Construction Method of HPLC Characteristic Chromatogram of Chuanminshen Radix, Slices, Standard Decoction and Its Formula Granules

Abstract

The present invention provides a method for constructing the HPLC characteristic chromatogram of Chuanminshen medicinal materials, cut pieces, standard decoctions and their formula granules, comprising: A) extracting the Chuanminshen raw materials with a solvent to obtain a test solution; B) determining the test solution by high performance liquid chromatography to obtain the HPLC characteristic chromatogram of the Chuanminshen raw materials; the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; mobile phase A is acetonitrile, and mobile phase B is 0.1% phosphoric acid, with gradient elution. The present invention adopts high performance liquid chromatography, selects acetonitrile - 0.1% phosphoric acid as the mobile phase for gradient elution, uses psoralen as the reference substance, and establishes the HPLC characteristic chromatogram of Chuanminshen medicinal materials, cut pieces, standard decoctions and their formula granules, which has good repeatability and precision, and the method is stable and reliable, and can control the quality of Chuanminshen medicinal materials, cut pieces, standard decoctions and their formula granules.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical detection, and particularly relates to a method for constructing HPLC characteristic fingerprints of Chuanminshen officinale, its cut crude drugs, standard decoctions and formula granules. Background Art

[0002] The dried root of Chuanminshen violaceum Sheh et Shan, a plant of the Umbelliferae family, has the effects of nourishing yin and relieving fever, and belongs to the liver and lung meridians, with the efficacy of liver heat attacking the head and eyes. The standard decoction is a freeze-dried powder prepared from the crude drug after processing according to a fixed preparation process.

[0003] In order to ensure the uniformity and stability of the quality of Chuanminshen officinale, its cut crude drugs and their standard decoctions, a new characteristic fingerprint method is established in this paper to control their quality. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for constructing HPLC characteristic fingerprints of Chuanminshen officinale, its cut crude drugs, standard decoctions and formula granules. The HPLC characteristic fingerprint method of Chuanminshen officinale, its cut crude drugs, standard decoctions and formula granules constructed by the present invention is stable and reliable, and can control the quality of Chuanminshen officinale, its cut crude drugs, standard decoctions and formula granules.

[0005] The present invention provides a method for constructing HPLC characteristic fingerprints of Chuanminshen officinale, its cut crude drugs, standard decoctions and formula granules, including:

[0006] A) Extracting the raw material of Chuanminshen officinale with a solvent to obtain a test solution;

[0007] B) Determining the test solution by high performance liquid chromatography to obtain the HPLC characteristic fingerprint of the raw material of Chuanminshen officinale.

[0008] The chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; mobile phase A is acetonitrile, and mobile phase B is 0.1% phosphoric acid, with gradient elution.

[0009] The method for constructing HPLC characteristic fingerprints of Chuanminshen officinale, its cut crude drugs, standard decoctions and formula granules provided by the present invention first takes the raw material of Chuanminshen officinale and extracts it with a solvent to obtain a test solution. The solvent is preferably 50% methanol.

[0010] According to the present invention, the raw material of Chuanminshen officinale is Chuanminshen officinale, its cut crude drugs, standard decoctions and formula granules.

[0011] According to the present invention, the preparation of the test solution of Chuanminshen medicinal materials / slices is specifically as follows: The Chuanminshen medicinal materials / slices are decocted with water for 20 - 30 min, and then 50% methanol is added for ultrasonic extraction; the ultrasonic power is 600 W, the frequency is 40 kHz; the extraction time is 20 - 40 min; more preferably 30 min.

[0012] According to the present invention, the preparation of the test solution of the standard decoction / formula granules of Chuanminshen is specifically as follows: The standard decoction / formula granules of Chuanminshen are ultrasonically extracted with 50% methanol, cooled, shaken well, and filtered to obtain the solution. The ultrasonic power is 600 W, the frequency is 40 kHz; the extraction time is 20 - 40 min; more preferably 30 min.

[0013] The ratio of the mass g of the Chuanminshen raw material to the volume mL of the solvent is (0.5 - 2):(25 - 50). Most preferably, for the medicinal materials and slices, it is 1.5:50, for the standard decoction it is 0.5:25, and for the formula granules it is 0.5:25.

[0014] The chromatographic peaks of the extraction solvent used in the present invention have a large amount of information and good effects.

[0015] The Chuanminshen raw material is Chuanminshen medicinal materials, Chuanminshen slices or decoctions. The present invention does not limit it, and the above raw materials can all be subjected to quality control and qualitative detection by the method of the present invention.

[0016] The present invention also includes the preparation of a reference solution: Psoralen is separately taken and dissolved with 50% methanol to obtain a reference solution of the reference substance.

[0017] The control medicinal materials are decocted with water, and then ultrasonically extracted with 50% methanol to obtain a reference solution of the control medicinal materials.

[0018] The reference solution of the reference substance and the reference solution of the control medicinal materials are determined by high performance liquid chromatography to obtain the chromatograms of the reference substance and the control medicinal materials respectively; and the components of the HPLC characteristic fingerprints of Chuanminshen medicinal materials, slices, standard decoctions and their formula granules are qualitatively determined according to the chromatograms of the reference substance and the control medicinal materials.

[0019] In the present invention, the mobile phase A is acetonitrile and the mobile phase B is 0.1% phosphoric acid solution for gradient elution.

[0020] The gradient elution in the present invention is preferably specifically as follows:

[0021] 0 - 3 min, mobile phase A: 10 - 14%, mobile phase B: 90 - 86%;

[0022] 3 - 15 min, mobile phase A: 14% - 17%, mobile phase B: 86% - 83%;

[0023] 15-30min, phase A: 17%-40%, phase B: 83%-60%;

[0024] 30-40min, phase A: 40%-80%, phase B: 60%-20%.

[0025] The present invention has good baseline separation under the above elution gradient, good separation of each peak and stable baseline.

[0026] C18 column, specifications: 2.7μm, 3.0×150mm; column temperature: 25℃.

[0027] The chromatographic column of the present invention has symmetrical chromatographic peaks and good separation under the above 25°C condition.

[0028] The mobile phase flow rate is preferably 0.3 ml / min.

[0029] The present invention found that the chromatographic peaks were better separated and the peak shapes were more symmetrical at a flow rate of 0.3 ml / min, which was the most preferred solution.

[0030] The detection wavelength of the present invention is preferably 335 nm.

[0031] The inventors found that at 335 nm, the chromatographic information was rich, each component had good absorption, the response value was moderate, the peak separation was good, and the baseline was stable.

[0032] The injection volume of the present invention is preferably 10 μL.

[0033] The beneficial effect of the present invention is that under one liquid chromatography condition, the substance group of Sichuan ming ginseng medicinal materials, decoction pieces, standard decoctions and formula granules thereof is controlled by fingerprint spectrum, and the fingerprint spectrum is positioned by psoralen; the cost of detection can be greatly reduced and qualitative detection can be realized.

[0034] The similarity of HPLC characteristic spectra of Sichuan ming ginseng medicinal materials, decoction pieces, standard decoction and formula granules was evaluated by the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint, and the HPLC standard characteristic spectra of Sichuan ming ginseng medicinal materials, decoction pieces, standard decoction and formula granules were obtained, which consisted of 10 characteristic peaks, including peak 3: caffeic acid peak, peak 7 (S): psoralen peak, peak 8: 5,8-dimethoxypsoralen peak.

[0035] In the characteristic spectrum of the standard decoction of Sichuan ming ginseng, psoralen is used as the reference peak S, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.22 (peak 1), 0.24 (peak 2), 0.30 (peak 3), 0.45 (peak 4), 0.84 (peak 5), 0.88 (peak 6), 1.11 (peak 8), 1.20 (peak 9), and 1.22 (peak 10).

[0036] Quality judgment criteria: Take the samples of Chuanminshen medicinal materials, cut pieces, standard decoctions and their formula granules, and operate according to the same method as above to obtain the characteristic chromatograms of Chuanminshen medicinal materials, cut pieces, standard decoctions and their formula granules. Use the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" (2012 edition) of the Pharmacopoeia Commission of the People's Republic of China to analyze the standard characteristic chromatograms and sample characteristic chromatograms of Chuanminshen medicinal materials, cut pieces, standard decoctions and their formula granules, and the similarity is greater than 0.90.

