fig.18 This is a chromatogram comparing different gradients of the fragrant vine in comparative example 1. DETAILED DESCRIPTION

[0070] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0071] The preparation method of the freeze-dried powder of the standard decoction of Caulis Sinensis of the present invention is as follows: take Caulis Sinensis medicinal materials, remove impurities, wash slightly, moisten thoroughly, cut into thick slices, and dry to obtain Caulis Sinensis slices. Take 100g of Caulis Sinensis slices, place them in an electric ceramic pot, add water and boil twice: the first time, add 12 times the amount of water (by mass ratio) to soak for 30min, boil over high heat and keep it slightly boiling for 30min, filter the decoction through a 200-mesh sieve while hot, and set aside; the second time, add 10 times the amount of water (by mass ratio), heat and boil over high heat and keep it slightly boiling for 20min, and filter the decoction through a 200-mesh sieve while hot. Combine the two decoctions, concentrate under reduced pressure at 65°C to about 120g of extract, and dispense them into 6mL~14mL containers, with a sub-packaging volume of 2mL~3mL per bottle, freeze-dry after sub-packaging, take out, roll aluminum cover, and get it. Properties of the freeze-dried powder of Qingxiangteng standard decoction: yellow-brown to brown powder; slight odor and light taste. The preparation process complies with the relevant provisions of the "Technical Requirements for Quality Control and Standardization of Traditional Chinese Medicine Formula Granules".

[0072] Example 1 Establishment of UPLC characteristic spectrum method for standard decoction of Rhizoma Cibotii

[0073] 1. Instruments and reagents

[0074] 1.1 Instruments

[0075] Shimadzu LC-30A high performance liquid chromatograph (Shimadzu Corporation);

[0076] Agilent 1260 chromatograph high performance liquid chromatograph (Agilent, USA);

[0077] KM-5000DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.);

[0078] Mettler-Toledo Instruments (Shanghai) Co., Ltd. (ME104E, MS1050U);

[0079] MWO-ABS20-DW ultrapure water machine (Mingwo Technology);

[0080] Chromatographic column: ACQUITY UPLC HSS T3 C18; (2.1 mm × 100 mm, 1.8 μm).

[0081] 1.2 Reagents

[0082] Methanol was of analytical grade (batch number: 20220824, Sinopharm Group Co., Ltd.), ethanol was of analytical grade (batch number: 20221124, Sinopharm Group Co., Ltd.), methanol was of chromatographic grade (batch number: 220852283, American Tiandi Co., Ltd.), acetonitrile was of chromatographic grade (batch number: 22115202, American Tiandi Co., Ltd.), phosphoric acid was of chromatographic grade (Tianjin Komiou Chemical Reagent Co., Ltd.), and water was purified water (homemade).

[0083] 1.3 Drug testing

[0084] Ferulic acid (purity 99.4%, China Food and Drug Inspection Institute, batch number: 110773-201915), Qingxiangteng reference medicinal materials (Zhuhai Anzhe Biotechnology Co., Ltd., batch number: 23041); Qingxiangteng standard decoction (lyophilized powder), batch numbers: QXT-BT230255~QXT-BT230267, QXT-BT230284~QXT-BT230288 (among which, QXT-BT230255~QXT-BT230257, QXT-BT230261~QXT-BT230263 are from Yueyang County, Hunan, QXT-BT230258~QXT-BT230260, QXT-BT230264~QXT-BT230267 are from Pingjiang County, Hunan, and QXT-BT230284~QXT-BT230288 are from Xiangyin County, Hunan).

[0085] 2 Chromatographic conditions and system suitability test

[0086] 2.1 Proposed chromatographic conditions

[0087] Octadecylsilane bonded silica gel is used as the filler (column length is 100 mm, inner diameter is 2.1 mm, particle size is 1.8 μm); acetonitrile is used as mobile phase A, 0.1% phosphoric acid aqueous solution is used as mobile phase B, and gradient elution is performed according to the provisions in the following table; the flow rate is 0.3 mL / min; the column temperature is 30°C; the detection wavelength is 336 nm; the theoretical plate number calculated based on the ferulic acid peak should be not less than 2000.

[0088]

[0089] The percentage sign in the table means volume percentage.

