Method for constructing characteristic chromatogram of radix clematidis or derivative products thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a construction method of a specific chromatogram of radix clematidis or a derivative product thereof. According to the construction method of the specific chromatogram of the radix clematidis or the derivative product of the radix clematidis, provided by the invention, nine common characteristic peaks are obtained by taking octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A and one of a phosphoric acid-containing aqueous solution, a formic acid-containing aqueous solution and a glacial acetic acid-containing aqueous solution as a mobile phase B through a specific gradient elution procedure, and the specific chromatogram of the radix clematidis or the derivative product of the radix clematidis is obtained. The effective separation of common characteristic peaks is realized, the separation degree is good, the obtained characteristic spectrum has more characteristic peaks, the baseline separation is better, and the positioning and qualitative analysis are easier; the precision, the stability and the repeatability are relatively good; the identified characteristic component chlorogenic acid provides a basis for quality detection and content determination of the radix clematidis or the derivative products thereof.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine detection technology, specifically relating to a method for constructing a characteristic spectrum of Yunweiling or its derivative products. Background Technology

[0002] Yunweiling is the dried root and rhizome of *Inula nervosa* Wall. ex. DC, a plant belonging to the genus *Inula* of the Asteraceae family. Current quality reports on Yunweiling mainly focus on the determination of volatile oil content; no relevant literature has established quality control methods using characteristic spectroscopy, thin-layer chromatography, or other techniques. Improving the efficiency of quality testing for Yunweiling or its derivatives and effectively controlling the quality of finished products is an urgent issue to be addressed. Summary of the Invention

[0003] Therefore, the first objective of this invention is to provide a method for constructing a characteristic spectrum of Yunweiling or its derivative products. Based on the characteristics of Yunweiling or its derivative products, this method establishes a characteristic spectrum of the product with a large number of characteristic peaks and high separation, obvious characteristics, good precision, stability and repeatability, and can be used for comprehensive quality control of Yunweiling or its derivative products.

[0004] To this end, the present invention provides the following technical solution.

[0005] This invention provides a method for constructing characteristic chromatograms of Yunweiling or its derivatives, including detection using high-performance liquid chromatography (HPLC), with chromatographic conditions including: Using octadecylsilane-bonded silica gel as the packing material, acetonitrile as mobile phase A, and one of the following as mobile phase B: an aqueous solution containing phosphoric acid, an aqueous solution containing formic acid, or an aqueous solution containing glacial acetic acid, the gradient elution program includes: From 0 to 15 minutes, the volume percentage of mobile phase A changes from 5% to 14%, and the volume percentage of mobile phase B changes from 95% to 86%. Over 15-25 minutes, the volume percentage of mobile phase A changes from 14% to 20%, and the volume percentage of mobile phase B changes from 86% to 80%. Over 25-35 minutes, the volume percentage of mobile phase A changes from 20% to 22%, and the volume percentage of mobile phase B changes from 80% to 78%. Over 35-45 minutes, the volume percentage of mobile phase A changes from 22% to 30%, and the volume percentage of mobile phase B changes from 78% to 70%.

[0006] In one optional implementation, the chromatographic conditions include at least one of the following: (1) The detection wavelength is 325-329 nm; preferably, the wavelength is 327 nm; (2) The flow rate is 0.20-0.30 mL / min; preferably, the flow rate is 0.2 mL / min; (3) Column temperature 20-30℃; (4) The injection volume is 1-5 μL; (5) The mobile phase B is an aqueous solution containing phosphoric acid. Preferably, the concentration of phosphoric acid in the aqueous solution is 0.05-0.2% v / v; preferably, the concentration of phosphoric acid is 0.1% v / v.

[0007] In one optional embodiment, the preparation of the test solution is further included, comprising: weighing the test sample, extracting it with a solvent, separating the solid and liquid, and taking the liquid, which is the test solution.

[0008] In one optional embodiment, the preparation of the test solution satisfies at least one of the following: A. The ratio of the mass of the Yunweiling test sample to the volume of the solvent is (0.2-1.0):(10-50); the unit of the mass of the test sample is g, and the unit of the volume of the solvent is mL; B. The extraction method is ultrasonic extraction, heating reflux, or oscillation extraction; ultrasonic extraction is preferred. C. Extraction time is ≥20 min; D. The solid-liquid separation is selected from filtration; E. The solvent is selected from one or more of methanol and water; preferably, it is a methanol aqueous solution with a volume percentage of 50-100% v / v, and more preferably, it is a methanol aqueous solution with a volume percentage of 70-90% v / v.

[0009] In an optional embodiment, the construction method further includes the step of preparing a reference solution using chlorogenic acid and a solvent, and the step of detecting the reference solution using high performance liquid chromatography in the construction method to obtain a reference spectrum of the reference standard. Preferably, the concentration of chlorogenic acid reference standard in the chlorogenic acid reference solution is 10-40 μg / mL; more preferably, it is 10 μg / mL. Preferably, the solvent used in the preparation of the reference solution is selected from methanol or an aqueous methanol solution with a volume fraction of not less than 50-100% v / v; more preferably, the solvent used in the preparation of the reference solution is selected from an aqueous methanol solution with a volume fraction of 80% v / v. Preferably, the construction method further includes the step of preparing a reference herb solution using Yunweiling reference herb, and the step of obtaining a reference herb reference spectrum by detecting the reference herb solution using high performance liquid chromatography in the construction method.

[0010] In one optional embodiment, the Yunweiling or its derivative products include one or more of Yunweiling medicinal materials, Yunweiling processed slices, or Yunweiling preparations.

