Fingerprint spectrum detection method for traditional Chinese medicine powder of Jingfenean pills and application of fingerprint spectrum detection method

Abstract

The invention relates to a detection method and application of a fingerprint spectrum of traditional Chinese medicine powder of a Jingyanping pill, in particular to an HPLC (High Performance Liquid Chromatography) method for detecting the fingerprint spectrum of the traditional Chinese medicine powder of the Jingyanping pill. The invention further discloses a method for establishing the control fingerprint spectrum of the traditional Chinese medicine powder of the Jianjing Pingan pill and a quality control method of the traditional Chinese medicine powder of the Jianjing Pingan pill. The HPLC method is accurate and reliable, can be used for quality control of the traditional Chinese medicine powder of the Jingfenean pill, and is of great significance to sustainable development of medicines.

Description

Technical Field

[0001] This application belongs to the technical field of the quality analysis of Yuzhi Ping'an Pills, and relates to an HPLC method for detecting the fingerprint of traditional Chinese medicine powder in Yuzhi Ping'an Pills, including the establishment of this method, the establishment and application of the reference fingerprint. Background Art

[0002] The formula composition of Yuzhi Ping'an Pills is as follows: Atractylodes lancea (parched) 104g, Tangerine peel 104g, Magnolia officinalis (roasted) 104g, Licorice root 104g, Hawthorn (charred) 104g, Laofanzhi Wan Ying Shenqu 104g, Germinated barley (parched) 104g, Immature bitter orange (parched) 67.2g, Galangal 52g, Amomum kravanh 52g, Amomum tsao-ko 52g, Nutmeg 52g, Aquilaria sinensis 67.2g, Aucklandia lappa 52g, Santalum album 67.2g, Eugenia caryophyllata 52g. The formula preparation method is as follows: For the above sixteen flavors, take Atractylodes lancea, Tangerine peel, Magnolia officinalis, Licorice root, Hawthorn, Laofanzhi Wan Ying Shenqu, Germinated barley, and Immature bitter orange, decoct with water twice, 2 hours for the first time and 1 hour for the second time, combine the decoction liquids, filter, concentrate the filtrate into a thick paste, and powder the remaining eight flavors of Galangal, etc. into fine powder, mix with the above thick paste, dry, powder into fine powder, make pills with water, coat with carbon black, and dry to make 450g, thus obtaining the product. Yuzhi Ping'an Pills have the effects of warming the middle-jiao and regulating the stomach, promoting qi circulation to relieve pain, suppressing nausea and vomiting, and promoting digestion and removing retention. It can be used for symptoms such as motion sickness, nausea and vomiting, disharmony between the stomach and intestines, fullness and oppression in the chest and diaphragm, anorexia due to food stagnation, abdominal distension and pain, and loose stools.

[0003] Among them, the eight flavors of Galangal, Amomum kravanh, Amomum tsao-ko, Nutmeg, Aquilaria sinensis, Aucklandia lappa, Santalum album, and Eugenia caryophyllata are powdered into fine powder and then called the traditional Chinese medicine powder of Yuzhi Ping'an Pills.

[0004] At present, the inspection items of Yuzhi Ping'an Pills only include appearance, identification, inspection, and determination of the content of Eugenia caryophyllata (calculated as eugenol) by gas chromatography, and cannot truly and comprehensively reflect the internal quality.

[0005] In order to comprehensively and effectively control the internal quality of the traditional Chinese medicine powder of Yuzhi Ping'an Pills and ensure the stability and uniformity of product quality, it is necessary to develop a new method for controlling the quality of the traditional Chinese medicine powder of Yuzhi Ping'an Pills. Summary of the Invention

[0006] In order to comprehensively and effectively control the internal quality of the Yuzhi Ping'an Pills traditional Chinese medicine powder and ensure the stable and uniform product quality, the inventors of this application have deeply carried out research on the high performance liquid chromatography (HPLC) fingerprint method of the Yuzhi Ping'an Pills traditional Chinese medicine powder (prepared by pulverizing Aucklandia lappa, Aquilaria sinensis, Santalum album, Syzygium aromaticum, Amomum kravanh, Myristica fragrans, Alpinia galanga, and Alpinia katsumadai), studied the extraction method of the test sample, column temperature, mobile phase, similarity, etc., and verified the specificity, precision, stability, repeatability, etc., and established an HPLC method for detecting the fingerprint of the Yuzhi Ping'an Pills traditional Chinese medicine powder, including the method for establishing the control fingerprint and its application. The method is accurate and reliable, can be used for the quality control of the Yuzhi Ping'an Pills traditional Chinese medicine powder, and is of great significance for the sustainable development of drugs.

[0007] On the one hand, the present invention provides an HPLC method for detecting the fingerprint of the Yuzhi Ping'an Pills traditional Chinese medicine powder, which is detected under the following chromatographic conditions:

[0008] Chromatographic column: Agilent ZORBAX SB-Aq, 250mm, 4.6*5um,

[0009] Sample injection volume: 10ul,

[0010] Flow rate: 0.6ml / min,

[0011] Column temperature: 30°C,

[0012] Detection wavelength: 200nm,

[0013] Mobile phase: 0.3% phosphoric acid aqueous solution (A) - acetonitrile (B)

[0014] The gradient elution conditions are as follows:

[0015]

[0016] In the present invention, the Yuzhi Ping'an Pills traditional Chinese medicine powder can be obtained as follows: according to the corresponding proportion of the prescription, take Aucklandia lappa, Aquilaria sinensis, Santalum album, Syzygium aromaticum, Amomum kravanh, Myristica fragrans, Alpinia galanga, and Alpinia katsumadai, mix them evenly and powder them, and pass through an 80-mesh sieve. However, the present invention is not limited thereto.