[0090] 2.2 Solution preparation

[0091] (1) Preparation of reference solution: Take an appropriate amount of ferulic acid reference substance, accurately weigh it, and add 50% methanol aqueous solution to make a solution containing 10 μg per 1 mL.

[0092] (2) Preparation of control medicinal material solution: Take 0.1 g of the control medicinal material of Caulis Sinensis, place it in a stoppered conical flask, add 25 ml of 50% ethanol, and ultrasonically treat (500 W, frequency 40 kHz) for 30 minutes. Take it out, cool it, shake it well, filter it, and take the filtrate as the control medicinal material reference solution.

[0093] (3) Preparation of test solution: Take about 0.1 g of the powder of the product, weigh it accurately, place it in a stoppered conical flask, add 25 ml of 50% ethanol accurately, weigh it, extract it by ultrasonic for 30 minutes, let it cool, weigh it again, make up the lost weight with 50% ethanol, shake it well, filter it, and obtain the solution.

[0094] 2.3 Determination method

[0095] Take 1 μL of the obtained reference solution, reference medicinal material solution and test solution respectively, inject them into the high performance liquid chromatograph, and measure to obtain the result.

[0096] 3. Investigation of chromatographic conditions

[0097] 3.1 Investigation of the optimal absorption wavelength

[0098] Based on the proposed experimental conditions, the diode array detector was used to perform full wavelength scanning on the test solution of the standard decoction of Qingxiangteng, and the chromatograms of the test solution at wavelengths of 210nm, 254nm, and 320nm were extracted. The results are as follows: Figures 1 to 3 shown.

[0099] The results show that when the detection wavelength is 320nm, the chromatographic peak information is larger, the chromatogram baseline is more stable, and the response values ​​of each characteristic peak are larger, so the detection wavelength is determined to be 320nm.

[0100] 3.2 Elution gradient investigation

[0101] The chromatograms of the three different gradients of the safflower were investigated. The gradient elution is shown in the table below. The other chromatographic conditions were determined in the same way as in "2.1". The results are shown in the table below. Figure 4 shown.

[0102] Gradient elution 1

[0103]

[0104] Gradient elution 2

[0105]

[0106] Gradient elution 3

[0107]

[0108] The results showed that when gradient elution 3 was selected, the separation was better, so gradient elution 3 was finally selected.

[0109] 3.3 Column temperature investigation

[0110] The column temperature was set to 28℃, 30℃, and 32℃ respectively. The other chromatographic conditions were the same as those in "2.1". The results are shown in Table 1. Figure 5 shown.

[0111] Table 1 Relative retention time results of different column temperature tests

[0112]

[0113] The results showed that when the column temperature was (30±2)℃, the RSD differences of the relative retention times of the six characteristic peaks were small, and the column temperature durability results met the method validation requirements, so the proposed column temperature was 28-32℃.

[0114] 3.4 Flow rate investigation

[0115] The flow rates were set to 0.28 mL / min, 0.30 mL / min, and 0.32 mL / min, respectively. The other chromatographic conditions were the same as those in "2.1". The results are shown in Table 2. Figure 6 shown.

[0116] Table 2 Relative retention time results at different flow rates

[0117]

[0118] The results showed that when the mobile phase flow rate varied within (0.3±0.02) ml / min, the relative retention time RSD of the six characteristic peaks was less than 3%, and the durability results at different flow rates met the method validation requirements; therefore, the proposed flow rate was 0.28-0.32 mL / min.

[0119] 3.5 Double retention time

[0120] Take a portion of the above test solution and detect the characteristic spectrum of twice the retention time. The results show that there are no other chromatographic peaks of the test sample after 30 minutes, and the overall performance of this method is good. Figure 7 shown.

[0121] In summary, the chromatographic conditions and system suitability test of the characteristic spectrum of the standard decoction of Qingxiangteng were determined as follows: octadecylsilane bonded silica gel was used as the filler (column length was 100 mm, inner diameter was 2.1 mm, and particle size was 1.8 μm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid aqueous solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the detection wavelength was 320 nm, the flow rate was 0.3 ml / min, the column temperature was 30°C, and the number of theoretical plates calculated based on the ferulic acid peak should be no less than 2000.