[0011] In one optional embodiment, the Yunweiling preparation includes at least one of Yunweiling decoction pieces and Yunweiling formula granules; preferably, the Yunweiling standard decoction includes at least one of Yunweiling standard decoction concentrated extract, Yunweiling standard decoction freeze-dried powder, and Yunweiling standard decoction aqueous solution.

[0012] This invention also provides a method for determining the content of active ingredients in Yunweiling or its derivative products, comprising: Take the test solution and the reference solution, and detect them by high performance liquid chromatography in the above-mentioned method for constructing the characteristic chromatogram of Yunweiling or its derivatives; The reference standard includes chlorogenic acid.

[0013] The present invention also provides a quality testing method for Yunweiling or its derivative products, including the step of comparing the feature spectrum of the product to be tested with the feature spectrum of Yunweiling or its derivative products; The feature map of the product under test is obtained according to the above construction method; The feature map of Yunweiling or its derivative products is selected from any one of the following (1)-(4): (1) It has 9 common characteristic peaks. The peak corresponding to the reference peak of chlorogenic acid is the S peak. The relative retention time of peaks 2-9 and S peak is within ±10% of the specified value. The specified values ​​of peaks 2-9 are as follows: 1.102, 1.165, 2.821, 2.914, 3.110, 3.196, 3.501, 5.052. (2) It has 9 common characteristic peaks, one of which corresponds to the retention time of the chlorogenic acid reference peak. The peak corresponding to the chlorogenic acid reference peak is the S peak. The relative retention times of peaks 2-9 and S peak are within ±10% of the specified values. The specified values ​​of peaks 2-9 are as follows: 1.102, 1.165, 2.821, 2.914, 3.110, 3.196, 3.501, 5.052. (3) Characteristic chromatograms of Yunweiling and / or its derivatives obtained by using a single batch or multiple batches of Yunweiling and / or its derivatives as test samples according to the above construction method; (4) Using multiple batches of Yunweiling and / or its derivatives as test samples, the characteristic spectra obtained according to the above construction method are used to make a control characteristic spectra by means of the average or median.

[0014] In one alternative implementation, peak 1 (S) corresponds to chlorogenic acid.

[0015] The technical solution of this invention has the following advantages: 1. The method for constructing the characteristic spectrum of Yunweiling or its derivatives provided by this invention uses octadecylsilane-bonded silica gel as a filler, acetonitrile as mobile phase A, and one of the following: an aqueous solution containing phosphoric acid, an aqueous solution containing formic acid, or an aqueous solution containing glacial acetic acid as mobile phase B. Through a specific gradient elution procedure, nine common characteristic peaks are obtained, and effective separation of the common characteristic peaks is achieved. The separation degree is good, and the obtained characteristic spectrum has more characteristic peaks and better baseline separation, making it easier to locate and qualitatively analyze. It also has good precision, stability, and repeatability. The identified chlorogenic acid is a characteristic component, providing a basis for the quality detection and content determination of Yunweiling or its derivatives.

[0016] 2. The quality testing method for Yunweiling or its derivative products provided by the present invention selects S-peak chlorogenic acid as the internal reference peak in the fingerprint spectrum, and determines 9 common characteristic peaks in the fingerprint spectrum of Yunweiling or its derivative products. The relative retention time of each common characteristic peak is calculated based on S-peak chlorogenic acid, which is beneficial for comprehensive quality testing and overall quality control of Yunweiling or its derivative products, thereby helping to improve the safety and stability of Yunweiling or its derivative products. Attached Figure Description

[0017] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0018] Figure 1 shows the characteristic chromatogram of Yunweiling standard decoction (lyophilized powder); specifically, Figure 1-(1) is the test sample; Figure 1-(2) is chlorogenic acid; Figure 2 The chromatogram of gradient condition 1 in Experimental Example 1; Figure 3 The chromatogram of gradient condition 2 in Experiment Example 1; Figure 4 The chromatogram of gradient condition 3 in Experiment Example 1; Figure 5 The chromatogram for the detection wavelength of 327 nm in Experiment Example 1 is shown. Figure 6 The chromatogram for the detection wavelength of 205 nm in Experiment Example 1 is shown. Figure 7 The chromatogram for the detection wavelength of 254 nm in Experiment Example 1 is shown. Figure 8 The chromatogram for the detection wavelength of 280 nm in Experiment Example 1 is shown. Figure 9 The characteristic chromatograms of 15 batches of Yunweiling decoction standard decoction (lyophilized powder) in Experimental Example 3 (batch numbers from bottom to top are: YWL2020001, YWL2020002, YWL2020003, YWL2020004, YWL2020005, YWL2020006, YWL2020007, YWL2020008, YWL2020009, YWL2020010, YWL2020011, YWL2020012, YWL2020013, YWL2020014, YWL2020015) are shown below. Figure 10 For reference characteristic chromatograms; where peak 1: chlorogenic acid; Figure 11 This is a confirmation diagram of the peaks of Yunweiling standard decoction (lyophilized powder) and chlorogenic acid in Experiment Example 3; Figure 12 The chromatograms for precision in Experiment Example 4 are shown below (from bottom to top: Precision 1, Precision 2, Precision 3, Precision 4, Precision 5, Precision 6, and Control Characteristic Chromatogram). Figure 13 The chromatograms for repeatability in Experiment Example 4 are shown below (from bottom to top: repeatability 1, repeatability 2, repeatability 3, repeatability 4, repeatability 5, repeatability 6, and control characteristic chromatogram). Figure 14 The chromatograms for intermediate precision in Experiment Example 4 are shown below (from bottom to top: intermediate precision 1, intermediate precision 2, intermediate precision 3, and control characteristic chromatogram). Figure 15 The chromatograms for stability in Experiment Example 4 are shown below (from bottom to top: stability 1, stability 2, stability 3, stability 4, stability 5, stability 6, and control characteristic chromatogram). Figure 16 This is the chromatogram of the negative blank solution in Experiment Example 4; Figure 17 This is the overall chromatogram for Experiment Example 4. Detailed Implementation