[0037] The method provided by the present invention can effectively monitor the quality of Chuanminshen medicinal materials, cut pieces, standard decoctions and their formula granules in different batches, making their quality stable. The method has the characteristics of high precision and good reproducibility, which is conducive to comprehensively monitoring the quality of products.

[0038] The characteristic chromatograms of Chuanminshen medicinal materials, cut pieces, standard decoctions and their formula granules established by the present invention use psoralen as the reference substance, pay attention to the order of each characteristic peak and its correlation with the medicinal materials and intermediate products, and can comprehensively evaluate the overall quality characteristics of the products. The method is scientific and reliable.

[0039] The newly established characteristic chromatogram method of the present invention can detect the components with relatively large polarity in Chuanminshen and its standard decoctions. And the preparation method of the test sample is simple and easy to operate, and there are relatively many identified characteristic peaks. It can accurately and reliably detect the characteristic chromatograms of Chuanminshen and its preparations. It can effectively detect and control the authenticity, quality consistency and stability of Chuanminshen and its preparations. It provides a basis for effectively controlling and comprehensively evaluating the quality of Chuanminshen standard decoctions. Ensure the uniformity and stability of the quality of Chuanminshen and its standard decoctions.

[0040] The present invention provides a method for constructing the HPLC characteristic chromatograms of Chuanminshen medicinal materials, cut pieces, standard decoctions and their formula granules, including: A) extracting the Chuanminshen raw materials with a solvent to obtain a test solution; B) determining the test solution by high performance liquid chromatography to obtain the HPLC characteristic chromatogram of the Chuanminshen raw materials; the chromatographic conditions of the high performance liquid chromatography are: the chromatographic column is a C18 column; mobile phase A is acetonitrile, and mobile phase B is 0.1% phosphoric acid, with gradient elution. The present invention uses high performance liquid chromatography, selects acetonitrile - 0.1% phosphoric acid as the mobile phase for gradient elution, uses psoralen as the reference substance, and establishes the HPLC characteristic chromatograms of Chuanminshen medicinal materials, cut pieces, standard decoctions and their formula granules. The method has good repeatability and precision, and is stable and reliable, and can control the quality of Chuanminshen medicinal materials, cut pieces, standard decoctions and their formula granules. Description of the Drawings

[0041] Figure 1 3D diagram of Chuanminshen standard decoction;

[0042] Figure 2 Chromatograms of Chuanminshen standard decoction at different wavelengths;

[0043] Figure 3 For flow rate investigation;

[0044] Figure 4 Column temperature investigation;

[0045] Figure 5 Results of extraction solvent investigation;

[0046] Figure 6 Extraction method investigation;

[0047] Figure 7 Extraction time investigation;

[0048] Figure 8 Chromatographic peak identification;

[0049] Figure 9 Investigation of different chromatographic columns;

[0050] Figure 10 Characteristic chromatogram of Chuanminshen standard decoction;

[0051] Figure 11 Control characteristic chromatogram of Chuanminshen standard decoction;

[0052] Figure 12 3D map of Chuanminshen medicinal materials.

[0053] Figure 13 Results of flow rate investigation;

[0054] Figure 14 Results of column temperature investigation;

[0055] Figure 15 Results of extraction solvent investigation;

[0056] Figure 16 Investigation of extraction method;

[0057] Figure 17 Investigation of extraction time;

[0058] Figure 18 Chromatographic peak identification;

[0059] Figure 19 Investigation of chromatographic column durability;

[0060] Figure 20-1 Characteristic chromatogram of Chuanminshen medicinal materials;

[0061] Figure 20-2 Characteristic chromatogram of Chuanminshen medicinal materials;

[0062] Figure 21-1 Control characteristic chromatogram of Chuanminshen medicinal materials;

[0063] Figure 21-2; Characteristic Chromatogram of Chuanminshen Slices for Control

[0064] Figure 22 Is the characteristic chromatogram of Chuanminshen slices

[0065] Figure 23 Is the characteristic chromatogram of Chuanminshen slices

[0066] Figure 24 Ultraviolet Absorption Spectrogram of Finished Chuanminshen Products

[0067] Figure 25 Chromatograms of Chuanminshen Formula Granules at Different Wavelengths

[0068] Figure 26 Flow Rate Investigation

[0069] Figure 27 Chromatogram of Column Temperature Investigation

[0070] Figure 28 Investigation of Extraction Solvent

[0071] Figure 29 Investigation of Extraction Method

[0072] Figure 30 Investigation of Extraction Time

[0073] Figure 31 Is for Chromatographic Peak Identification

[0074] Figure 32 Different Chromatographic Columns

[0075] Figure 33 Verification Diagram of Characteristic Chromatograms of 3 Batches of Chuanminshen Formula Granules

[0076] Figure 34 Is the Characteristic Chromatogram of Chuanminshen Formula Granules for Control

[0077] Figure 35 Is the Comparison Diagram under Different Gradient Conditions of Comparative Example 1 and Comparative Example 2

[0078] Figure 36 Result Diagram of Comparison with Different Mobile Phases Detailed Implementation Modes

[0079] The present invention provides a method for constructing the HPLC characteristic chromatogram of Chuanminshen medicinal materials, cut pieces, standard decoctions and their formula granules. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they all fall within the scope of protection of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate alterations and combinations to the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0080] In order to further illustrate the present invention, the following takes the embodiments as examples to describe in detail a method for constructing the HPLC characteristic chromatogram of Chuanminshen medicinal materials, cut pieces, standard decoctions and their formula granules provided by the present invention.

[0081] High performance liquid chromatograph: Agilent ultra-high performance liquid chromatograph

[0082] Electronic balance: ME204E / 02, MS205DU, XP26 (Mettler Toledo Instruments Co., Ltd.);

[0083] Ultra-pure water machine: Cell type 1810A (Shanghai Moller Scientific Instruments Co., Ltd.);

[0084] Ultrasonic cleaner: Model KQ5200DB (600W, 40KHz; Kunshan Ultrasonic Instruments Co., Ltd.);

[0085] Chromatographic column: C18; preferably InfinityLab Poroshell 120EC-C18 (column length is 150mm, inner diameter is 3.0mm, particle size is 2.7μm).

[0086] Acetonitrile (chromatographic pure, Sigma-Aldrich Shanghai Trading Co., Ltd.), phosphoric acid (chromatographic pure, Tianjin Kemiou Chemical Reagent Co., Ltd.), methanol (analytical pure, Chengdu Kelong Chemical Reagent Co., Ltd.), the water for the mobile phase is self-made ultra-pure water in the laboratory, and the rest are all self-made pure water in the laboratory.

[0087] Psoralen reference substance (National Institutes for Food and Drug Control, batch number: 110739-201617; purity: 99.7%);

[0088] Caffeic acid reference substance (National Institutes for Food and Drug Control, batch number: 110885-201703, purity: 99.7%);

[0089] 5,8-Dimethoxypsoralen reference substance (Chengdu Push Bio-Tech Co., Ltd., batch number: PS010566, purity: 99.71%);

[0090] Chuanshencan reference medicinal material (Shanghai Hongyong Biotechnology Co., Ltd., 230100 - 202304).

[0091] Freeze-dried powder of standard decoction of Chuanshencan (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: BT-1, BT-2, BT-3, BT-4, BT-5, BT-6, BT-7, BT-8, BT-9, BT-10, BT-11, BT-12, BT-13, BT-14, BT-15, BT-16, BT-17, BT-18, BT-19, BT-20, BT-21).

[0092] Chuanshencan medicinal materials: YC-1, YC-2, YC-3, YC-4, YC-5, YC-6, YC-7, YC-8, YC-9, YC-10, YC-11, YC-12, YC-13, YC-14, YC-15, YC-16, YC-17, YC-18, YC-19, YC-20, YC-21.

[0093] Chuanshencan cut pieces: YP-1, YP-2, YP-3, YP-4, YP-5, YP-6, YP-7, YP-8, YP-9, YP-10, YP-11, YP-12, YP-13, YP-14, YP-15, YP-16, YP-17, YP-18, YP-19, YP-20, YP-21.

[0094] Chuanshencan formula granules KL-1, KL-2, KL-3.