[0122] Specify the gradient elution program:

[0123]

[0124] 4 Investigation of the preparation method of test solution

[0125] 4.1 Investigation of extraction methods

[0126] Compare the two extraction methods of heating reflux and ultrasonic extraction. Take an appropriate amount of Qingxiangteng sample, weigh two portions of powder, each about 0.1g, accurately weigh, put in a stoppered conical bottle, accurately add 25ml of 50% ethanol, weigh the weight, reflux one portion for 30 minutes, and ultrasonically treat one portion (power 250W, frequency 40kHz) for 30 minutes, let cool, weigh again, make up the lost weight with 50% ethanol, shake well, filter, and get it. According to the above-determined chromatographic conditions, select the appropriate extraction method according to the parameters of each characteristic peak (peak shape, separation degree, symmetry, total peak area and other parameters).

[0127] According to the chromatographic conditions under "2.1", the "total peak area / sample weight" of the six characteristic peaks was calculated to compare the effects of different extraction methods on the characteristic spectrum of the standard decoction of Qingxiangteng, and the best extraction solvent was selected. The results are shown in Table 3. Figure 8 .

[0128] Table 3 Characteristic spectra of Qingxiangteng samples - different extraction methods

[0129]

[0130] The results showed that there was no significant difference between the total peak area of ​​the characteristic spectrum of reflux extraction and ultrasonic extraction. Taking all factors into consideration, ultrasonic extraction was selected as the extraction method for the preparation of the characteristic spectrum of the sample of Qingxiangteng.

[0131] 4.2 Extraction solvent investigation

[0132] The extraction solvents for the preparation of the characteristic spectrum of the Qingxiang Teng sample were investigated, and the extraction efficiency of the characteristic spectrum of the Qingxiang Teng sample was investigated when methanol, 50% ethanol, 30% methanol, 50% methanol and water were used as the extraction solvents.

[0133] Take an appropriate amount of Qingxiangteng sample, weigh five portions of powder, each about 0.1g, accurately weigh, put in a stoppered conical bottle, accurately add 25ml of 50% ethanol, weigh the weight, ultrasonically extract for 30 minutes, cool, weigh again, make up the lost weight with 50% ethanol, shake well, filter, and obtain. According to the above chromatographic conditions, select the appropriate type of extraction solvent according to the parameters of each characteristic peak (peak shape, separation degree, symmetry, total peak area and other parameters).

[0134] According to the chromatographic conditions under "2.1", the "total peak area / sample weight" of the six characteristic peaks was calculated to compare the effects of different extraction solvents on the characteristic spectrum of the standard decoction of Qingxiangteng, and the best extraction method was selected. The results are shown in Table 4. Fig. 9 .

[0135] Table 4 Characteristic spectrum of scutellaria baicalensis samples - suitability parameters of different extraction solvent systems

[0136]

[0137] The results showed that the extraction efficiency of 50% methanol, 50% ethanol and 30% methanol was basically the same; taking comprehensive considerations, in order to ensure sufficient extraction and the convenience of organic solvent preservation and compliance with content determination, 50% ethanol was selected as the extraction solvent for the preparation of the characteristic spectrum of the sample of Qingxiangteng.

[0138] 4.3 Extraction time investigation

[0139] Different extraction times will affect the extraction efficiency of each characteristic peak. Compare the extraction efficiency of the six proposed characteristic peaks of the characteristic spectrum of the Qingxiangteng sample at different extraction times, and determine the appropriate time comprehensively based on the parameters of each characteristic peak. Take an appropriate amount of Qingxiangteng sample, weigh three portions of powder, each about 0.1g, accurately weigh, place in a stoppered conical flask, accurately add 25ml of 50% ethanol, weigh the weight, ultrasonically extract for 20, 30, and 40 minutes respectively, cool, weigh again, make up the lost weight with 50% ethanol, shake well, filter, and obtain. Perform detection according to the chromatographic conditions determined above. Compare the changes in the chromatographic peaks of each extraction time result (based on parameters such as peak shape, separation effect, symmetry, and total peak area) to select the appropriate extraction time.

[0140] According to the chromatographic conditions under "2.1", the "total peak area / sample weight" of the six characteristic peaks was calculated to compare the effects of different extraction times on the characteristic spectrum of the Qingxiangteng standard decoction and select the best extraction time. The results are shown in Table 5. Fig.10 .