[0019] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0020] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0021] The test sample used in the following examples and experimental cases is Yunweiling standard decoction (lyophilized powder). The specific preparation method of Yunweiling standard decoction (lyophilized powder) is as follows: Take 100g of Yunweiling slices (batch number: YWL202001), place them in a clay pot, add 12 times the amount of water and soak for 30 minutes. First, bring to a boil over high heat (500W), then simmer over low heat (200W) for 30 minutes. Filter while hot through a 200-mesh filter cloth, and quickly cool the filtrate to room temperature. Add 10 times the amount of water to the dregs, bring to a boil over high heat, then simmer over low heat for 25 minutes. Filter while hot through a 200-mesh filter cloth, and quickly cool the filtrate to room temperature. Combine the two filtrates and weigh the filtrate. Concentrate the filtrate under reduced pressure to form a concentrated extract, place it in a freeze dryer until dry, remove it, grind it into powder, weigh it, and dispense it into vials to obtain Yunweiling standard decoction (lyophilized powder).

[0022] The main instruments and reagents involved in this invention are as follows: 1. Instruments, reagents and reagents Chromatograph 1: Agilent 1290 chromatography system, including G7104A quaternary pump and G7117A DAD detector; Chromatograph 2: Agilent 1290 Infinity II chromatography system, including G7104A quaternary pump and G7114B VWD detector; Chromatographic columns: Agilent Eclipse Plus C18 column (50 mm length, 2.1 mm inner diameter, 1.8 μm particle size); Welch Ultimate XB C18 column (50 mm length, 2.1 mm inner diameter, 1.8 μm particle size).

[0023] SQP electronic balance; DHG-9245A electric thermostatic drying oven; Milli-Q IQ 7000 pure water machine; XM-P101H ultrasonic cleaner.

[0024] 2. Reagents and reagents: Chlorogenic acid reference standard (batch number: 110753-202119, purchased from the National Institutes for Food and Drug Control, purity 96.3%). Methanol and acetonitrile were of chromatographic grade, water was Wahaha purified water, and all other reagents were of analytical grade.

[0025] Yunweiling Standard Decoction (Freeze-dried Powder) (Batch Nos.: YWL2020001, YWL2020002, YWL2020003, YWL2020004, YWL2020005, YWL2020006, YWL2020007, YWL2020008, YWL2020009, YWL2020010, YWL2020011, YWL2020012, YWL2020013, YWL2020014, YWL2020015).

[0026] Example 1 This embodiment provides a method for constructing a feature map of Yunweiling or its derivative products, including: (1) Preparation of test solution: Using Yunweiling standard decoction (lyophilized powder) as the test sample, take about 0.5g of the test sample powder, accurately weigh it, place it in a stoppered conical flask, add 25mL of 80% v / v methanol aqueous solution, weigh it, sonicate (power 250W, frequency 40kHz) for 30 minutes, take it out, cool it, make up the lost weight with 80% v / v methanol aqueous solution, shake it well, filter it, and take the filtrate to obtain the solution. Preparation of reference solution: Take an appropriate amount of chlorogenic acid reference standard, accurately weigh it, and add 80% v / v methanol aqueous solution to prepare a solution containing 10 μg per 1 mL, thus obtaining the chlorogenic acid reference solution.

[0027] (2) Take 1 μL each of the test solution and the reference solution and determine them by high performance liquid chromatography. The chromatographic conditions are as follows: use octadecylsilane-bonded silica gel as the stationary phase (Agilent Eclipse Plus C). 18 The column length is 50 mm, the inner diameter is 2.1 mm, and the particle size is 1.8 μm. Acetonitrile is used as mobile phase A, and 0.1% (v / v) phosphoric acid solution is used as mobile phase B. Gradient elution is performed according to the specifications in the table below. The flow rate is 0.2 mL per minute; the column temperature is 25 °C; and the detection wavelength is 327 nm. The theoretical plate number, calculated based on the chlorogenic acid peak, should not be less than 5000.

[0028] Table 1 Gradient Elution Table

[0029] The results are shown in Table 2 and Figure 1. From Figure 1-(1) to Figure 1-(2), it can be seen that the characteristic spectrum of Yunweiling standard decoction (lyophilized powder) has 9 characteristic peaks. All 9 characteristic peaks were effectively separated. Each characteristic peak has a good peak shape, stable baseline, short detection time, and uniform peak height or peak area. Peak 1 corresponds to the retention time of the chlorogenic acid reference peak. The peak corresponding to the retention time of the chlorogenic acid reference peak is the S peak. The relative retention times of peaks 2-9 and S peak were calculated. Their relative retention times are within ±10% of the specified values. The specified values ​​are: 1.102 (peak 2), 1.165 (peak 3), 2.821 (peak 4), 2.914 (peak 5), 3.110 (peak 6), 3.196 (peak 7), 3.501 (peak 8), and 5.052 (peak 9). Among them, peak 1 corresponds to chlorogenic acid.