[0095] Example 1 HPLC characteristic fingerprint of standard decoction of Chuanshencan

[0096] 1.1 Chromatographic conditions and system suitability test Use octadecylsilane chemically bonded silica gel as the filler (column length is 150 mm, inner diameter is 3.0 mm, particle size is 2.7 μm); use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the regulations in the following table; column temperature is 25 °C; detection wavelength is 335 nm, and the number of theoretical plates calculated by the psoralen peak should be not less than 5000.

[0097]

[0098] Preparation of reference substance solution Weigh about 1.5 g of Chuanshencan reference medicinal material, place it in a stoppered conical flask, add 50 ml of water, decoct for 30 minutes, centrifuge, take the supernatant, evaporate to dryness, add 10 ml of 50% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the subsequent filtrate as the reference medicinal material reference substance solution. Separately weigh an appropriate amount of psoralen reference substance, accurately weigh it, and make a solution containing 10 μg per 1 ml with 50% methanol as the reference substance reference substance solution.

[0099] Preparation of the test solution: Take about 0.5 g of the powder of this product, add 25 ml of 50% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool to room temperature, shake well, filter, and take the subsequent filtrate, that is obtained.

[0100] Assay method: Accurately pipette 5 μl each of the reference medicinal material reference solution and the test solution, inject into the liquid chromatograph, and determine, that is obtained.

[0101] 1.2 Wavelength selection

[0102] On the basis of "Chromatographic conditions and system suitability test", use a diode array detector to perform a full wavelength scan on the test solution respectively, and extract the chromatograms of the test solution at wavelengths of 220 nm, 240 nm, 280 nm, 300 nm, 335 nm, and 350 nm respectively. The results are shown in Figure 1 、 2 。 Figure 1 3D map of the standard decoction of Chuanminshen;

[0103] Figure 2 Chromatograms of the standard decoction of Chuanminshen at different wavelengths; The results show that when the detection wavelength is 335 nm, the chromatographic peak information is relatively large. Therefore, the detection wavelength of the characteristic fingerprint method for the standard decoction of Chuanminshen is determined to be 335 nm.

[0104] 1.3 Flow rate investigation

[0105] On the basis of "Chromatographic conditions and system suitability test", investigate the flow rates of 0.2 ml / min, 0.3 ml / min, and 0.4 ml / min respectively. See Figure 3 。 Figure 3 For the flow rate investigation.

[0106] The results show that the RSD of the relative retention time of each characteristic peak at different flow rates is between 0.3% and 18.3%, indicating that this method is relatively feasible. When the flow rates are 0.2 ml / min and 0.4 ml / min, the resolution of chromatographic peak 8 is poor. When the flow rate is 0.3 ml / min, the chromatogram peak shapes are symmetrical and the resolutions meet the requirements. Therefore, according to the convention, a flow rate of 0.3 ml / min is selected for subsequent investigation.

[0107] 1.4 Column temperature investigation

[0108] On the basis of "Chromatographic conditions and system suitability test", investigate the column temperatures of 25 °C, 30 °C, and 35 °C respectively. See Figure 4 。 Figure 4 For the column temperature investigation.

[0109] The results showed that when the column temperature was 25 °C, the peak shapes in the chromatograms were all symmetrical and the resolution met the requirements. Therefore, a column temperature of 25 °C was selected for subsequent investigations.

[0110] To sum up, the chromatographic conditions and system suitability test for the characteristic chromatogram of the standard decoction of Chuanminshen are tentatively as follows: For the chromatographic conditions and system suitability test, octadecylsilane chemically bonded silica gel is used as the filler (column length is 150 mm, inner diameter is 3.0 mm, particle size is 2.7 μm); acetonitrile is used as mobile phase A, and 0.1% phosphoric acid solution is used as mobile phase B, and gradient elution is carried out according to the regulations in the following table; the column temperature is 25 °C; the detection wavelength is 335 nm, and the theoretical plate number calculated based on the psoralen peak should be not less than 5000.

[0111]

[0112] 1.5 Investigation on the preparation of the test solution

[0113] 1.5.1 Investigation on the extraction solvent

[0114] Take 6 portions of the freeze-dried powder of the standard decoction of Chuanminshen (BT-1), about 0.5 g each, add 25 ml of water, 70% methanol, methanol, 50% methanol, and ethanol respectively, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate to obtain the test solution. Inject the sample for determination according to the results of "4 Chromatographic conditions and system suitability test", and the results are shown in Figure 5 。 Figure 5 Results of the investigation on the extraction solvent.

[0115] The results showed that in the test solution extracted with 50% methanol, both the peak shape and resolution of the chromatographic peaks were better. Therefore, 50% methanol was selected as the extraction solvent for the preparation of the test solution in the determination of the characteristic chromatogram of Chuanminshen medicinal materials.

[0116] 1.5.2 Investigation on the extraction method

[0117] Take 2 portions of the freeze-dried powder of the standard decoction of Chuanminshen (BT-1), about 0.5 g each, add 25 ml of 50% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) and reflux treat for 30 minutes respectively, let it cool, shake well, filter, and take the subsequent filtrate to obtain the test solution. Inject the sample for determination according to the results of "1.4 Chromatographic conditions and system suitability test", and the results are shown in Figure 6 。 Figure 6 Results of the investigation on the extraction method.

[0118] From Figure 6 it can be seen that there is no significant difference in the final effects between reflux extraction and ultrasonic extraction. Therefore, ultrasonic extraction, which is more convenient to operate, was selected as the extraction method for the preparation of the test solution in the determination of the characteristic chromatogram of the standard decoction of Chuanminshen.

[0119] 1.5.3 Investigation on the extraction time

[0120] Take 3 portions (about 0.5 g) of the freeze-dried powder of the standard decoction of Chuanminshen (BT-1), place them in a stoppered conical flask, add 25 ml of 50% methanol, and ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, 30 minutes, and 40 minutes respectively. Let it cool, shake well, filter, and take the subsequent filtrate to obtain the solution. Inject and determine according to the results of "1.4 Chromatographic Conditions and System Suitability Test", and the results are shown in Figure 7 。 Figure 7 Investigation of extraction time.

[0121] From Figure 7 it can be seen that when the ultrasonic extraction time is 20 minutes, 30 minutes, and 40 minutes, there is no obvious difference in the peak separation in the chromatogram. However, in order to ensure that the solution can be fully extracted. Therefore, the extraction time of 30 minutes is selected as the extraction time for the preparation of the test solution in the determination of the characteristic chromatogram of the standard decoction of Chuanminshen.

[0122] In summary, the preparation method of the test solution for the characteristic chromatogram of the standard decoction of Chuanminshen is determined as follows: Take about 0.5 g of this product, add 25 ml of 50% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0123] 1.6 Methodology investigation

[0124] 1.6.1 Chromatographic peak identification

[0125] Preparation of the reference medicinal material solution: Take 1.5 g of the reference medicinal material of Chuanminshen, place it in a conical flask, add 50 ml of water, decoct for 30 minutes, centrifuge, take the supernatant, evaporate to dryness, dissolve the residue in 10 ml of 50% methanol solution, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate as the reference medicinal material reference solution.

[0126] Preparation of the reference solution: Weigh appropriate amounts of caffeic acid, psoralen, and 5,8-dimethoxypsoralen reference substances accurately, and dissolve them in 50% methanol respectively to prepare solutions containing 10 μg per 1 ml as the reference substance reference solutions.

[0127] Preparation of the test solution: Prepare the test solution of the standard decoction of Chuanminshen according to the results of "1.5 Investigation of the Preparation of the Test Solution".

[0128] Preparation of the negative control solution: Prepare the negative control solution without the standard decoction of Chuanminshen according to the results of "1.5 Investigation of the Preparation of the Test Solution". Inject and determine according to the results of "1.4 Chromatographic Conditions and System Suitability Test", and the results are shown in Figure 8 。 Figure 8 Chromatographic peak identification.

[0129] 1.6.2 Precision test

[0130] Take 6 parts of the freeze-dried powder of the standard decoction of Chuanminshen (BT-1), prepare the test solution according to the results of "5 Preparation and Investigation of Test Solutions", and inject and determine according to the results of injecting continuously 6 times under the conditions of "1.4 Chromatographic Conditions and System Suitability Test". The results are shown in Table 1.

[0131] Table 1 Precision Investigation - Retention Time of Characteristic Peaks

[0132]

[0133] The results show that the RSD values of the retention times of each characteristic peak are between 0.1% and 0.2%, indicating that the precision of this instrument is good.