[0141] Table 5 Characteristic spectra of scutellaria baicalensis samples - system suitability parameters at different extraction times

[0142]

[0143] The results showed that there was no significant difference in the peak area of ​​each characteristic peak and the total peak area after conversion of the sampling volume when the extraction time was 20 minutes, 30 minutes and 40 minutes. The ultrasonic time had little effect on the spectrum. In order to allow the freeze-dried powder to completely dissolve, the ultrasonic time was finally selected as 30 minutes.

[0144] 4.4 Investigation of solvent dosage

[0145] Take an appropriate amount of Qingxiangteng sample, weigh three portions of powder, each of which is about 15ml, 25ml, and 35ml respectively, weigh accurately, put in a stoppered conical bottle, accurately add 25ml of 50% ethanol, weigh the weight, ultrasonically extract for 30 minutes, cool, weigh again, make up the lost weight with 50% ethanol, shake well, filter, and you are done. Perform the test according to the chromatographic conditions determined above. Compare the changes in the chromatographic peaks of the results of each extraction time (based on parameters such as peak shape, separation effect, symmetry, total peak area, etc.) to select the appropriate amount of solvent.

[0146] According to the chromatographic conditions under "2.1", the "total peak area / sample weight" of the six characteristic peaks was calculated to compare the effects of different solvent dosages on the characteristic spectrum of the Qingxiangteng standard decoction and select the best extraction method. The results are shown in Table 6. Fig.11 .

[0147] Table 6 Characteristic spectrum of Qingxiangteng samples - suitability parameters of different solvent measurement systems

[0148]

[0149] The results showed that a comparison of the total peak areas of the test samples with solvent dosages of 15ml, 25ml and 35ml revealed no significant difference after sampling volume conversion, and the extraction efficiency of the proposed characteristic peaks in the test samples with solvent dosages of 15ml, 25ml and 35ml was basically the same, indicating that the above-mentioned extraction parameters can stably extract 0.1g of each characteristic peak substance. Taking all factors into consideration, 25ml was determined as the solvent dosage for the characteristic spectrum of the Qingxiangteng sample.

[0150] In summary, the main parameters of the test solution preparation method are determined as follows: take about 0.1 g of the product powder, accurately weigh it, put it in a stoppered conical flask, accurately add 25 ml of 50% ethanol, weigh the weight, ultrasonically extract (power 500 W, frequency 40 kHz) for 30 minutes, cool, weigh the weight again, make up the lost weight with 50% ethanol, shake well, filter, and obtain.

[0151] 5. Validation of feature map methodology

[0152] 5.1 Specificity inspection

[0153] Take appropriate amounts of the above-mentioned control medicinal material solution, reference substance solution, test solution, and blank solvent (50% ethanol in water), perform sampling and measurement according to the chromatographic conditions under "2.1", and record the chromatogram. The results show that the baselines of the chromatograms of the Qingxiangteng control medicinal material, reference substance, and test sample are stable, the chromatographic peak separation and symmetry are good, the retention time of each chromatographic peak in the test sample is basically the same as that of the corresponding chromatographic peaks in the reference substance and control medicinal material, and there is no interference from the blank solvent. See the results for details. Fig.12 .

[0154] 5.2 Precision test

[0155] Weigh about 0.1g of freeze-dried powder of standard decoction of Qingxiangteng (batch number QXT-BT230255), prepare according to the test sample method, inject 6 times continuously and record the chromatogram, take peak 3 as the reference peak, calculate the relative retention time and relative peak area of ​​each characteristic peak, the results are shown in the table below, the relative retention time of each characteristic peak is less than 1%, and the relative peak area RSD is less than 5%, the test shows that the precision of this method is good. The results are shown in Table 7 and Table 8.

[0156] Table 7 Precision test results of Qingxiangteng standard decoction (relative retention time)

[0157]

[0158] Table 8 Precision test results of standard decoction of Qingxiangteng (relative peak area)

[0159]

[0160] 5.3 Repeatability test

[0161] Weigh about 0.1g of freeze-dried powder of standard decoction of Qingxiangteng (batch number BT230255), prepare 6 copies in parallel according to the test sample method, inject samples separately, record the chromatogram and integrate it, take peak 3 as the reference peak, calculate the relative retention time and relative peak area of ​​each characteristic peak, and the results are shown in the table below. The relative retention time of each characteristic peak is less than 1%, and the relative peak area RSD is less than 5%. The experiment shows that this method has good repeatability. The results are shown in Tables 9 and 10.