[0030] Table 2 System Adaptability Results

[0031] Experimental Example 1 I. Investigation of chromatographic conditions 1. Selection of mobile phase gradient Take the same sample solution prepared according to Example 1, and inject 1 μL; use octadecylsilane-bonded silica gel as the filler (Agilent Eclipse Plus C). 18 The column was 50 mm long, with an inner diameter of 2.1 mm and a particle size of 1.8 μm. Acetonitrile was used as mobile phase A, and 0.1% (v / v) phosphoric acid solution was used as mobile phase B. Gradient elution was performed according to the specifications in Tables 3-5. The flow rate was 0.2 mL per minute; the column temperature was 25 °C; and the detection wavelength was 327 nm. The results are shown in Tables 6 and 7. Figure 2-4 .

[0032] Table 3 Gradient Condition 1

[0033] Table 4 Gradient Condition 2

[0034] Table 5 Gradient Condition 3

[0035] Table 6 Results of investigation on different mobile phase gradients

[0036] From Table 6 and Figure 2-4 It can be seen that gradient elution using gradient condition 1 provides rich chromatographic information, numerous major chromatographic peaks, good resolution, a relatively stable baseline, and a reasonable analysis time. Therefore, gradient condition 1 is tentatively selected as the elution gradient, and further screening and investigation of subsequent conditions will be conducted.

[0037] 2. Examination of the detection wavelength In addition to the detection wavelength, the chromatographic conditions determined according to the investigation of the mobile phase gradient in section 1 were used. The same sample solution prepared according to Example 1 was measured at different detection wavelengths of 205 nm, 254 nm, 280 nm, and 327 nm, with the detection wavelength as the variable. The resulting chromatograms are shown in Table 7. Figure 5-8 As shown.

[0038] Table 7 Results of different detection wavelengths

[0039] From Table 7 and Figure 5-8 It is known that, compared with other wavelengths, the detection wavelength of 327nm has more chromatographic peaks, a larger peak response, and a more uniform distribution; therefore, the present invention selects 327nm as the optimal detection wavelength for characteristic spectrum determination.

[0040] 3. Investigation of the mobile phase Apart from the mobile phase, under the chromatographic conditions determined according to section 2, which investigated the detection wavelength, the same sample solution prepared according to Example 1 was tested under different mobile phases (acetonitrile-0.1% v / v formic acid aqueous solution, acetonitrile-0.1% v / v phosphoric acid aqueous solution, acetonitrile-0.1% v / v glacial acetic acid aqueous solution, acetonitrile-0.05% v / v phosphoric acid aqueous solution, acetonitrile-0.2% v / v phosphoric acid aqueous solution), with the mobile phase as the variable. The resulting chromatograms are shown in Table 8.

[0041] Table 8 Results of investigation of different mobile phase systems

[0042] As shown in Table 8, different mobile phases have little impact on the characteristic spectrum. Therefore, the mobile phase with relatively good separation effect is tentatively selected as acetonitrile-0.1% v / v phosphoric acid aqueous solution for subsequent condition screening.

[0043] 4. Flow velocity assessment Except for the flow rate, the chromatographic conditions determined according to the investigation of the mobile phase in section 3 were used. The same sample solution prepared according to Example 1 was measured at flow rates of 0.20 mL / min, 0.25 mL / min, and 0.30 mL / min, respectively. The resulting chromatograms are shown in Table 9.

[0044] Table 9 Results of investigation at different flow velocities

[0045] As shown in Table 9, the chromatographic separation effect is relatively good when the flow rate is 0.20 mL / min. Therefore, the flow rate is tentatively set at 0.20 mL / min for subsequent condition screening.

[0046] 5. Examination of column temperature Apart from column temperature, the chromatographic conditions determined according to the investigation of flow rate in section 4 were used. With column temperature as the variable, the same sample solution prepared according to Example 1 was measured at column temperatures of 20℃, 25℃, and 30℃ respectively. The resulting chromatograms are shown in Table 10.

[0047] Table 10 Results of investigation at different column temperatures

[0048] As shown in Table 10, the column temperature has little effect on the separation of chromatographic peaks. Therefore, the column temperature is tentatively set at 25℃ for subsequent condition screening.

[0049] 6. Examination of injection volume Except for the injection volume, under the chromatographic conditions determined according to the investigation of column temperature in section 5, with the injection volume as the variable, 1 μL and 2 μL of the same test solution prepared according to Example 1 were accurately pipetted and measured. The resulting chromatograms are shown in Table 11.

[0050] Table 11 Comparison of chromatographic peak parameters with different injection volumes

[0051] As shown in Table 11, different injection volumes have little effect on the chromatographic peak response, and the peak shape, peak height and peak width are good. Therefore, the injection volume is tentatively set at 1 μL for subsequent condition screening.

[0052] 7. Determination of optimal chromatographic conditions Using octadecylsilane-bonded silica gel as a filler (Agilent Eclipse Plus C) 18 The column was 50 mm long, with an inner diameter of 2.1 mm and a particle size of 1.8 μm. Acetonitrile was used as mobile phase A, and 0.1% (v / v) phosphoric acid solution was used as mobile phase B. Gradient elution was performed according to the specifications in Table 12. The flow rate was 0.2 mL per minute, the column temperature was 25 °C, and the detection wavelength was 327 nm. The injection volume was 1 μL.