[0017] In the present invention, the percentages in the mobile phase and gradient elution are volume percentages. For example, 95% refers to 95 volume %. For example, when the mobile phases A and B are 95:5, it means that 95 volumes of mobile phase A are mixed with 5 volumes of mobile phase B.

[0018] In the present invention, the 0.3% phosphoric acid aqueous solution is prepared as follows: Pipette 3.0 ml of phosphoric acid, add ultrapure water to 1000 ml, shake well, filter, and degas. When necessary, the mobile phase can be enlarged or reduced in the same proportion to prepare mobile phases of different volumes.

[0019] In the HPLC method for detecting the fingerprint spectrum of the traditional Chinese medicine powder of Yuzhi Ping'an Pills, the test solution of the traditional Chinese medicine powder of Yuzhi Ping'an Pills for detection can be prepared as follows: Take about 3 g of the powder of the traditional Chinese medicine powder of Yuzhi Ping'an Pills, accurately weigh it, accurately add 50 ml of 50% methanol aqueous solution, tightly stopper, weigh, shake well, let stand overnight, perform ultrasonic treatment (power 500 W, frequency 40 kHz) for 60 minutes, cool, weigh again, make up the lost weight with 50% methanol aqueous solution, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0020] In the present invention, the 50% methanol aqueous solution is prepared as follows: Transfer 500 ml of methanol, add ultrapure water to 1000 ml, shake well, filter, and degas.

[0021] The HPLC method for detecting the fingerprint spectrum of the traditional Chinese medicine powder of Yuzhi Ping'an Pills of the present invention may further include the step of selecting characteristic peaks in the fingerprint spectrum of the traditional Chinese medicine powder of Yuzhi Ping'an Pills, wherein the chromatographic peaks with better separation of the main components are selected as characteristic peaks. In some embodiments, a total of 23 characteristic peaks are determined.

[0022] The HPLC method for detecting the fingerprint spectrum of the traditional Chinese medicine powder of Yuzhi Ping'an Pills of the present invention may further include the steps of attributing and identifying the characteristic peaks in the fingerprint spectrum of the traditional Chinese medicine powder of Yuzhi Ping'an Pills, including:

[0023] (1) Preparation of the test solution and reference solution of each medicinal material

[0024] Respectively take cloves, costus root, agarwood, sandalwood, amomum kravanh, tsaoko amomum fruit, galangal fruit, nutmeg, crush them and sieve through No. 5 sieve, each weigh about 1 g, accurately weigh, accurately add 50 ml of 50% methanol aqueous solution, tightly stopper, weigh, let stand overnight and then perform ultrasonic treatment (power 500 W, frequency 40 kHz) for 60 minutes, cool, weigh again, make up the lost weight with 50% methanol aqueous solution, shake well, filter, and take the subsequent filtrate to obtain the test solution of each medicinal material;

[0025] Take an appropriate amount of cardamom pure reference substance and prepare a reference solution with a concentration of 70 μg / ml;

[0026] Take an appropriate amount of costunolide reference substance and prepare a reference solution with a concentration of 70 μg / ml;

[0027] (2) Detection of the chromatograms of each medicinal material and reference substance

[0028] Use the HPLC method for detecting the fingerprint spectrum of the traditional Chinese medicine powder of Yuzhi Ping'an Pills according to the present invention to detect the chromatograms of each medicinal material and reference substance;

[0029] (3) Identification and attribution of common peaks:

[0030] The attribution and identification of characteristic peaks in the fingerprint of the traditional Chinese medicine powder of Yuzhi Ping'an Pills are completed by comparing with the chromatograms of each medicinal material and reference substance.

[0031] On the other hand, the present invention provides a method for establishing a reference fingerprint of the traditional Chinese medicine powder of Yuzhi Ping'an Pills, the method comprising:

[0032] (1) Preparation of test solution:

[0033] Take about 3 g of the powder of the traditional Chinese medicine powder of Yuzhi Ping'an Pills, accurately weigh, accurately add 50 ml of 50% methanol aqueous solution, tightly stopper, weigh, shake well, let stand overnight, ultrasonically treat (power 500 W, frequency 40 kHz) for 60 minutes, cool, weigh again, make up the lost weight with 50% methanol aqueous solution, shake well, filter, take the subsequent filtrate to obtain the test solution;

[0034] (2) HPLC detection:

[0035] Precisely pipette 10 μl of the test solutions of n batches respectively, and detect to obtain the test solution fingerprint by the HPLC method for detecting the fingerprint of the traditional Chinese medicine powder of Yuzhi Ping'an Pills according to the present invention;

[0036] (3) Generating a reference fingerprint:

[0037] Generate a reference fingerprint based on the common peaks in the fingerprints of n batches of test solutions, wherein, select the chromatographic peaks with better resolution among the main components in the fingerprint of the traditional Chinese medicine powder of Yuzhi Ping'an Pills as characteristic peaks to determine the common peaks, and n≥10.