[0162] Table 9 Repeatability test results of Qingxiangteng standard decoction (relative retention time)

[0163]

[0164] Table 10 Repeatability test results of Qingxiangteng standard decoction (relative peak area)

[0165]

[0166] 5.4 Stability test

[0167] Weigh about 0.1g of freeze-dried powder of standard decoction of Qingxiangteng (batch number QXT-BT230255), prepare it according to the test sample method, inject samples at different time points within 24 hours, record and integrate the chromatogram, take peak 3 as the reference peak, calculate the relative retention time and relative peak area of ​​each characteristic peak, and the results are shown in the table below. The relative retention time of each characteristic peak is less than 1%, and the relative peak area RSD is less than 5%. The experiment shows that the test solution has good stability within 24 hours. The results are shown in Tables 11 and 12.

[0168] Table 11 Stability test results of Qingxiangteng standard decoction (relative retention time)

[0169]

[0170] Table 12 Stability test results of standard decoction of Qingxiangteng (relative peak area)

[0171]

[0172] In summary: the RSD of the relative retention time of each characteristic peak meets the requirements in the above investigations. This method is good, and the above 6 characteristic peaks will be included in the subsequent investigation.

[0173] 4.5 Establishment of characteristic spectra and identification of common peaks

[0174] The HPLC fingerprints of 18 batches of standard decoctions of Qingxiangteng were imported into the "Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System Software (2012 Edition)" to generate overlay diagrams and reference characteristic spectra, and 6 common peaks with good separation, large peak area and good peak shape were selected as characteristic peaks. Through comparison with reference substances, peak 3 is ferulic acid. Ferulic acid is the main active ingredient of Qingxiangteng and has stable chemical properties. Its chromatographic peak has good separation and large peak area among all chromatographic peaks. Therefore, peak 3 (ferulic acid) was selected as the reference peak of the characteristic spectrum, and the relative retention times of peaks 1, 2, 4, 5 and 6 were calculated. The results are shown in Table 13. The characteristic spectrum of ferulic acid reference substance is shown in Fig.13 , see the reference herbal medicine characteristic spectrum Fig.14 , 18 The characteristic spectrum superposition of the batch of Qingxiangteng standard decoction is shown in Figure 15, and the comparative characteristic spectrum of Qingxiangteng standard decoction is shown in Fig.16 .

[0175] Table 13 Relative retention time of 18 batches of freeze-dried powder samples of standard decoction of Rhizoma Cibotii

[0176]

[0177] 5.6 Establishment of characteristic graph standards

[0178] In summary, the standard for determining the characteristic spectrum is: 6 characteristic peaks should be present in the chromatogram of the test sample, and should correspond to the retention time of the 6 characteristic peaks in the chromatogram of the reference medicinal material, among which the retention time of peak 3 should correspond to the retention time of the reference solution. The peak corresponding to the peak of the ferulic acid reference is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value, and the specified value is 0.77 (peak 1), 0.89 (peak 2), 1.33 (peak 4), 1.39 (peak 5), 1.95 (peak 6).

[0179] Example 2 Establishment of UPLC content determination method for standard decoction of Caulis Sinensis

[0180] Based on the construction method of the UPLC characteristic spectrum of the standard decoction of the Herba Lycopodii in Example 1, Example 2 also conducted the following methodological investigations and established a UPLC content determination method for the standard decoction of the Herba Lycopodii, specifically including:

[0181] 1 Linear relationship investigation

[0182] In this verification, the reference solutions with ferulic acid concentrations of 1.0799E-07mg / ml, 2.6999E-06mg / ml, 6.7497E-05mg / ml, 0.001687414mg / ml, 0.04218536mg / ml, and 0.2109268mg / ml were prepared. The concentration of the reference solution was used as the horizontal coordinate and the corresponding peak area was used as the vertical coordinate to draw a standard curve. The results showed that the concentration of ferulic acid was in the range of 1.0799E-07mg / ml to 0.2109268mg / ml, that is, the ferulic acid content of the test sample was in the range of 1.0799E-07mg / ml to 0.2109268mg / ml. The regression equation was y=9,853,192.9747x+19,122.9834, R2=99991. The results are shown in Table 14, and the standard curve is shown in Table 14. Fig.15 .