[0053] Table 12 Gradient Elution Table

[0054] II. Optimization of the preparation method of the test sample solution 1. Examination of extraction methods Using Yunweiling standard decoction (lyophilized powder) as the test sample, take about 0.5 g of the test sample powder, accurately weigh it, place it in a stoppered conical flask, add 25 mL of 80% v / v methanol aqueous solution, weigh it, and perform ultrasonic treatment for 30 minutes (power 250 W, frequency 40 kHz) and shaking extraction (30 s, 1 time / s). Take it out, cool it, make up the weight loss with 80% v / v methanol aqueous solution, shake it well, filter it, and take the filtrate. Determine it according to the optimal chromatographic conditions determined above. The obtained chromatogram is shown in Table 13.

[0055] Table 13 Chromatogram parameters of standard decoction (lyophilized powder) of Yunweiling medicinal slices under different extraction methods

[0056] As shown in Table 13, different extraction methods have little impact on the characteristic chromatograms, and the obtained chromatographic peak information is large, the peak shape is good, and the separation effect is good. Ultrasonic extraction is more convenient, so ultrasonic extraction is tentatively selected for subsequent condition screening.

[0057] 2. Examination of extraction time Except for the extraction time, the preparation method of the test solution determined according to the extraction method in section 1 was investigated. With the extraction time as the variable, the optimal chromatographic conditions determined above were used to perform ultrasonic extraction for 20 min, 30 min and 40 min respectively, and the resulting chromatograms are shown in Table 14.

[0058] Table 14 Chromatogram parameters of standard decoction (lyophilized powder) of Yunweiling medicinal slices at different extraction times

[0059] As shown in Table 14, different extraction times have little impact on the feature map. Therefore, the extraction time is tentatively set at 30 min for subsequent condition screening.

[0060] 3. Investigation of extraction solvents Except for the extraction solvent, the test solution was prepared according to the method determined in section 2, which focused on the extraction time. The extraction solvent was used as a variable, and the optimal chromatographic conditions determined above were used to determine the chromatographic results. The extraction solvents were 50% v / v methanol aqueous solution, 80% v / v methanol aqueous solution, methanol, and purified water, respectively. The resulting chromatograms are shown in Table 15.

[0061] Table 15 Chromatographic parameters of standard decoction (lyophilized powder) of Yunweiling medicinal slices under different extraction solvents

[0062] As shown in Table 15, different extraction solvents have a significant impact on the characteristic chromatograms. When water is used as the solvent, the response and information of the characteristic peaks are less. The chromatogram of the test solution prepared with 80% v / v methanol aqueous solution has more chromatographic peak information, better peak shape, and better separation effect. Therefore, 80% v / v methanol aqueous solution is tentatively selected as the extraction solvent for subsequent condition screening and investigation.

[0063] 4. Investigation into the amount of extraction solvent added Except for the amount of extraction solvent added, the test solution was prepared according to the method determined in section 3, which investigated the extraction solvent. The amount of extraction solvent added was used as a variable. Under the optimal chromatographic conditions determined above, 10 mL, 25 mL, and 50 mL of 80% v / v methanol aqueous solution were used for determination. The chromatograms obtained are shown in Table 16.

[0064] Table 16 Comparison of chromatographic peak areas with different extraction solvent amounts

[0065] As shown in Table 16, the amount of different extraction solvents added has little effect on the characteristic chromatograms, and the separation of each characteristic peak is not significantly different. Therefore, the amount of extraction solvent added is tentatively set at 25 mL for subsequent condition screening.

[0066] 5. Examination of sample size In addition to the sample amount, the test solution was prepared according to the method determined by the investigation of the amount of extraction solvent added in section 4. The sample amount was used as a variable. The test solution was prepared by taking 0.25g, 0.5g and 1g of sample under the optimal chromatographic conditions determined above. The chromatograms obtained are shown in Table 17.

[0067] Table 17 Comparison of Sampling Amounts, Chromatographic Peak System Adaptability Parameters for Yunweiling Medicinal Herbs Standard Decoction (Freeze-dried Powder)

[0068] As shown in Table 17, different sample amounts have little impact on the characteristic spectrum, and the separation of each characteristic peak is not significantly different. Therefore, the sample amount is tentatively set at 0.5g for subsequent condition screening.

[0069] 6. Determination of the preparation method for the test solution The method for preparing the test solution of Yunweiling decoction pieces (lyophilized powder) is as follows: Take 0.50g of this product, accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of 80% v / v methanol solution, weigh it, extract it by sonication for 30 minutes, cool it, make up the weight loss with 80% v / v methanol aqueous solution, shake it well, filter it, and the solution is obtained.

[0070] Construction of characteristic chromatograms of batch 215 of Yunweiling decoction standard decoction (lyophilized powder) in Experiment Example 2 Fifteen batches of Yunweiling decoction standard decoction (lyophilized powder) samples were taken as test samples (YWL2020001, YWL2020002, YWL2020003, YWL2020004, YWL2020005, YWL2020006, YWL2020007, YWL2020008, YWL2020009, YWL2020010, YWL2020011, YWL2020012, YWL2020013, YWL2020014, YWL2020015), and test sample solutions were prepared according to the method in Example 1.

[0071] The above solution was analyzed using the high-performance liquid chromatography method of Example 1, and characteristic chromatograms of 15 batches of Yunweiling decoction pieces (lyophilized powder) were obtained. The results are shown in Tables 18-19. The relative retention times of all 18 batches of Chuanmutong granules were within ±10% of the specified values, which met the requirements.

[0072] Table 1815 shows the results of relative retention time determination of characteristic chromatograms of Yunweiling decoction pieces (lyophilized powder).