[0038] In the above step (3), there is no particular limitation on the method for generating a reference fingerprint based on the test solution fingerprint chromatogram, and conventional methods in the art can be used. For example, the test solution fingerprint chromatogram of the traditional Chinese medicine powder of Yuzhi Ping'an Pills measured can be imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" promulgated by the Pharmacopoeia Commission of the People's Republic of China, set one of the test solution chromatograms as the reference chromatogram, the standard chromatogram generation method is, for example, the median method, and at the same time, for example, the multi-point calibration method is used to establish the reference fingerprint of the traditional Chinese medicine powder of Yuzhi Ping'an Pills.

[0039] In some embodiments, 23 common peaks are determined in the above step (3).

[0040] In some embodiments, the reference fingerprint of the traditional Chinese medicine powder of Yuzhi Ping'an Pills is generally as Figure 16 shown.

[0041] On the other hand, the present invention provides a quality control method for the traditional Chinese medicine powder of Yuzhi Ping'an Pills, the method comprising the following steps:

[0042] (1) Preparation of test solution:

[0043] Take about 3 g of the traditional Chinese medicine powder of Yuzhi Ping'an Pills, accurately weigh it, accurately add 50 ml of 50% methanol aqueous solution, tightly stopper, weigh, shake well, let stand overnight, ultrasonically treat (power 500 W, frequency 40 kHz) for 60 minutes, cool, weigh again, make up the lost weight with 50% methanol aqueous solution, shake well, filter, take the continuous filtrate to obtain the test solution;

[0044] (2) Fingerprint determination:

[0045] Precisely pipette 10 μL of the test solution, and determine it by the HPLC method for detecting the fingerprint of the traditional Chinese medicine powder of Yuzhi Ping'an Pills according to the present invention to obtain the fingerprint of the test traditional Chinese medicine powder of Yuzhi Ping'an Pills;

[0046] (3) Qualification determination:

[0047] Calculate the similarity between the fingerprint of the test traditional Chinese medicine powder of Yuzhi Ping'an Pills and the reference fingerprint. If the similarity is greater than or equal to 0.9, it is a qualified product.

[0048] The similarity can be calculated by importing the fingerprint of the test traditional Chinese medicine powder sample of Yuzhi Ping'an Pills and the reference fingerprint of the traditional Chinese medicine powder of Yuzhi Ping'an Pills into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" promulgated by the National Pharmacopoeia Commission for comparison. If the similarity is greater than or equal to 0.9, it is a qualified product; otherwise, it is an unqualified product.

[0049] As confirmed by the experimental results, in the chromatogram obtained by the method for detecting the fingerprint of the traditional Chinese medicine powder of Yuzhi Ping'an Pills according to the present invention, the number of peaks is large, the sensitivity is high, the baseline is stable, the noise is small, the response is sensitive, the response value is large, and the resolution is good. In addition, the blank solvent does not interfere with the test of the test solution, and the specificity is good. The precision, stability and reproducibility of the method are good.

[0050] The present invention has been described in detail above, but the above embodiments are essentially illustrative only and are not intended to limit the present invention. In addition, the present invention is not limited by any theory described in the foregoing prior art or the invention content or the following examples.

[0051] Unless otherwise expressly stated, the numerical ranges in the entire application document include any sub-ranges therein and any numerical values incremented by the smallest sub-unit of the given values therein. Unless otherwise expressly stated, the numerical values in the entire application document represent an approximate measure or limitation of the scope of embodiments including minor deviations from the given values, having approximately the mentioned values, and having the exact values mentioned. Except for the working examples provided at the end of the detailed description, all numerical values of parameters (e.g., quantities or conditions) in this application document (including the appended claims) should be understood to be modified by the term "about" in all cases, whether or not "about" actually appears before the numerical value. "About" means that the stated numerical value allows for some imprecision (some approximation to the exact value; approximately or reasonably close to the value; approximate). If the imprecision provided by "about" is not understood in this ordinary meaning in the art, then "about" as used herein means at least the variations that can be produced by the ordinary methods of measuring and using these parameters. For example, "about" can include variations of less than or equal to 10%, less than or equal to 5%, less than or equal to 4%, less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, or less than or equal to 0.1%, and in some aspects, less than or equal to 0.01%.

[0052] The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text. Without departing from the scope of the technical idea and technical essence of the present invention, various modifications, variations, or substitutions can be made to the technical solutions of the present invention, and these modified, varied, or substituted technical solutions are still included within the scope of the present invention.

[0053] It will be understood that the words or terms used in the specification and claims should not be construed as having the meanings defined in a common dictionary. It will be further understood that based on the principle that the inventor can appropriately define the meanings of the words or terms to best explain the present invention, these words or terms should be construed as having meanings consistent with their meanings in the context of the relevant field and the technical idea of the present invention.

[0054] Beneficial effects

[0055] In the present invention, by studying the extraction method, column temperature, mobile phase, similarity, etc., and verifying the specificity, precision, stability, repeatability, etc., an HPLC fingerprint method for Zhiyuzhipingan Pills traditional Chinese medicine powder is established to ensure the stable and uniform quality of the product.