[0183] Table 14 Results of investigation on linear relationship of ferulic acid

[0184]

[0185] The results showed that under the above chromatographic conditions, the injection concentration of ferulic acid in the range of 1.0799E-07 mg / ml to 0.2109268 mg / ml showed a good linear relationship with the peak area value.

[0186] 2 Repeatability test

[0187] Take the same batch of Qingxiangteng dry powder, prepare 6 test solutions in parallel according to the method of "Preparation of test solution", determine the chromatographic conditions, and test the sample. The data show that the repeatability test results of the Qingxiangteng dry powder content determination in the sample have a mean ferulic acid content of 0.715 mg / g, and the RSD value is less than 4% (Chinese Pharmacopoeia 2020 Edition General Principles 9101), with good repeatability. The experimental results are shown in Table 15.

[0188] Table 15 Methodological verification of ferulic acid content determination in vine powder - experimental data on method repeatability of vine powder

[0189]

[0190] The results showed that the average RSD of ferulic acid content in the standard decoction of Rhizoma Cibotii was less than 3% after 6 measurements, indicating that the repeatability of this method was good.

[0191] 3. Precision test

[0192] It is used to evaluate the repeatability of the response value of the chromatographic system during continuous injection. The same reference solution was taken to determine the chromatographic conditions and injected 6 times continuously. The data showed that the RSD value of the instrument's repeated measurement results was less than 2%, and the instrument had good repeatability. The experimental results are shown in Table 16.

[0193] Table 16 Methodological verification of ferulic acid content determination in dried powder of Fragrant vine - precision experimental data

[0194]

[0195] 4. Stability test

[0196] Take the same batch of dried powder of Fragrant vine, prepare the test sample according to the method of “Preparation of test sample solution”, determine the chromatographic conditions, inject and detect within 24 hours, record the chromatogram, and examine the effect of different storage time on the test results of ferulic acid content. The experimental results are shown in Table 17.

[0197] Table 17 Effect of the test solution placement time on the test results - Ferulic acid

[0198]

[0199] The above results show that when the test personnel inject the sample within 24 hours, the retention time RSD is less than 2%, and the peak area RSD is less than 2%, so the solution stability of this method is good and meets the method validation requirements.

[0200] 5. Intermediate precision test

[0201] The same batch of samples were prepared according to the method of "Preparation of test sample solution" and tested on Shimadzu LC-30A HPLC and Agilent 1260 HPLC respectively according to the determined chromatographic conditions. The experimental results are shown in Table 18.

[0202] Table 18 Methodological verification of determination of ferulic acid content in dried powder of Fragrant vine - experimental data of intermediate precision of different instruments

[0203]

[0204]

[0205] The results showed that the RSD values ​​of the content of the dried powder of Ampelopsis ternata were less than 4% when Shimadzu LC-30A HPLC and Agilent 1290 HPLC were used to determine the content of the dried powder of Ampelopsis ternata. The results showed that the intermediate precision of different instruments of this method was good.

[0206] 6. Sample recovery test

[0207] Weigh 9 samples of dried powder of Aconitum carmichaelii with known content, the ferulic acid content is 0.715 mg / g, each sample is about 0.05 g, accurately weighed, and appropriate amount of ferulic acid reference substance was added. The samples were treated according to the preparation method of the test solution, determined according to the law, and the chromatograms were recorded and integrated. The results are shown in Table 19. The recovery rate RSD is less than 5%. The experiment shows that the spiked recovery rate of this method is better.

[0208] Table 19 Sample recovery test results

[0209]

[0210] 7. Sample content determination

[0211] 18 batches of freeze-dried powder of standard decoction of Qingxiangteng were taken and measured according to the above method. The content of ferulic acid was calculated by external standard method. The results are shown in Table 20. The content of ferulic acid ranged from 0.47 to 2.75 mg / g, with a mean of 1.26 mg / g and a mean ±3SD range of -0.69 to 3.21. Based on the data distribution of 18 batches of standard decoctions, the deviation of 18 batches of samples was large, and the SD algorithm could be used, with the lower limit being the lowest value of the measured value. The limit range of ferulic acid content is: Ferulic acid (C 2.5) is contained in each 1g freeze-dried powder of standard decoction of Qingxiangteng. 10 H 10 O4) should be 0.47mg~3.21mg.