[0134] 1.6.3 Repeatability Investigation

[0135] Take 6 parts of the freeze-dried powder of the standard decoction of Chuanminshen (BT-1), prepare the test solution according to the results of "1.5 Preparation and Investigation of Test Solutions", and inject and determine according to the results of "1.4 Chromatographic Conditions and System Suitability Test". The results are shown in Table 2.

[0136] Table 2 is the repeatability investigation - ratio of relative retention times of characteristic peaks

[0137]

[0138] The results show that the RSD values of the relative retention times of each characteristic peak are between 0.1% and 0.2%, indicating that the repeatability of this method is good.

[0139] 1.6.4 Intermediate Precision Investigation

[0140] Take the same test sample (batch number: BT1), prepare the test solution by different personnel (A, B) at different times (Ⅰ, Ⅱ) according to the results of "5 Preparation and Investigation of Test Solutions", and inject and determine on instruments a and b respectively according to the results of "1.4 Chromatographic Conditions and System Suitability Test". The results are shown in Table 3.

[0141] Table 3 Intermediate Precision - Relative Retention Time

[0142]

[0143]

[0144] As can be seen from Table 3, under different test solution preparation personnel and different test solution preparation times, the RSD of the relative retention times of each characteristic peak is between 0.4% and 6.2%, indicating that the applicability of this method is good.

[0145] 1.6.5 Durability Investigation of Chromatographic Column

[0146] On the basis of the above-mentioned experimental conditions, three chromatographic columns with different numbers, InfinityLab Poroshell120EC-C18 (column length: 150 mm, inner diameter: 3.0 mm, particle size: 2.7 μm), were investigated respectively. The results are shown in Figure 9 and Table 4. Figure 9 Investigation of different chromatographic columns.

[0147] Table 4 Chromatographic column durability investigation - Relative retention time of characteristic peaks

[0148]

[0149] The results showed that when the samples were detected using the above three chromatographic columns, the RSD of the relative retention time of the characteristic peaks was between 0.1% and 1.2%, indicating good durability of the chromatographic column for this method.

[0150] 1.6.6 Stability

[0151] Prepare one sample solution according to the results of "5 Preparation and investigation of sample solutions", and inject samples for determination at 0 h, 2 h, 4 h, 8 h, 16 h, and 24 h respectively according to the results of "1.4 Chromatographic conditions and system suitability test". The results are shown in Table 5.

[0152] Table 5 24-hour stability investigation - Retention time of characteristic peaks

[0153]

[0154] The results showed that the RSD of the corresponding retention time of the characteristic peaks was between 0.1% and 0.2%, and the sample solution was relatively stable within 24 hours.

[0155] 1.6.7 Determination of characteristic peaks and establishment of reference chromatogram

[0156] 1.6.7.1 Verification results of 21 batches of standard decoctions of Chuanminshen

[0157] Prepare the sample solutions of 21 batches of standard decoctions according to the results of "5 Preparation and investigation of sample solutions", and inject samples for determination and analysis according to the results of "4 Chromatographic conditions and system suitability test", and calculate the relative retention time and relative peak area ratio. The results are shown in Figure 10 , Table 6. Figure 10 It is the characteristic chromatogram of the standard decoction of Chuanminshen.

[0158] (Batch numbers from bottom to top are: BT-1, BT-2, BT-3, BT-4, BT-5, BT-6, BT-7, BT-8, BT-9, BT-10, BT-11, BT-12, BT-13, BT-14, BT-15, BT-16, BT-17, BT-18, BT-19, BT-20, BT-21)

[0159] Table 6 Relative retention times of 21 batches of standard decoctions of *Chuanminshen violaceum*

[0160]

[0161] According to the principle of stable relative retention time, all batches of samples can be detected and the peaks are relatively high, a total of 10 peaks with good repeatability were selected as characteristic peaks. The relative retention times of each characteristic peak and the S peak were calculated. Finally, it was stipulated that: 10 characteristic peaks should be presented in the characteristic chromatogram of the test sample, the peak corresponding to the psoralen reference substance peak was the S peak, and the relative retention times of peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, and peak 10 and the S peak were calculated, and their relative retention times should be within the range of ±10% of the specified values. The specified values were: 0.22 (peak 1), 0.24 (peak 2), 0.30 (peak 3), 0.45 (peak 4), 0.84 (peak 5), 0.88 (peak 6), 1.11 (peak 8), 1.20 (peak 9), 1.22 (peak 10).

[0162] Using the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (2012 Edition), the 21 batches of standard decoctions of *Chuanminshen violaceum* were synthesized, and a reference chromatogram of the characteristic chromatogram of the standard decoction of *Chuanminshen violaceum* was established, as shown in Figure 11 . Figure 11 It was the reference characteristic chromatogram of the standard decoction of *Chuanminshen violaceum*; peak 3: caffeic acid peak, peak 7 (S): psoralen peak,: peak 8: 5,8-dimethoxypsoralen peak.

[0163] Example 2 HPLC characteristic chromatograms of *Chuanminshen violaceum* herbs and cut pieces

[0164] 2.1 Prescribed chromatographic conditions

[0165] Chromatographic conditions and system suitability test: Octadecylsilane chemically bonded silica gel was used as the filler (column length was 150 mm, inner diameter was 3.0 mm, particle size was 2.7 μm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was carried out according to the regulations in the following table; the column temperature was 25°C; the detection wavelength was 335 nm, and the number of theoretical plates calculated by the psoralen peak should be not less than 5000.

[0166]

[0167]

[0168] Preparation of the reference solution: Weigh approximately 1.5 g of the reference medicinal material of Chuanminshen, place it in a stoppered conical flask, add 50 ml of water, decoct for 30 minutes, centrifuge, take the supernatant, evaporate to dryness, add 10 ml of 50% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the subsequent filtrate as the reference medicinal material reference solution. Separately, weigh an appropriate amount of the psoralen reference substance accurately, dissolve it in 50% methanol to prepare a solution containing 10 μg per 1 ml as the reference substance reference solution.

[0169] Preparation of the test solution: Take an appropriate amount of the powder of this product (passed through No. 3 sieve), weigh approximately 1.5 g, add 50 ml of water, decoct for 30 minutes, centrifuge, take the supernatant, evaporate to dryness, add 10 ml of 50% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0170] Determination method: Accurately pipette 5 μl each of the reference solution and the test solution, inject them into the liquid chromatograph for determination to obtain the results.

[0171] 2.2 Chromatographic conditions and system suitability test

[0172] 2.2.1 Wavelength selection

[0173] Based on the "Chromatographic conditions proposed in 2.1", use a diode array detector to perform a full wavelength scan on the test solution respectively, and extract the chromatograms of the test solution at wavelengths of 220 nm, 250 nm, 270 nm, and 300 nm respectively, as shown in Figure 12 . Figure 12 It is the 3D map of the Chuanminshen medicinal material.

[0174] The results show that when the detection wavelength is 335 nm, the chromatographic peak information is relatively large. Therefore, the detection wavelength of the characteristic fingerprint method for the Chuanminshen medicinal material is finally determined to be 335 nm.

[0175] 2.2.2 Flow rate investigation

[0176] Based on the "Chromatographic conditions proposed in 2.1", investigate the separation effect of the chromatographic peaks in the test solution at three flow rates of 0.2 ml / min, 0.3 ml / min, and 0.4 ml / min respectively. The results are shown in Figure 13 and Table 7. Figure 13 It is the result of the flow rate investigation.

[0177] Table 7 Flow rate investigation - relative retention time

[0178]

[0179] The results showed that when the flow rate was 0.3 ml / min, the chromatographic peak shape was symmetrical and the resolution was good. Therefore, the flow rate of 0.3 ml / min was used for subsequent investigation.

[0180] 2.2.3 Column temperature investigation

[0181] On the basis of the "2.1 Prescribed chromatographic conditions", the separation effects of three column temperatures of 25 °C, 30 °C, and 35 °C on the chromatographic peaks in the test solution were investigated respectively. The results are shown in Figure 14 , Table 8. Figure 14 It is the result of the column temperature investigation.

[0182] Table 8 Column temperature investigation - Relative retention time

[0183]

[0184] Figure 14 It was shown that at the column temperature of 25 °C, the chromatographic peak shape was symmetrical and the resolution was good. Therefore, 25 °C was selected as the column temperature for the determination of the characteristic chromatogram of Chuanminshen Radix.