[0056] Traditional Chinese medicine cannot simply use the content of one or several active ingredients to indicate its quality and efficacy like chemical drugs. By establishing a fingerprint, comprehensively indicating the characteristics and proportions of its main chemical components, it provides a comprehensive and quantifiable quality evaluation method for products to comprehensively evaluate the internal quality of drugs, enabling more comprehensive improvement in drug quality control, ensuring stable, uniform, and controllable product quality, safe and effective drug use, and also being of great significance for the high-quality and sustainable development of products. Description of the Drawings

[0057] Figure 1 Showing the chromatograms of test solution prepared with different extraction solvents.

[0058] Figure 2 Showing the 3D ultraviolet absorption spectrum of sample S1.

[0059] Figures 3 - 8 Respectively showing the chromatograms obtained from Experiments 1 - 6 in 2.2.2 of the examples.

[0060] Figures 9 - 11 Respectively showing the chromatograms obtained in 2.2.3 of the examples.

[0061] Figure 12 Showing the chromatograms of the blank solvent and the test solutions of sample S1, clove, costus root, agarwood, sandalwood, amomum kravanh, tsaoko amomum fruit, galangal fruit, and nutmeg obtained in the specificity test of 2.3.1 in the examples.

[0062] Figure 13 Showing the chromatograms of the test solution of sample S3 and the reference solutions of cardamonin and costunolide obtained in the specificity test of 2.3.1 in the examples.

[0063] Figure 14 Showing the chromatograms obtained in the intermediate precision test of 2.3.5 in the examples.

[0064] Figure 15 Showing the superimposed chromatograms of samples S1 to S12 in 2.4 of the examples.

[0065] Figure 16 Showing the control fingerprint of the traditional Chinese medicine powder of Yuzhi Ping'an Pills obtained in 2.4 of the examples. Detailed Description of the Invention

[0066] The present invention will be described in detail below in combination with the technical solutions and the drawings, but the present invention is not limited thereto.

[0067] Examples

[0068] 1 Instruments and Reagents

[0069] 1.1 Instruments

[0070] Agilent 1260 Infinity II high performance liquid chromatograph (Agilent Technologies, Inc.), using an Agilent ZORBAX SB-Aq (250 mm × 4.6 mm × 5 μm) chromatographic column;

[0071] BL-600 electronic balance (Xiamen Bailuns Electronic Technology Co., Ltd.);

[0072] XS105 DualRange Mettler Toledo electronic analytical balance (Mettler Toledo Co., Ltd.);

[0073] Constant temperature three-frequency numerically controlled ultrasonic cleaner (Dongguan Keqiao Ultrasonic Equipment Co., Ltd.).

[0074] 1.2 Reagents

[0075] Cardamonin reference substance (Batch number: 110763-201604; Content: Calculated as 99.8%; Source: National Institutes for Food and Drug Control).

[0076] Costunolide (Batch number: 111524-202312; Content: Calculated as 99.6%; Source: National Institutes for Food and Drug Control)

[0077] Methanol (chromatographic grade, Sigma-Aldrich), acetonitrile (chromatographic grade, Sigma-Aldrich), water (distilled water, Watson), methanol, ethanol, ethyl acetate (analytical grade, Sinopharm Chemical Reagent Co., Ltd.).

[0078] 1.3 Samples

[0079] Twelve batches of the traditional Chinese medicine powder of Yuzhi Ping'an Pills were provided by Xiamen Traditional Chinese Medicine Factory Co., Ltd., and were respectively named as samples S1 to S12.

[0080] 2 Methods and Results

[0081] 2.1 Preparation of test solution

[0082] Take the traditional Chinese medicine powder sample S1 of Yuzhi Ping'an Pills as the test sample, and investigate the preparation method of the test solution from two aspects of extraction solvent and extraction method, and select the optimal preparation conditions.

[0083] 2.1.1 Selection of extraction solvent

[0084] Chromatographic conditions:

[0085] Detection wavelength: 252 nm; Column temperature: 30 °C; Flow rate: 0.8 ml / min; Injection volume: 10 μl;

[0086] Chromatographic column: Agilent ZORBAX SB-Aq 250mm 4.6*5um

[0087] Mobile phase: Acetonitrile (A) - Ultra-pure water (B)

[0088] The gradient elution conditions are as follows:

[0089] Time (min) A Acetonitrile (%) B Ultra-pure water (%) 0-22 15-33 85-67 22-40 33 67 40-42 33-35 67-65 42-50 35 65 50-60 35-90 65-10 60-61 90-15 10-85 60-60 15 85 。

[0090] Using methanol, ethanol, 50% methanol aqueous solution, 50% ethanol aqueous solution, and ethyl acetate as extraction solvents respectively, the test solution was prepared as follows: Take about 2 g of the test sample powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of the extraction solvent, stopper it, weigh it, ultrasonically treat it (power 500 W, frequency 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with the corresponding solvent, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0091] Using the above liquid phase elution analysis method to conduct HPLC detection on the test solutions prepared with different extraction solvents, the obtained chromatograms are as Figure 1 shown.

[0092] Figure 1 The results showed that the chromatogram of the test solution prepared by extraction with 50% methanol aqueous solution had a stable baseline and relatively more identifiable peaks. Therefore, 50% methanol aqueous solution was selected as the extraction solvent.