[0073] Table 19. Results of relative peak area determination of characteristic spectra of 15 batches of Yunweiling decoction pieces (lyophilized powder)

[0074] Experimental Example 3: Establishment of Control Feature Maps (1) Generation of comparative feature maps Fifteen batches of Yunweiling decoction standard powder (lyophilized powder) were taken as test samples (YWL2020001, YWL2020002, YWL2020003, YWL2020004, YWL2020005, YWL2020006, YWL2020007, YWL2020008, YWL2020009, YWL2020010, YWL2020011, YWL2020012, YWL2020013, YWL2020014, YWL2020015). Test solutions were prepared according to the method in Example 1, and the results are shown in Example 2.

[0075] The above solution was analyzed using the high-performance liquid chromatography method described in Example 1. The results are shown in Tables 18-21 and... Figure 9The results showed that the characteristic chromatograms of 15 batches of Yunweiling decoction standard powder (lyophilized) contained 9 characteristic peaks. The characteristic chromatograms of the 15 batches of Yunweiling decoction standard powder (lyophilized) were fitted using the fingerprint chromatogram similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission to generate control characteristic chromatograms. Through HPLC identification of the characteristic peaks, 9 common characteristic peaks with suitable response values, good separation, and high purity were obtained. The chromatographic peak numbers were rearranged according to the order of the chromatographic peaks as peak 1-peak 9. Figure 10 As shown.

[0076] Table 20: Relative Retention Time of Standard Decoctions (Freeze-dried Powder) of Yunweiling Herbal Pieces

[0077] Table 21. Comparison of relative peak areas in standard decoctions (lyophilized powder) of Yunweiling medicinal slices.

[0078] (3) Identification of characteristic peaks in the comparative characteristic spectrum Through Table 22 and Figure 11 It can be seen that, based on the peak identification and reference standard positioning results, peak 1 is confirmed to be chlorogenic acid; The main chemical components of Yunweiling include volatile oils, triterpenoids, flavonoids, and phenylpropanoids; however, its main active ingredients are organic acids. The volatile oil components of Yunweiling include compounds such as thymol and thymol isobutyrate, but these are oils and insoluble in water, and therefore do not represent the main components of the standard Yunweiling decoction. Based on the results of standard decoction research and literature review, chlorogenic acid is considered the pharmacodynamic basis of the standard Yunweiling decoction. Chlorogenic acid has high water solubility, resulting in a relatively high transfer rate. Therefore, its characteristic peaks are easily observed and are correspondingly high when establishing characteristic spectra. Thus, organic acids are considered the main chemical components of the characteristic spectra of Yunweiling decoction pieces. Therefore, chlorogenic acid is selected as the S peak to calculate the relative retention times of each characteristic peak.

[0079] Table 22 Qualitative results of chlorogenic acid retention time

[0080] In summary, the chromatogram of the test sample should show 9 characteristic peaks, among which peak 1 corresponds to the retention time of the corresponding reference peak. The peak corresponding to the chlorogenic acid reference peak is peak S. Calculate the relative retention times of peaks 2-9 with peak S. The relative retention times should be within ±10% of the specified values, which are: 1.102 (peak 2), 1.165 (peak 3), 2.821 (peak 4), 2.914 (peak 5), 3.110 (peak 6), 3.196 (peak 7), 3.501 (peak 8), and 5.052 (peak 9). Peak 1 represents chlorogenic acid.

[0081] Experiment Example 4: Methodological Validation 1. Precision Take the same sample solution of Yunweiling standard decoction (lyophilized powder) prepared according to the method of Example 1, and inject it repeatedly 6 times. Record the chromatograms, and determine the relative retention time and relative peak area of ​​9 characteristic peaks. The results are shown in Tables 23-24. The chromatogram of the precision experiment is shown in Table 23-24. Figure 12 As shown in Tables 23-24 and Figure 12 It can be seen that the relative retention time and relative peak area of ​​the nine characteristic peaks are all less than 2% RSD, indicating good precision.

[0082] Table 23 Results of Instrument Precision and Relative Retention Time Tests

[0083] Table 24 Results of Instrument Precision Relative Peak Area Test

[0084] 2. Repeatability test Take the same sample of Yunweiling standard decoction (lyophilized powder) and repeat the sample 6 times according to the method in Example 1. Perform chromatographic analysis under the chromatographic conditions described in Example 1, and record the chromatograms. The results are shown in Tables 25-26. The chromatograms of the repeatability experiments are shown below. Figure 13 As shown in Tables 25-26 and Figure 13 It can be seen that the relative retention time and relative peak area of ​​the nine characteristic peaks are all less than 2% by RSD, indicating good repeatability.

[0085] Table 25 Results of the method repeatability relative retention time test (n=6)

[0086] Table 26 Results of the method repeatability relative peak area test (n=6)

[0087] 3. Intermediate precision (for different operators) Three inspectors, at different times and using the same equipment, determined the relative retention times and relative peak areas of the nine common peaks of the same batch of Yunweiling standard decoction (lyophilized powder) according to the test solution preparation method and chromatographic conditions described in Example 1. The results are shown in Tables 27-28. The chromatograms of the intermediate precision experiment (different operators) are shown below. Figure 14 As shown in Tables 27-28 and Figure 14 It can be seen that the RSD of the relative retention time and relative peak area of ​​the nine characteristic peaks are all less than 2%, indicating good intermediate precision (different operators).