[0185] To sum up, the chromatographic conditions and system suitability test for the characteristic chromatogram of Chuanminshen Radix were tentatively set as follows: using octadecylsilane chemically bonded silica gel as the filler (column length 150 mm, inner diameter 3.0 mm, particle size 2.7 μm); using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and performing gradient elution according to the regulations in the following table; column temperature 25 °C; detection wavelength 335 nm, and the theoretical plate number calculated by the psoralen peak should be not less than 5000.

[0186]

[0187]

[0188] 2.3 Preparation investigation of the test solution

[0189] 2.3.1 Investigation of extraction solvents

[0190] Take 6 portions of Chuanminshen Radix (YC-1), about 1.0 g for each portion, place them in a stoppered conical flask, add 50 ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 10 ml of methanol, ethanol, 30% methanol, 50% methanol, 70% methanol, and water respectively to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the subsequent filtrate to obtain the solution. Inject the sample for determination according to the results of "4 Chromatographic conditions and system suitability test". The results are shown in Figure 15 . Figure 15 It is the result of the extraction solvent investigation.

[0191] The results showed that the peak shapes and resolutions of the chromatographic peaks in the test solution extracted with 50% methanol were both good. Therefore, 50% methanol was selected as the extraction solvent for preparing the test solution in the determination of the characteristic chromatogram of Chuanminshen Radix.

[0192] 2.3.2 Investigation of extraction methods

[0193] Take 3 portions of Chuanminshen Radix (YC-1). Add 50 ml of water to one portion, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 10 ml of 50% methanol, and ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes respectively. Add 10 ml of 50% methanol to the other two portions, ultrasonically treat (power 600 W, frequency 40 kHz) and decoct for 30 minutes respectively. Let it cool, shake well, filter, and take the subsequent filtrate to obtain the solution. Inject and determine according to the results of "2.2 Chromatographic conditions and system suitability test". The results are shown in Figure 16 . Figure 16 This is for the investigation of extraction methods.

[0194] The results showed that there was no significant difference in the final effects between reflux extraction and ultrasonic extraction, but more peaks appeared during decoction. Therefore, decoction was selected as the extraction method for preparing the test solution in the determination of the characteristic chromatogram of Chuanminshen Radix.

[0195] 2.3.3 Investigation of extraction time

[0196] Take 3 portions of Chuanminshen Radix (YC-1), each portion about 1.5 g, place in a stoppered conical flask, add 50 ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 10 ml of 50% methanol, and ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, 30 minutes, and 40 minutes respectively. Let it cool, shake well, filter, and take the subsequent filtrate to obtain the solution. Inject and determine according to the results of "2.2 Chromatographic conditions and system suitability test". The results are shown in Figure 17 . Figure 17 This is for the investigation of extraction time.

[0197] From Figure 17 it can be seen that when the ultrasonic extraction time was 20 minutes, 30 minutes, and 40 minutes, there was no obvious difference in the peak separation in the chromatogram. To ensure sufficient extraction, the extraction time of 30 minutes was selected as the extraction time for preparing the test solution in the determination of the characteristic chromatogram of Chuanminshen Radix.

[0198] To sum up, the preparation method of the test solution for the characteristic chromatogram of Chuanminshen Radix was determined as follows: Take an appropriate amount of the powder of this product (sieved through No. 3 sieve), take about 1.5 g, place in a stoppered conical flask, add 50 ml of water, decoct for 30 minutes, centrifuge, take the supernatant, evaporate to dryness, add 10 ml of 50% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0199] 2.4 Methodological Investigation

[0200] 2.4.1 Chromatographic Peak Identification

[0201] Preparation of reference solution: Weigh appropriate amounts of caffeic acid, psoralen, and 5,8-dimethoxypsoralen reference substances accurately, and dissolve them in 50% methanol to prepare a solution containing 10 μg per 1 ml as the reference solution.

[0202] Preparation of reference medicinal material solution: Take about 1.5 g of the reference medicinal material of Chuanminshen, place it in a stoppered conical flask, add 50 ml of water, decoct for 30 minutes, centrifuge, take the supernatant, evaporate to dryness, add 10 ml of 50% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the subsequent filtrate as the reference medicinal material reference solution.

[0203] Preparation of test solution: Prepare the test solution of Chuanminshen medicinal material according to the results of "2.3 Investigation on the Preparation of Test Solution".

[0204] Preparation of negative control solution: Prepare the negative control solution without Chuanminshen medicinal material according to the results of "2.3 Investigation on the Preparation of Test Solution". Inject and determine according to the results of "2.2 Chromatographic Conditions and System Suitability Test", and the results are shown in Figure 18 . Figure 18 Chromatographic peak identification. It can be seen from Figure 18 that a total of 10 characteristic peaks are pointed out. Among them, the peak corresponding to peak 3 is the caffeic acid peak, the peak corresponding to peak 7 is the psoralen peak, and the peak corresponding to peak 8 is the 5,8-dimethoxypsoralen peak.

[0205] 2.4.2 Precision Test

[0206] Take the Chuanminshen medicinal material (YC-1) and prepare the test solution according to the results of "2.3 Investigation on the Preparation of Test Solution". Inject and determine according to the results of "2.2 Chromatographic Conditions and System Suitability Test" for 6 consecutive times, 5 μl each time, and calculate the relative retention time and relative peak area of each characteristic peak. The results are shown in Table 9.

[0207] Table 9 Precision Investigation - Retention Time

[0208]

[0209]

[0210] The results show that the RSD of the retention time of each characteristic peak is between 0.02% and 0.16%, indicating that the precision of this instrument is good.

[0211] 2.4.3 Repeatability Investigation

[0212] Take 6 parts of Chuanmingcan medicinal materials (YC-1), prepare the test solution according to the results of "2.3 Preparation and Investigation of Test Solutions", and inject and determine according to the results of "2.2 Chromatographic Conditions and System Suitability Test". The results are shown in Table 10.

[0213] Table 10 Repeatability Investigation - Relative Retention Time

[0214]

[0215] The results show that the RSD of the relative retention time of each characteristic peak is between 0.0% and 0.1%, indicating that this method has good repeatability.

[0216] 2.4.4 Intermediate Precision Investigation

[0217] Take the same test solution (batch number: YC-1), prepare the test solution by different personnel (A, B) at different times (Ⅰ, Ⅱ) according to the results of "5 Preparation and Investigation of Test Solutions", and inject and determine on instruments a and b respectively according to the results of "2.2 Chromatographic Conditions and System Suitability Test". The results are shown in Table 11.

[0218] Table 11 Intermediate Precision - Relative Retention Time

[0219]

[0220]

[0221] It can be seen from the results that under different test solution preparation personnel and different test solution preparation times, the RSD of the relative retention time of each characteristic peak is between 0.4% and 6.3%, indicating that this method has good applicability.

[0222] 2.4.5 Chromatographic Column Durability Investigation

[0223] On the basis of the above experimental conditions, three chromatographic columns with different numbers, Agilent InfinityLab Poroshell120 EC-C18 (column length 150 mm, inner diameter 3.0 mm, particle size 2.7 μm), were investigated respectively, and the results are shown in Figure 19 , Table 12. Figure 19 Chromatographic Column Durability Investigation.

[0224] Table 12 Chromatographic Column Durability Investigation - Relative Retention Time Ratio

[0225]

[0226] The results show that when the samples are detected with the above 3 chromatographic columns, the RSD of the relative retention time of each characteristic peak is 0.1% - 1.2%, indicating that this method has good durability for different chromatographic columns.

[0227] 2.4.6 Stability

[0228] According to the results of "2.3 Preparation of test solution", a sample solution was prepared, and according to the results of "2.2 Chromatographic conditions and system suitability test", the sample was injected and tested at 0h, 2h, 4h, 8h, 16h, and 24h. The results are shown in Table 13.

[0229] Table 13 Stability Study-Retention Time

[0230]

[0231] The results showed that the RSD of the retention time of each characteristic peak was between 0.01% and 0.19%, indicating that the test solution had good stability and could be used for detection within 24 hours.