[0093] 2.1.2 Selection of extraction methods

[0094] The following 5 methods were used for extraction respectively to investigate the effects of different extraction methods.

[0095] Extraction method 1:

[0096] Take about 3 g of the test sample powder, accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of 50% methanol aqueous solution, stopper it, weigh it, let it stand overnight and then ultrasonically treat it (power 500 W, frequency 40 kHz) for 60 minutes, let it cool, weigh it again, make up the lost weight with 50% methanol aqueous solution, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0097] Extraction method 2:

[0098] Except that standing overnight was modified to soak at room temperature for 3 h, the others were the same as extraction method 1.

[0099] Extraction method 3:

[0100] Except that standing overnight was modified to soak at room temperature for 40 minutes and ultrasonically treat for 0.5 h, the others were the same as extraction method 1.

[0101] Extraction method 4:

[0102] Except for not standing overnight and directly performing ultrasonic treatment for 60 minutes, the others are the same as extraction method 1.

[0103] Extraction method 5:

[0104] Except for not standing overnight and directly performing ultrasonic treatment for 0.5 h, the others are the same as extraction method 1.

[0105] Chromatographic conditions:

[0106] Chromatographic column: Agilent ZORBAX SB-Aq, 250 mm, 4.6 * 5 μm,

[0107] Sample injection volume: 10 μl, flow rate: 0.6 ml / min, column temperature: 30 °C, detection wavelength: 200 nm, sample injection volume: 10 μl;

[0108] Mobile phase: 0.3% phosphoric acid aqueous solution (A) - acetonitrile (B)

[0109] Gradient elution conditions are as follows:

[0110] Time (min) A 0.3% Aqueous phosphoric acid solution (%) B Acetonitrile (%) 0-10 95 5 10-15 95-85 5-15 15-30 85-65 15-35 30-55 65-55 35-45 55-90 55-35 45-65 90-95 35-95 65-5 95-100 95 5

[0111] Using the above liquid phase elution analysis method, HPLC detection was performed on the test solution prepared by different extraction methods. By comparing peak 1, which has the smallest peak area among the 23 common peaks in the obtained chromatogram, it was found that the peak area of peak 1 in the chromatogram obtained by standing overnight and ultrasonic treatment for 1 h is the largest. Therefore, this extraction method was selected.

[0112] 2.1.3 Finally determined extraction method

[0113] The preparation method of the finally determined test solution is as follows: Take about 3 g of the traditional Chinese medicine powder of Yuzhi Ping'an Pills, accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of 50% methanol aqueous solution, tightly stopper it, weigh it, stand overnight and then perform ultrasonic treatment (power 500 W, frequency 40 kHz) for 60 minutes, cool it, weigh it again, make up the lost weight with 50% methanol aqueous solution, shake well, filter, and take the continuous filtrate to obtain.

[0114] 2.2 Selection of chromatographic conditions

[0115] Take the traditional Chinese medicine powder sample S1 of Yuzhi Ping'an Pills as the test sample, prepare the test solution according to the preparation method of the test solution finally determined in 2.1.3, and conduct the following experiments.

[0116] 2.2.1 Determination of detection wavelength

[0117] The test solution prepared from sample S1 was scanned at 190 - 400 nm using a multi - wavelength detector, and its 3D ultraviolet absorption spectrum ( Figure 2 ) showed that the sample had more absorption peaks at 190 - 230 nm. Therefore, 190 nm, 200 nm, 210 nm, and 230 nm were selected for subsequent experiments.

[0118] 2.2.2 Selection of mobile phase, corresponding elution gradient and flow rate

[0119] Experiments were carried out using HPLC (High Performance Liquid Chromatography) in combination with the wavelength selection results. Some of the experiments and their results are as follows. Experiment 1:

[0120] Chromatographic conditions:

[0121] Detection wavelength: 210 nm; Column temperature: 30 °C; Flow rate: 0.8 ml / min; Injection volume: 10 μl; Chromatographic column: Agilent ZORBAX SB - Aq 250 mm 4.6 * 5 μm

[0122] Mobile phase: ultrapure water (A) - methanol (B)

[0123] Gradient elution conditions are shown in Table 1.

[0124] Table 1

[0125] Time (min) A Ultra-pure water (%) B Methanol (%) 0-10 90 10 10-40 90-75 10-25 40-70 75 25 70-100 75-33 25-67 100-101 33-90 67-10 101-107 90 10

[0126] The obtained chromatogram is shown in Figure 3 .

[0127] Experiment 2:

[0128] Chromatographic conditions:

[0129] Detection wavelength: 210 nm; Column temperature: 30 °C; Flow rate: 0.8 ml / min; Injection volume: 10 μl; Chromatographic column: Agilent ZORBAX SB - Aq 250 mm 4.6 * 5 μm

[0130] Mobile phase: ultrapure water (A) - methanol (B)

[0131] Gradient elution conditions are shown in Table 2.

[0132] Table 2

[0133] Time (min) A Ultra-pure water (%) B Methanol (%) 0-5 95 5 5-30 95-70 5-30 30-50 70 30 60-75 35 65 75-80 35-25 65-75 80-90 25 75 90--91 25-95 75-5 91-95 95 5

[0134] The obtained chromatogram is shown in Figure 4 .