[0212] Table 20 Determination results of ferulic acid in 18 batches of freeze-dried powder of standard decoction of Rhizoma Cibotii

[0213]

[0214]

[0215] Comparative Example 1 Comparison of different gradients of Fragrant Vine

[0216] Chromatographic conditions:

[0217] Octadecylsilane bonded silica gel was used as the filler (column length was 100 mm, inner diameter was 2.1 mm, particle size was 1.8 μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid aqueous solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the detection wavelength was 320 nm, the flow rate was 0.3 ml / min, the column temperature was 30°C, and the theoretical plate number calculated based on the ferulic acid peak should be no less than 2000.

[0218] The elution procedure of Comparative Example 1:

[0219]

[0220] Example 1 Gradient elution procedure:

[0221]

[0222] According to the above results, the elution procedure in Example 1 is gradient elution, while Comparative Example 1 is isocratic elution. Gradient elution has the characteristics of shortening the analysis cycle, improving separation ability, improving peak shape and less tailing; the sample peaks of gradient elution, regardless of the components that emerge first or later, their peak shapes can basically ensure sharp symmetry. In isocratic elution, the chromatographic peaks of the components that emerge later are wider and the separation is poorer. Therefore, the results show that the peak separation degree of Example 1 is high, the peaks are obvious, the baseline is stable, the measured components can be completely separated, and characteristic peaks with better peak shapes can be obtained. The test results are shown in Fig.18 , in the figure, S1: Qingxiang rattan (comparative example 1), S2: Qingxiang rattan (example 1), S3: reference substance (comparative example 1), S4: reference substance (example 1).

[0223] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for determining the content of ferulic acid in a standard decoction of Rhizoma Cibotii, characterized in that: Determined by high performance liquid chromatography, the conditions of the high performance liquid chromatography include: The chromatographic column is filled with octadecylsilane bonded silica gel; Acetonitrile was used as mobile phase A, 0.1% by volume phosphoric acid aqueous solution was used as mobile phase B, and gradient elution was performed; The conditions of the gradient elution are:

2. The content determination method according to claim 1, characterized in that: The volume percentage of the phosphoric acid aqueous solution is 0.1-0.3%.

3. The content determination method according to claim 1, characterized in that: The conditions of the high performance liquid chromatography also include at least one of the following i-iv: i. Flow rate: 0.28~0.32mL / min; ii. Column temperature: 28-32°C; iii. Injection volume: 1 μl; iv. Detection wavelength: 320nm.

4. The content determination method according to claim 1, characterized in that: It also includes the preparation of test solution and reference solution; Preferably, the mass concentration of the test solution is 3-6 mg / ml; Preferably, the mass concentration of the reference solution is 10-50 μg / ml.

5. The content determination method according to claim 1, characterized in that: The number of theoretical plates, calculated based on the ferulic acid peak, shall not be less than 2000.

6. The content determination method according to claim 4, characterized in that: The solvent of the test solution and the reference solution is a mixed solution of alcohol and water; Preferably, the solvent is a methanol aqueous solution with a volume percentage of 50-70% or an ethanol aqueous solution with a volume percentage of 50-70%.

7. A method for constructing a characteristic spectrum of a standard decoction of Rhizoma Cibotii, characterized in that: The reference substance solution, the reference medicinal material solution and the test solution are respectively measured by high performance liquid chromatography, and the common peaks are calibrated to obtain; the high performance liquid chromatography adopts the conditions described in any one of claims 1 to 6.

8. The construction method according to claim 7, characterized in that: The characteristic spectrum includes six characteristic peaks, among which peak 3 ferulic acid is taken as the reference peak, and the relative retention time of the remaining characteristic peaks and the reference peak is calculated. The relative retention time should be within ±10% of the specified value, and the specified value is: peak 1: 0.77, peak 2: 0.89, peak 4: 1.33, peak 5: 1.39, peak 6: 1.

95.

9. The construction method according to claim 7, characterized in that: The mass concentration of the control medicinal material solution is 3-6 mg / ml.

10. Use of the content determination method according to any one of claims 1 to 6 or the construction method according to any one of claims 7 to 9 in detecting the quality of a standard decoction of Rhizoma Cibotii.

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