[0232] 2.4.7 Determination of characteristic peaks and establishment of reference spectrum

[0233] 2.4.7.1 Verification results of 21 batches of Sichuan ginseng medicinal materials

[0234] According to the results of "5 Preparation of test solution", 21 batches of test solutions of medicinal materials were prepared, and according to the results of "4 Chromatographic conditions and system suitability test", the samples were injected, measured and analyzed, and the relative retention time and relative peak area ratio were calculated. The results are shown in Figure 20 and Table 14. Figure 20-1 Characteristic spectrum of Sichuan ginseng medicinal materials (the batch numbers from bottom to top are: YC-1, YC-2, YC-3, YC-4, YC-5, YC-6, YC-7, YC-8, YC-9, YC-10, and control medicinal materials); Figure 20-2 Characteristic spectrum of Sichuan ginseng medicinal materials (the batch numbers from bottom to top are: YC-11, YC-12, YC-13, YC-14, YC-15, YC-16, YC-17, YC-18, YC-19, YC-20, YC-21, and control medicinal materials).

[0235] Table 14 Relative retention time of 21 batches of Sichuan ginseng medicinal materials

[0236]

[0237]

[0238] According to the principle of stable relative retention time and relatively high common peaks detected in all batches of samples, 10 peaks with good repeatability were selected as characteristic peaks. The RSD of relative retention time of 10 characteristic peaks of 21 batches of Sichuan ginseng medicinal materials was between 0.0% and 0.1%, all less than 10%.

[0239] Final Specification: In the characteristic chromatogram of the test sample, 10 characteristic peaks should be presented and should correspond to the retention times of the 10 characteristic peaks in the reference medicinal material of the control crude drug. Among them, the peak corresponding to the psoralen reference substance is the S peak. Calculate the relative retention times of each characteristic peak to the S peak, and the relative retention times should be within ±10% of the specified values. The specified values are: 0.22 (peak 1), 0.24 (peak 2), 0.30 (peak 3), 0.45 (peak 4), 0.84 (peak 5), 0.88 (peak 6), 1.11 (peak 8), 1.20 (peak 9), 1.22 (peak 10).

[0240] The similarity evaluation system for traditional Chinese medicine chromatographic fingerprints (2012 version) was used to synthesize 21 batches of Chuanminshen medicinal materials, and the reference chromatogram of the characteristic chromatogram of Chuanminshen medicinal materials was established. See Figure 21-1 。 Figure 21-1 Reference characteristic chromatogram of Chuanminshen medicinal materials; Peak 3: Caffeic acid peak, Peak 7 (S): Psoralen peak, Peak 8: 5,8-Dimethoxypsoralen peak. The reference chromatogram of the characteristic chromatogram of Chuanminshen decoction pieces was established. As Figure 21-2 ; Reference characteristic chromatogram of Chuanminshen decoction pieces; Peak 3: Caffeic acid peak, Peak 7 (S): Psoralen peak, Peak 8: 5,8-Dimethoxypsoralen peak.

[0241] 2.4.7.2 Verification Results of 21 Batches of Chuanminshen Decoction Pieces

[0242] Prepare the test sample solutions of 21 batches of medicinal materials according to the results of "2.3 Preparation and Investigation of Test Sample Solutions", and inject and determine according to the results of "2.2 Chromatographic Conditions and System Suitability Tests", and calculate the relative retention times and relative peak area ratios. The results are shown in Figure 22 、 23 , Table 15. Figure 22 It is the characteristic chromatogram of Chuanminshen decoction pieces.

[0243] (From bottom to top, the batch numbers are: YP-1, YP-2, YP-3, YP-4, YP-5, YP-6, YP-7, YP-8, YP-9, YP-10, control crude drug); Figure 23 It is the characteristic chromatogram of Chuanminshen decoction pieces; from bottom to top, the batch numbers are: YP-11, YP-12, YP-13, YP-14, YP-15, YP-16, YP-17, YP-18, YP-19, YP-20, YP-21, control crude drug).

[0244] Table 15 Relative Retention Times of 21 Batches of Chuanminshen Decoction Pieces

[0245]

[0246]

[0247] According to the principle of stable relative retention time and relatively high common peaks that can be detected in each batch of samples, a total of 10 peaks with good repeatability were selected as characteristic peaks. The results showed that the relative standard deviations (RSDs) of the relative retention times of the 10 characteristic peaks in 21 batches of Chuanminshen Radix Praeparata slices were between 0.1% and 1.4%, all less than 2.0%.

[0248] Finally, it was stipulated that: the characteristic chromatogram of the test sample should show 10 characteristic peaks, and the retention times of these 10 characteristic peaks should correspond to those in the reference chromatogram of the control medicinal material. The peak corresponding to the psoralen reference was defined as the S peak. Calculate the relative retention times of each characteristic peak relative to the S peak, and the relative retention times should be within ±10% of the specified values. The specified values are: 0.22 (peak 1), 0.24 (peak 2), 0.30 (peak 3), 0.45 (peak 4), 0.84 (peak 5), 0.88 (peak 6), 1.11 (peak 8), 1.20 (peak 9), 1.22 (peak 10).

[0249] The similarity evaluation system for traditional Chinese medicine chromatographic fingerprints (2012 edition) was used to synthesize 21 batches of Chuanminshen Radix Praeparata slices, and a reference chromatogram of the characteristic chromatogram of Chuanminshen Radix Praeparata slices was established.

[0250] 2.5 Determination of the characteristic chromatogram method for Chuanminshen Radix Praeparata and its slices

[0251] Chromatographic conditions and system suitability test: Octadecylsilane chemically bonded silica gel was used as the filler (column length 150 mm, inner diameter 3.0 mm, particle size 2.7 μm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was carried out according to the regulations in the following table; the column temperature was 25°C; the detection wavelength was 335 nm, and the number of theoretical plates calculated based on the psoralen peak should be not less than 5000.

[0252]

[0253] Preparation of the reference solution: Weigh about 1.5 g of the control medicinal material of Chuanminshen Radix Praeparata, place it in a stoppered conical flask, add 50 ml of water, decoct for 30 minutes, centrifuge, take the supernatant, evaporate to dryness, add 10 ml of 50% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the subsequent filtrate as the reference solution of the control medicinal material. Separately, weigh an appropriate amount of psoralen reference substance accurately, dissolve it in 50% methanol to make a solution containing 10 μg per 1 ml as the reference solution of the reference substance.

[0254] Preparation of the test solution: Take an appropriate amount of the powder of this product (sieved through No. 3 sieve), weigh about 1.5 g, add 50 ml of water, decoct for 30 minutes, centrifuge, take the supernatant, evaporate to dryness, add 10 ml of 50% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the subsequent filtrate, that is obtained.

[0255] Determination method: Accurately pipette 5 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and perform the determination to obtain the results.

[0256] Example 3 HPLC characteristic fingerprint of Chuanminshen formula granules

[0257] 3.1 Prescribed chromatographic conditions

[0258] Chromatographic conditions and system suitability test: Use octadecylsilane chemically bonded silica gel as the filler (column length is 150 mm, inner diameter is 3.0 mm, particle size is 2.7 μm); use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the regulations in the following table; the flow rate is 0.3 ml per minute; the column temperature is 25 °C; the detection wavelength is 335 nm.

[0259]

[0260] Preparation of reference solution: Take 1.5 g of the reference medicinal material of Chuanminshen, place it in a conical flask, add 50 ml of water, decoct for 30 minutes, centrifuge, take the supernatant, evaporate to dryness, dissolve the residue in 10 ml of 50% methanol solution, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate as the reference medicinal material reference solution. Separately weigh an appropriate amount of psoralen reference substance, accurately weigh it, dissolve it in 50% methanol to make a solution containing 10 μg per 1 ml as the reference substance reference solution.

[0261] Preparation of test solution: Take an appropriate amount of this product, grind it finely, take 0.5 g, add 25 ml of 50% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0262] Determination method: Accurately pipette 5 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and perform the determination to obtain the results.

[0263] 3.2 Chromatographic conditions and system suitability test

[0264] 3.2.1 Wavelength selection

[0265] On the basis of the "3.1 Prescribed chromatographic conditions", use a diode array detector to perform full wavelength scanning on the test solution respectively, and extract the chromatograms of the test solution at wavelengths of 240 nm, 280 nm, 330 nm, 335 nm, and 350 nm respectively. See Figure 24 、 25 。 Figure 24 Ultraviolet absorption spectrum of finished product of Chuanminshen; Figure 25 Chromatograms of Chuanminshen formula granules at different wavelengths; The results show that when the detection wavelength is 335 nm, the chromatographic peak information is larger, the resolution is better, and the chromatogram baseline is more stable. Therefore, the detection wavelength is determined to be 335 nm.