[0135] Experiment 3:

[0136] Chromatographic conditions:

[0137] Detection wavelength: 210 nm; Column temperature: 30 °C; Flow rate: 0.8 ml / min; Injection volume: 10 μl; Chromatographic column: Agilent ZORBAX SB-Aq 250 mm 4.6 * 5 μm

[0138] Mobile phase: Ultra-pure water (A) - Methanol (B)

[0139] Gradient elution conditions are shown in Table 3.

[0140] Table 3

[0141] Time (min) A Ultra-pure water (%) B Methanol (%) 0-5 95 5 5-8 95-70 5-30 8-20 70 30 20-22 70-50 30-50 22-40 50 50 40-42 50-30 50-70 42-60 30 70 60-62 30-15 70-85 62-80 15 85 80-81 15-95 85-5 81-85 95 5

[0142] The obtained chromatogram is shown in Figure 5 .

[0143] Experiment 4:

[0144] Chromatographic conditions:

[0145] Detection wavelength: 190 nm / 210 nm; Column temperature: 30 °C; Flow rate: 0.8 ml / min; Injection volume: 10 μl; Chromatographic column: Agilent ZORBAX SB-Aq 250 mm 4.6 * 5 μm

[0146] Mobile phase: Ultra-pure water (A) - Methanol (B)

[0147] Gradient elution conditions are shown in Table 4.

[0148] Table 4

[0149] Time (min) A Ultra-pure water (%) B Methanol (%) 0-5 95 5 5-8 95-70 5-30 8-20 70 30 20-22 70-72 30-28 22-25 72-50 28-50 25-42 50 50 42-43 50-70 50-30 43-65 70 30 65-70 70-40 30-60 70-85 40 60 85-90 40-30 60-70 90-110 30 70 110-111 30-95 70-5 111-118 95 5

[0150] The obtained chromatogram is shown in Figure 6 .

[0151] Experiment 5:

[0152] Chromatographic conditions:

[0153] Detection wavelength: 230 nm / 210 nm; Column temperature: 30 °C; Flow rate: 0.8 ml / min; Injection volume: 10 μl;

[0154] Chromatographic column: Agilent ZORBAX SB-Aq 250 mm 4.6 * 5 μm

[0155] Mobile phase: Ultra-pure water (A) - Methanol (B)

[0156] Gradient elution conditions are shown in Table 5.

[0157] Table 5

[0158] Time (min) A Ultra-pure water (%) B Methanol (%) 0-5 95 5 5-8 95-70 5-30 8-20 70 30 20-22 70-72 30-28 22-25 72-50 28-50 25-50 50 50 50-55 50-40 50-60 55-60 40 60 60-62 40-45 60-55 62-75 45-30 55-70 75-80 30-40 70-60 80-100 40-30 60-70 100-101 30-95 70-5 101-108 95 5

[0159] The obtained chromatogram is shown in Figure 7 .

[0160] Experiment 6:

[0161] Prepare 0.3% phosphoric acid aqueous solution as follows: Pipette 3.0 ml of phosphoric acid, add ultrapure water to 1000 ml, shake well, filter, and degas. Chromatographic conditions:

[0162] Detection wavelength: 200 nm; Column temperature: 30 °C; Flow rate: 0.6 ml / min; Injection volume: 10 μl;

[0163] Chromatographic column: Agilent ZORBAX SB-Aq 250 mm 4.6*5 μm

[0164] Mobile phase: 0.3% phosphoric acid aqueous solution (A) - acetonitrile (B)

[0165] Gradient elution conditions are shown in Table 6.

[0166] Table 6

[0167] Time (min) A 0.3% Aqueous phosphoric acid solution (%) B Acetonitrile (%) 0-10 95 5 10-15 95-85 5-15 15-30 85-65 15-35 30-55 65-55 35-45 55-90 55-35 45-65 90-95 35-95 65-5 95-100 95 5

[0168] The obtained chromatogram is shown in Figure 8 .

[0169] Comparison Figures 3 to 8 It can be seen that under the chromatographic conditions of Experiment 6, the baseline is relatively stable, the number of identifiable chromatographic peaks is relatively large and the resolution is good. Therefore, the elution gradient, wavelength and flow rate of Experiment 6 are finally selected.

[0170] 2.2.3 Selection of column temperature

[0171] Except for selecting 20 °C, 30 °C, and 40 °C as the column temperature respectively, other chromatographic conditions are the same as those in Experiment 6. Examine the influence of different column temperatures on chromatographic peaks. The results are shown in Figure 9 (20 °C), Figure 10 (30 °C) and Figure 11 (40 °C).

[0172] Comparison Figures 9 - 11 It can be seen that the number of peaks is relatively large at a column temperature of 30 °C. Therefore, 30 °C is selected as the column temperature.

[0173] 2.2.4 Finally determined chromatographic conditions

[0174] Using high performance liquid chromatography, adopt Agilent ZORBAX SB-Aq (250 mm 4.6*5 μm) chromatographic column, injection volume is 10 μl, volume flow rate is 0.6 ml / min, column temperature is 30 °C, detection wavelength is 200 nm, mobile phase is 0.3% phosphoric acid aqueous solution - acetonitrile, and gradient elution conditions are as shown in Table 6.