[0088] Table 27 Results of intermediate precision relative retention time test (different operators)

[0089] Table 28 Intermediate Precision Relative Peak Area Test Results (Different Operators)

[0090] 4. Stability test Take the same sample solution prepared according to Example 1, and analyze it at 0, 4, 8, 12, 18, and 24 hours according to the chromatographic conditions described in Example 1. Record the chromatograms, determine the relative retention time and relative peak area of ​​the nine characteristic peaks, and analyze them. The results are shown in Tables 29-30. The chromatograms of the stability test are shown below. Figure 15 As shown in Tables 29-30 and Figure 15 It can be seen that the relative retention time and relative peak area of ​​the nine characteristic peaks are all less than 2%; indicating good stability and meeting the requirements of the determination.

[0091] Table 29 Results of the stability relative retention time test

[0092] Table 30 Results of Stability Relative Peak Area Test

[0093] 5. Daytime precision The same inspector took the same sample solution prepared according to Example 1 on different dates, and determined the relative retention time and relative peak area of ​​the nine common peaks under the chromatographic conditions described in Example 1. The results are shown in Tables 31-32. As can be seen from Tables 31-32, the RSD of the relative retention time and relative peak area of ​​the nine characteristic peaks are all less than 2%, indicating good inter-day precision.

[0094] Table 31 Results of Daytime Precision Relative Retention Time Test

[0095] Table 32 Results of Daytime Precision Relative Peak Area Test

[0096] 6. Exclusivity Accurately pipette 1 μL of the test solution (1 μL) and the negative control solution (purified water) (1 μL) obtained in Example 1, and inject them separately into the high-performance liquid chromatograph. Perform the test according to the chromatographic conditions of Example 1. Figure 16 As shown in Figure 1, the results indicate that the negative result has no interference.

[0097] 7. Integrity Take the same sample solution prepared according to Example 1, and inject and elute according to the gradient elution program in Table 33 under the chromatographic conditions described in Example 1. Record the chromatograms, and the results are shown in [Table 33]. Figure 17 .Depend on Figure 17 It can be seen that no other chromatographic peaks were eluted during the subsequent elution period, indicating that the characteristic peak information in the sample has been fully presented during the gradient elution time and will not affect subsequent injections.

[0098] Table 33 Gradient Elution Table

[0099] 8. Durability test 8.1 Investigation of different flow velocities Take the same sample solution prepared according to Example 1, and measure it at flow rates of 0.20 mL / min, 0.25 mL / min and 0.30 mL / min respectively under the chromatographic conditions described in Example 1. Record the chromatograms, measure the relative retention time and relative peak area at different flow rates, and analyze them. The results are shown in Tables 34-35. Table 34 Comparison of Relative Retention Time Measurement Results for Different Flow Rates

[0100] Table 35 Experimental results of relative peak area at different flow velocities

[0101] Different flow rates have a certain impact on the information content of chromatographic peaks and system adaptability parameters. After comparing and analyzing the above results, it was found that the flow rate change has a significant impact on the relative retention time and relative peak area. Therefore, the optimal flow rate selected in this invention is 0.20 mL / min.

[0102] 8.2 Investigation of different column temperatures Take the same sample solution prepared according to Example 1, and measure it at column temperatures of 20℃, 25℃ and 30℃ according to the chromatographic conditions described in Example 1. Record the chromatograms, measure the relative retention time and relative peak area at different column temperatures, and analyze them. The results are shown in Tables 36-37. Table 36 Comparison of relative retention time results at different column temperatures

[0103] Table 37 Results of relative peak area tests at different column temperatures

[0104] Different column temperatures have a certain impact on the information content of chromatographic peaks and system adaptability parameters. After comparing and analyzing the above results, it was found that changes in column temperature have a significant impact on the relative peak area. Therefore, this invention selects a column temperature of 25℃ as the optimal value.

[0105] 8.3 Investigation of different brands of chromatographic columns Take the same sample solution prepared according to Example 1, and chromatographically analyze it under the conditions described in Example 1 using different columns (Column 1: Agilent Eclipse Plus C). 18 Column 2: Welch Ultimate XB C 18 The chromatograms were recorded, and the relative retention times and relative peak areas of different chromatographic columns were determined and analyzed. The results are shown in Tables 38-39. Table 38 Results of relative peak area tests for different chromatographic columns

[0106] Table 39 Results of relative peak area tests for different chromatographic columns

[0107] Different chromatographic columns have a certain impact on the information content of chromatographic peaks and system adaptability parameters. After comparing and analyzing the above results, Agilent Eclipse Plus C 18 The chromatographic column provides good chromatographic resolution; therefore, this invention selects the Agilent Eclipse Plus C column. 18 (1.8 μm, 2.1 × 50 mm) is the optimal column size.

[0108] 8.4 Investigation of different chromatographs Take the same sample solution prepared according to Example 1, and perform the analysis under the chromatographic conditions described in Example 1 using different chromatograms (chromatogram 1: Waters ACQUITY UPLC H-Class PLUS chromatographic system; chromatogram 2: Agilent 1290 Infinity II chromatographic system). Record the chromatograms, determine the relative retention time and relative peak area of ​​different instruments, and perform analysis. The results are shown in Tables 40-41. Table 40 Results of intermediate precision relative retention time test (different liquid chromatographs)

[0109] Table 41 Results of intermediate precision relative peak area tests (different liquid chromatographs)

[0110] After comparing and analyzing the above results, it was found that the reproducibility of each chromatographic peak was good with different chromatograms, indicating that the method provided by the present invention has good robustness.