[0266] 3.2.2 Flow rate investigation

[0267] On the basis of the "Chromatographic conditions determined in 3.1", the separation effects of chromatographic peaks in the test solution were investigated at three flow rates of 0.2 ml / min, 0.3 ml / min, and 0.4 ml / min respectively. The results are shown in Figure 26 . Figure 26 Flow rate investigation; the results show that when the flow rate is 0.3 ml / min, the peak shape in the chromatogram is better and the distribution of each characteristic peak is moderate. Therefore, the flow rate is determined to be 0.3 ml / min.

[0268] 3.2.3 Column temperature investigation

[0269] On the basis of the "Chromatographic conditions determined in 3.1", the separation effects of chromatographic peaks in the test solution were investigated at four column temperatures of 25 °C, 30 °C, and 35 °C respectively. The results are shown in Figure 27 . Figure 27 Column temperature investigation chromatogram.

[0270] The results of column temperature investigation show that when the column temperature is 25 °C, the peak shapes in the chromatogram are relatively symmetric and the resolution is good. Therefore, 25 °C is finally determined as the column temperature for the characteristic chromatogram method of Chuanminshen formula granules.

[0271] To sum up, the chromatographic conditions and system suitability test for the characteristic chromatogram of Chuanminshen formula granules are determined as follows: using octadecylsilane chemically bonded silica gel as the filler (column length 150 mm, inner diameter 3.0 mm, particle size 2.7 μm), acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, and gradient elution is carried out according to the regulations in the following table: column temperature is 25 °C; flow rate is 0.3 ml per minute; detection wavelength is 335 nm.

[0272]

[0273]

[0274] 3.3 Preparation investigation of test solution

[0275] 3.3.1 Extraction solvent investigation

[0276] Take an appropriate amount of Chuanminshen formula granules (KL-1), grind them finely, take about 0.5 g, add 25 ml of water, ethanol, methanol, 70% methanol, 50% methanol, and 30% methanol respectively, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate to obtain the solution. Inject the sample for determination according to the results of "3.2 Chromatographic conditions and system suitability test". The results are shown in Figure 28 . Figure 28Investigation of extraction solvents; The results showed that when 50% methanol was used as the extraction solvent, the chromatographic peaks had a large amount of information and good resolution. Therefore, 50% methanol was selected as the extraction solvent.

[0277] 3.3.2 Investigation of extraction methods

[0278] Take an appropriate amount of Chuanminshen formula granules (KL-1), grind them finely, about 0.5 g, add 25 ml of 50% methanol for extraction, reflux and ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes respectively, cool, shake well, filter, take the subsequent filtrate, and then inject and determine according to the results of "3.2 Chromatographic conditions and system suitability test". The results are shown in Figure 29 . Figure 29 Investigation of extraction methods; The results showed that there was no significant difference in the amount of chromatographic peak information when the test samples were extracted by ultrasonic extraction and reflux extraction respectively. Since ultrasonic extraction is more convenient to operate, the extraction method of the test samples was determined to be ultrasonic extraction.

[0279] 3.3.3 Investigation of extraction time

[0280] Take an appropriate amount of Chuanminshen formula granules (KL-1), grind them finely, take 3 portions, each about 0.5 g, add 25 ml of 50% methanol, and perform ultrasonic treatment (power 600 W, frequency 40 kHz). The extraction time of the test samples was investigated at 20 minutes, 30 minutes, and 40 minutes respectively. Cool, shake well, filter, take the subsequent filtrate, and then inject and determine according to the results of "3.2 Chromatographic conditions and system suitability test". The results are shown in Figure 30 . Figure 30 Investigation of extraction time; The results showed that the extraction could be completed in 20 minutes. However, to ensure more complete extraction, the extraction time of the test samples was determined to be 30 minutes.

[0281] In summary, the preparation method of the test solution for the characteristic chromatogram of Chuanminshen formula granules was determined as follows: Take an appropriate amount of this product, grind it finely, take 0.5 g, add 25 ml of 50% methanol, perform ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the subsequent filtrate.

[0282] 3.4 Methodology investigation

[0283] 3.4.1 Chromatographic peak identification

[0284] Preparation of the reference medicinal material solution: Take 1.5 g of the reference medicinal material of Chuanminshen, place it in a conical flask, add 50 ml of water, decoct for 30 minutes, centrifuge, take the supernatant, evaporate to dryness, add 10 ml of 50% methanol solution to the residue, perform ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the subsequent filtrate as the reference medicinal material reference solution.

[0285] Preparation of reference solution: Appropriate amounts of caffeic acid, psoralen, and 5,8-dimethoxypsoralen reference substances were accurately weighed and dissolved in 50% methanol to prepare solutions containing 10 μg per 1 ml as reference solution for reference substances.

[0286] Preparation of test solution: The test solution of Chuanminshen formula granules was prepared according to the results of "3.3 Investigation on the preparation of test solution".

[0287] Preparation of negative control solution: The negative control solution without Chuanminshen formula granules was prepared according to the results of "3.3 Investigation on the preparation of test solution". It was injected for determination according to the results of "3.2 Chromatographic conditions and system suitability test", and the results are shown in Figure 31 . Figure 31 For chromatographic peak identification.

[0288] 3.4.2 Precision test

[0289] The test solution of Chuanminshen formula granules (KL-1) was prepared according to the results of "3.3 Investigation on the preparation of test solution" and injected for determination according to the results of "3.2 Chromatographic conditions and system suitability test", 5 μl each time for 6 consecutive times, and the relative retention times of each characteristic peak were calculated. The results are shown in Table 16.

[0290] Table 16 Precision investigation - retention time

[0291]

[0292] The results showed that the RSD values of the retention times of each characteristic peak were between 0.1% and 0.2%, indicating good precision of the instrument.

[0293] 3.4.3 Repeatability investigation

[0294] Six portions of Chuanminshen formula granules (KL-1) were taken and the test solutions were prepared according to the results of "3.3 Investigation on the preparation of test solution" and injected for determination according to the results of "3.2 Chromatographic conditions and system suitability test". The results are shown in Table 17.

[0295] Table 17 Repeatability investigation - relative retention time

[0296]

[0297] The results showed that the RSD% values of the relative retention times of each characteristic peak were between 0.1% and 0.2%, indicating good repeatability of the method.

[0298] 3.4.4 Intermediate precision investigation

[0299] Take the same test sample (batch number: KL-1) and prepare the test sample solution by different personnel (A, B) at different times (Ⅰ, Ⅱ) according to the results of "3.3 Preparation and Investigation of Test Sample Solution", and inject and measure on instruments a and b respectively according to the results of "3.2 Chromatographic Conditions and System Suitability Test", and the results are shown in Table 18.

[0300] Table 18 Investigation of Personnel and Time - Relative Retention Time

[0301]

[0302] The results show that when the same sample is determined by different personnel at different times, the RSD% values of the relative retention times of each characteristic peak are between 0.4% and 6.2%, indicating that the method has good repeatability.

[0303] 3.4.5 Investigation on the Durability of Chromatographic Column

[0304] On the basis of the above experimental conditions, three different numbered InfinityLab Poroshell 120 EC-C18 (column length 150 mm, inner diameter 3.0 mm, particle size 2.7 μm) chromatographic columns were investigated respectively, and the results are shown in Table 19. Figure 32 . Figure 32 Different chromatographic columns.

[0305] Table 19 Investigation on the Durability of Chromatographic Column - Ratio of Relative Retention Time

[0306]

[0307] As can be seen from the above table, when the sample is detected with the above 3 chromatographic columns, the RSD% values of the relative retention times of each characteristic peak are between 0.1% and 1.3%, indicating that the method has good durability for the chromatographic column.

[0308] 3.4.6 Stability

[0309] Prepare one test sample solution according to the results of "3.3 Preparation and Investigation of Test Sample Solution", and inject and measure at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h respectively according to the results of "3.2 Chromatographic Conditions and System Suitability Test". The results are shown in Table 20.

[0310] Table 20 Investigation on Stability - Retention Time

[0311]

[0312] The results show that the RSD of the retention time of the corresponding characteristic peak is 0.1 - 0.2%, indicating that the sample solution is stable within 24 hours.