[0175] 2.3 Methodological Investigation

[0176] 2.3.1 Specificity

[0177] Take clove, costus root, agalloch eaglewood, sandalwood, amomum kravanh, tsaoko amomum fruit, galangal fruit, and nutmeg respectively, pulverize them, sieve through No. 5 sieve, weigh about 1 g each, accurately weigh, place in a stoppered conical flask, accurately add 50 ml of 50% methanol aqueous solution, stopper tightly, weigh, let stand overnight, then perform ultrasonic treatment (power 500 W, frequency 40 kHz) for 60 minutes, cool, weigh again, make up the lost weight with 50% methanol aqueous solution, shake well, filter, and take the subsequent filtrate to obtain the test solution of each medicinal material.

[0178] Prepare the test solutions of the traditional Chinese medicine powder samples S1 and S3 of Yuzhi Ping'an Pills according to the preparation method of the test solution finally determined in 2.1.3.

[0179] Detect the blank solvent and the test solutions of sample S1, clove, costus root, agalloch eaglewood, sandalwood, amomum kravanh, tsaoko amomum fruit, galangal fruit, and nutmeg respectively according to the chromatographic conditions finally determined in 2.2.4. The chromatograms are as Figure 12 shown.

[0180] Compare Figure 12 The chromatogram of the test sample S1 in it with the chromatograms of each medicinal material, it can be confirmed that peak 3 in the chromatogram of the test sample S1 is the characteristic peak unique to clove, peak 19 is the characteristic peak unique to costus root, peak 8 is the characteristic peak unique to agalloch eaglewood, peak 22 is the characteristic peak unique to sandalwood, peak 1 is the characteristic peak unique to amomum kravanh, peak 21 is the characteristic peak unique to tsaoko amomum fruit, peak 11 is the characteristic peak unique to galangal fruit, and peak 14 is the characteristic peak unique to nutmeg.

[0181] Take appropriate amounts of cardamom pure reference substance and costunolide reference substance respectively and prepare a reference solution with a concentration of 70 μg / ml.

[0182] Detect the test solution of sample S3 and the above reference solution according to the chromatographic conditions finally determined in 2.2.4. The chromatograms are shown in Figure 13 , and perform chromatographic peak localization.

[0183] Compare Figure 13 The chromatograms in it, it can be confirmed that peak 17 is cardamom pure, and peak 19 is costunolide.

[0184] In summary, the fingerprint established under the chromatographic conditions of the present invention can effectively separate and identify the medicinal materials of clove, costus root, agalloch eaglewood, sandalwood, amomum kravanh, tsaoko amomum fruit, galangal fruit, and nutmeg and control their internal quality.

[0185] 2.3.2 Repeatability

[0186] Accurately weigh 6 portions of the test sample of the traditional Chinese medicine powder of Yuzhi Ping'an Pills, numbered 1 to 6 respectively. Prepare the test solution according to the final determined preparation method of the test solution in 2.1.3, and then detect it under the final determined chromatographic conditions in 2.2.4, and record the chromatogram.

[0187] Taking peak 1 as the reference peak, calculate the relative retention time and relative peak area of each common peak. The results are shown in Table 7 below.

[0188] Table 7

[0189]

[0190] The results in Table 7 show that the RSD of the relative retention time of each common peak ≤ 0.42%, and the RSD of the relative peak area of each common peak ≤ 4.52%, indicating that the method has good repeatability.

[0191] 2.3.3 Instrument precision

[0192] Take 1 portion of the test solution under the repeatability investigation item, detect it under the final determined chromatographic conditions in 2.2.4, inject the sample repeatedly 6 times, numbered 1 to 6 respectively, and record the chromatograms of each injection. Taking peak 1 as the reference peak, calculate the relative retention time and relative peak area of each common peak. The results are shown in Table 8.

[0193] Table 8

[0194]

[0195] Table 8 shows that the RSD of the relative retention time of each common peak ≤ 0.23%, and the RSD of the relative peak area of each common peak ≤ 4.30%, indicating that the instrument precision is good and meets the requirements of the fingerprint. The relative retention time and relative peak area of each common peak are shown in the following table.

[0196] 2.3.4 Stability

[0197] Accurately weigh 1 portion of the test sample (sample S3), prepare the test solution according to the final determined preparation method of the test solution in 2.1.3, and then detect it under the final determined chromatographic conditions in 2.2.4. Inject the sample at 0h, 5h, 10h, 15h, 20h, and 25h after the test solution is prepared (i.e., the injection times are 0h, 5h, 10h, 15h, 20h, and 25h), and record the chromatograms. Taking peak 1 as the reference peak, calculate the relative retention time and relative peak area of each common peak. The results are shown in Table 9.

[0198] Table 9

[0199]

[0200] Table 9 results showed that the relative retention time RSD of each common peak was ≤0.30%, and the relative peak area RSD of each common peak was ≤4.27%, indicating that the test solution was basically stable for at least 24 hours.