[0111] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing a feature map of Yunweiling or its derivative products, characterized in that, This includes detection using high-performance liquid chromatography (HPLC), with chromatographic conditions including: Using octadecylsilane-bonded silica gel as the packing material, acetonitrile as mobile phase A, and one of the following as mobile phase B: an aqueous solution containing phosphoric acid, an aqueous solution containing formic acid, or an aqueous solution containing glacial acetic acid, the gradient elution program includes: From 0 to 15 minutes, the volume percentage of mobile phase A changes from 5% to 14%, and the volume percentage of mobile phase B changes from 95% to 86%. Over 15-25 minutes, the volume percentage of mobile phase A changes from 14% to 20%, and the volume percentage of mobile phase B changes from 86% to 80%. Over 25-35 minutes, the volume percentage of mobile phase A changes from 20% to 22%, and the volume percentage of mobile phase B changes from 80% to 78%. Over 35-45 minutes, the volume percentage of mobile phase A changes from 22% to 30%, and the volume percentage of mobile phase B changes from 78% to 70%.

2. The construction method according to claim 1, characterized in that, The chromatographic conditions include at least one of the following: (1) The detection wavelength is 325-329 nm; preferably, the wavelength is 327 nm; (2) The flow rate is 0.20-0.30 mL / min; preferably, the flow rate is 0.2 mL / min; (3) Column temperature 20-30℃; (4) The injection volume is 1-5 μL; (5) The mobile phase B is an aqueous solution containing phosphoric acid. Preferably, the concentration of phosphoric acid in the aqueous solution is 0.05-0.2% v / v; preferably, the concentration of phosphoric acid is 0.1% v / v.

3. The construction method according to claim 1 or 2, characterized in that, It also includes the preparation of the test sample solution, including: weighing the test sample, extracting with solvent, separating solid and liquid, and taking the liquid, which is the test sample solution.

4. The construction method according to claim 3, characterized in that, The preparation of the test solution satisfies at least one of the following: A. The ratio of the mass of the Yunweiling test sample to the volume of the solvent is (0.2-1.0):(10-50); the unit of the mass of the test sample is g, and the unit of the volume of the solvent is mL; B. The extraction method is ultrasonic extraction, heating reflux, or oscillation extraction; ultrasonic extraction is preferred. C. Extraction time is ≥20 min; D. The solid-liquid separation is selected from filtration; E. The solvent is selected from one or more of methanol and water; preferably, it is a methanol aqueous solution with a volume percentage of 50-100% v / v, and more preferably, it is a methanol aqueous solution with a volume percentage of 70-90% v / v.

5. The construction method according to any one of claims 1-4, characterized in that, The construction method further includes the step of preparing a reference solution by adding solvent to chlorogenic acid, and the step of obtaining a reference standard reference spectrum by detecting the reference solution by high performance liquid chromatography according to the construction method. Preferably, the concentration of chlorogenic acid reference standard in the chlorogenic acid reference solution is 10-40 μg / mL; more preferably, it is 10 μg / mL. Preferably, the solvent used in the preparation of the reference solution is selected from methanol or an aqueous methanol solution with a volume fraction of not less than 50-100% v / v; more preferably, the solvent used in the preparation of the reference solution is selected from an aqueous methanol solution with a volume fraction of 80% v / v. Preferably, the construction method further includes the step of preparing a reference herb solution using Yunweiling reference herb, and the step of obtaining a reference herb reference spectrum by detecting the reference herb solution using high performance liquid chromatography in the construction method.

6. The construction method according to any one of claims 1-5, characterized in that, The Yunweiling or its derivative products include one or more of Yunweiling medicinal materials, Yunweiling processed slices, or Yunweiling preparations.

7. The construction method according to claim 6, characterized in that, The Yunweiling preparation includes at least one of Yunweiling decoction pieces and Yunweiling formula granules; preferably, the Yunweiling standard decoction includes at least one of Yunweiling standard decoction concentrated extract, Yunweiling standard decoction freeze-dried powder, and Yunweiling standard decoction aqueous solution.

8. A method for determining the content of active ingredients in Yunweiling or its derivative products, characterized in that, include: Take the test solution and the reference solution, and detect them respectively using the high performance liquid chromatography method in the method for constructing the characteristic spectrum of Yunweiling or its derivative products as described in any one of claims 1-7; The reference standard includes chlorogenic acid.

9. A quality testing method for Yunweiling or its derivative products, characterized in that, This includes the step of comparing the feature map of the product under test with the feature map of Yunweiling or its derivative products; The feature map of the product under test is obtained by the construction method according to any one of claims 1-7; The feature map of Yunweiling or its derivative products is selected from any one of the following (1)-(4): (1) It has 9 common characteristic peaks, and the peak corresponding to the reference peak of chlorogenic acid is the S peak. The relative retention times of peaks 2-9 and peak S are within ±10% of the specified values. The specified values ​​of peaks 2-9 are as follows: 1.102、1.165、2.821、2.914、3.110、3.196、3.501、5.052； (2) It has 9 common characteristic peaks, one of which corresponds to the retention time of the chlorogenic acid reference peak. The peak corresponding to the chlorogenic acid reference peak is peak S. The relative retention times of peaks 2-9 and peak S are within ±10% of the specified values. The specified values ​​of peaks 2-9 are as follows: 1.102、1.165、2.821、2.914、3.110、3.196、3.501、5.052； (3) Characteristic chromatograms of Yunweiling and / or its derivatives obtained by the construction method according to any one of claims 1-7 using single or multiple batches of Yunweiling and / or its derivatives as test samples; (4) Using multiple batches of Yunweiling and / or its derivatives as test samples, the characteristic spectra obtained by the construction method according to any one of claims 1-7 are used to make a control characteristic spectra by means of average or median.

10. The quality testing method for Yunweiling or its derivative products according to claim 9, characterized in that, Peak 1 (S) corresponds to chlorogenic acid.

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