[0313] 3.5 Determination of Characteristic Peaks and Establishment of Control Chromatogram

[0314] 3.5.1 Validation Results of 3 Batches of Chuanminshen Formula Granules

[0315] Prepare the test solution of 3 batches of formula granules according to the results of "3.3 Preparation and Investigation of Test Solution", and inject the sample for determination and analysis according to the results of "3.2 Chromatographic Conditions and System Suitability Test", and calculate the relative retention time. The results are shown in Figure 11 , Table 21, Figure 33 . Figure 33 Validation Diagram of the Characteristic Chromatogram of 3 Batches of Chuanminshen Formula Granules

[0316] Table 21 Relative Retention Time of 3 Batches of Chuanminshen Formula Granules

[0317]

[0318] According to the principle of stable relative retention time, all batches of samples can be detected and the peaks are relatively high, a total of 10 peaks with good repeatability were selected as characteristic peaks. The peak corresponding to the psoralen reference peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. Its relative retention time should be within the range of ±10% of the specified value. The specified values are: 0.22 (peak 1), 0.24 (peak 2), 0.30 (peak 3), 0.45 (peak 4), 0.84 (peak 5), 0.88 (peak 6), 1.11 (peak 8), 1.21 (peak 9), 1.22 (peak 10).

[0319] Use the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition) to synthesize 3 batches of Chuanminshen formula granules, and establish a reference chromatogram of the characteristic chromatogram of Chuanminshen formula granules Figure 34 It is the reference characteristic chromatogram of Chuanminshen formula granules, in which peak 3: caffeic acid; peak 7 (S): psoralen; peak 8: 5,8-dimethoxypsoralen

[0320] 3.6 Determination of the Method for the Characteristic Chromatogram of Chuanminshen Formula Granules

[0321] Chromatographic Conditions and System Suitability Test Use octadecylsilane chemically bonded silica gel as the filler (column length is 150 mm, inner diameter is 3.0 mm, particle size is 2.7 μm); use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the regulations in the following table; the flow rate is 0.3 ml per minute; the column temperature is 25°C; the detection wavelength is 335 nm. The number of theoretical plates calculated based on the psoralen peak should be not less than 5000

[0322]

[0323]

[0324] Preparation of the reference solution: Weigh accurately 1.5 g of the reference medicinal material of Chuanminshen, add 50 ml of water, decoct for 30 minutes, cool, centrifuge, take the supernatant, evaporate to dryness, dissolve the residue in 10 ml of 50% methanol, and ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the consecutive filtrate as the reference medicinal material reference solution. Separately, weigh accurately an appropriate amount of psoralen reference substance, dissolve it in 50% methanol to prepare a solution containing 10 μg per 1 ml as the reference substance reference solution.

[0325] Preparation of the test solution: Take an appropriate amount of this product, grind it finely, weigh 0.5 g, add 25 ml of 50% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the consecutive filtrate, which is the test solution.

[0326] Determination method: Accurately pipette 5 μl each of the reference solution and the test solution, inject them into the liquid chromatograph for determination, and that's it.

[0327] Comparative example 1

[0328]

[0329] The results are as Figure 35 shown. Figure 35 shown in the figure. It is a comparison chart of the gradient change conditions of comparative example 1 and comparative example 2 and the gradient determined in this patent when the mobile phase is acetonitrile - 0.1% phosphoric acid. It can be seen from Figure 35 the figure that in comparative example 1, all substances are piled up together before 2 minutes, and there are no obvious characteristic peaks. In comparative example 2, four obvious characteristic peaks appear, but the overall baseline fluctuates greatly. For the method selected in this patent, the baseline is relatively flat, and there are ten peaks with good resolution that can be used as characteristic peaks.

[0330] Comparative example 3

[0331] Under the condition that the mobile phase gradient remains unchanged, different mobile phases were selected for investigation, and acetonitrile - 0.1% phosphoric acid, acetonitrile - 0.1% glacial acetic acid, methanol - 0.1% phosphoric acid, and methanol - 0.1% glacial acetic acid were respectively selected for investigation. The results are as Figure 36 shown. It can be seen from the figure that when using methanol - 0.1% phosphoric acid and methanol - 0.1% glacial acetic acid as the mobile phase, different degrees of loss occur in all 10 characteristic peaks; acetonitrile - 0.1% phosphoric acid and acetonitrile - 0.1% glacial acetic acid can better separate the 10 characteristic peaks, but due to the easy volatility of glacial acetic acid and its poorer stability compared to phosphoric acid, finally, acetonitrile - 0.1% phosphoric acid was selected as the mobile phase.

[0332] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for constructing the HPLC characteristic chromatogram of Chuanminshen medicinal materials, decoction pieces, standard decoctions and their formula granules, comprising: A) Solvent extraction of Chuanminshen raw materials to obtain the test solution; specifically for the preparation of the test solution of Chuanminshen medicinal materials / decoction pieces: decoct Chuanminshen medicinal materials / decoction pieces in water for 20 - 30 min, and then add 50% methanol for ultrasonic extraction; Specifically for the preparation of the test solution of Chuanminshen standard decoctions / formula granules: ultrasonically extract Chuanminshen standard decoctions / formula granules with 50% methanol, cool to room temperature, shake well, filter, and obtain the solution; B) Determine the test solution by high performance liquid chromatography to obtain the HPLC characteristic chromatogram of Chuanminshen raw materials; it also includes the preparation of reference substance solutions of reference substances and reference medicinal materials: Respectively take psoralen, dissolve it with 50% methanol to obtain the reference substance solution of the reference substance; Take the reference medicinal material, decoct it with water, and then ultrasonically extract it with 50% methanol to obtain the reference substance solution of the reference medicinal material; Determine the reference substance solution of the reference substance and the reference medicinal material by high performance liquid chromatography to obtain the chromatograms of the reference substance and the reference medicinal material respectively; and qualitatively determine the components of the HPLC characteristic chromatograms of Chuanminshen medicinal materials, decoction pieces, standard decoctions and their formula granules according to the chromatograms of the reference substance and the reference medicinal material; The chromatographic conditions of the high performance liquid chromatography are: the chromatographic column is a C18 column; mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid, and gradient elution is used; The chromatographic column is a C18 column with a specification of 2.7 μm, 3.0×150 mm; the column temperature is 25 °C; the detection wavelength is 335 nm; The specific gradient elution is as follows: 0 - 3 min, mobile phase A: 10 - 14%, mobile phase B: 90 - 86%; 3 - 15 min, mobile phase A: 14% - 17%, mobile phase B: 86% - 83%; 15 - 30 min, mobile phase A: 17% - 40%, mobile phase B: 83% - 60%; 30 - 40 min, mobile phase A: 40% - 80%, mobile phase B: 60% - 20%.

2. The method according to claim 1, characterized in that The flow rate of the mobile phase is 0.3 mL / min; the injection volume is 10 μL.

3. The method according to claim 1, wherein Use the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints to evaluate the similarity of the HPLC characteristic chromatograms of Chuanminshen medicinal materials, decoction pieces, standard decoctions and their formula granules, and obtain the HPLC characteristic chromatograms of Chuanminshen medicinal materials, decoction pieces, standard decoctions and their formula granules composed of 10 characteristic peaks, among which peak 3: caffeic acid peak, peak 7(S): psoralen peak, peak 8: 5,8 - dimethoxypsoralen peak.

4. The method according to claim 3, characterized in that In the characteristic chromatogram of the Chuanminshen standard decoction, taking psoralen as the reference peak S peak, calculate the relative retention time of each characteristic peak to the S peak, and the relative retention time is within ±10% of the specified value. The specified values are respectively: 0.22 (peak 1), 0.24 (peak 2), 0.30 (peak 3), 0.45 (peak 4), 0.84 (peak 5), 0.88 (peak 6), 1.11 (peak 8), 1.20 (peak 9), 1.22 (peak 10).

5. The method according to claim 1, characterized in that, The Chuanminshen raw materials in step A) are Chuanminshen medicinal materials, decoction pieces, standard decoctions and their formula granules.

6. The method according to claim 1, wherein The ultrasonic power is 600 W and the frequency is 40 kHz; the extraction time is 20 - 40 min; The ratio of the mass g of the Chuanminshen raw material to the volume mL of the solvent is (0.5 - 2):(25 - 50).

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