[0201] 2.3.5 Intermediate precision

[0202] Take the test sample (sample S3), by different analysts A and B, use different Agilent 1260 Infinity II high performance liquid chromatographs in the laboratory and different batches of Agilent ZORBAX SB-Aq (250mm 4.6*5um) chromatographic columns, and prepare the test solution according to the preparation method of the test solution finally determined in 2.1.3. Then, detect according to the chromatographic conditions finally determined in 2.2.4, record the chromatogram, see Figure 14 .

[0203] Use the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition)" to analyze and process the obtained chromatogram results, calculate the similarity, and the results are shown in Table 11.

[0204] Table 11

[0205] Inspector A Inspector B Similarity 0.996 0.996

[0206] The obtained similarity was greater than 0.90, and the intermediate precision test was passed.

[0207] 2.4 Establishment of the control fingerprint of Yuzhi Ping'an Pills traditional Chinese medicine powder

[0208] Take the test samples S1 to S12 of Yuzhi Ping'an Pills traditional Chinese medicine powder, prepare the test solution according to the preparation method of the test solution finally determined in 2.1.3, and then detect according to the chromatographic conditions finally determined in 2.2.4, record the chromatogram, and the results are shown in Figure 15 .

[0209] Use the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition)" to analyze and process the obtained chromatogram results, set sample S1 as the reference chromatogram, automatically match the chromatograms of the other 11 batches of test samples with the reference chromatogram, generate the control fingerprint of Yuzhi Ping'an Pills traditional Chinese medicine powder, see Figure 16 , and then calculate the similarity, and the results are shown in Table 12.

[0210] Table 12 Similarity calculation values of 12 batches of Yuzhi Ping'an Pills traditional Chinese medicine powder

[0211] S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 Similarity 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000

[0212] The similarity between the 12 batches of the imperial peace pills traditional Chinese medicine powder and the fingerprint spectrum is equal to 1.000, indicating good similarity, suggesting that the source of its raw materials is relatively stable, the process parameters are controlled relatively consistently, and the stability and uniformity of its products are basically guaranteed.

Claims

1. An HPLC method for detecting the fingerprint of the traditional Chinese medicine powder of Yuzhi Ping'an Pills, which is detected under the following chromatographic conditions: Chromatographic column: Agilent ZORBAX SB-Aq, 250mm, 4.6 * 5um, Sample injection volume: 10ul, Flow rate: 0.6ml / min, Column temperature: 30 °C, Detection wavelength: 200nm, Mobile phase: 0.3% phosphoric acid aqueous solution (A) - acetonitrile (B) The gradient elution conditions are as follows:

2. The HPLC method according to claim 1, wherein The test solution of the traditional Chinese medicine powder of Yuzhi Ping'an Pills for detection is prepared as follows: Take about 3g of the traditional Chinese medicine powder of Yuzhi Ping'an Pills, accurately weigh it, accurately add 50ml of 50% methanol aqueous solution, tightly stopper, weigh, shake well, stand overnight, ultrasonically treat for 60 minutes (ultrasonic power 500W, frequency 40kHz), cool, weigh again, make up the lost weight with 50% methanol aqueous solution, shake well, filter, and take the subsequent filtrate to obtain.

3. The HPLC method according to claim 1, which further includes the step of selecting characteristic peaks in the fingerprint of the traditional Chinese medicine powder of Yuzhi Ping'an Pills, wherein the chromatographic peaks with better resolution of the main components are selected as characteristic peaks.

4. The HPLC method according to claim 3, wherein A total of 23 characteristic peaks are determined.

5. The HPLC method according to claim 3, which further includes the steps of attributing and identifying the characteristic peaks in the fingerprint of the traditional Chinese medicine powder of Yuzhi Ping'an Pills, including: (1) Preparation of the test solutions and reference solutions of each medicinal material Respectively take cloves, costus root, agarwood, sandalwood, cardamom, tsaoko amomum fruit, galangal, nutmeg, crush them and pass through a No. 5 sieve, weigh about 1g each, accurately weigh, accurately add 50ml of 50% methanol aqueous solution, tightly stopper, weigh, stand overnight and then ultrasonically treat for 60 minutes (ultrasonic power 500W, frequency 40kHz), cool, weigh again, make up the lost weight with 50% methanol aqueous solution, shake well, filter, and take the subsequent filtrate to obtain the test solutions of each medicinal material; Take an appropriate amount of cardamomin reference substance and prepare a reference solution with a concentration of 70ug / ml; Take an appropriate amount of costunolide reference substance and prepare a reference solution with a concentration of 70ug / ml; (2) Detection of the chromatograms of each medicinal material and reference substance Use the HPLC method described in claim 1 to detect the chromatograms of each medicinal material and reference substance; (3) Identification and attribution of common peaks: The attribution and identification of the characteristic peaks in the fingerprint of the traditional Chinese medicine powder of Yuzhi Ping'an Pills are completed by comparing with the chromatograms of each medicinal material and reference substance.

6. The HPLC method according to claim 2 or 5, wherein The 50% methanol aqueous solution is prepared as follows: Transfer 500ml of methanol, add ultrapure water to 1000ml, shake well, filter, and degas.

7. The HPLC method according to claim 1, wherein The 0.3% phosphoric acid aqueous solution is prepared as follows: Transfer 3.0ml of phosphoric acid, add ultrapure water to 1000ml, shake well, filter, and degas.

8. A method for establishing a reference fingerprint of the traditional Chinese medicine powder of Yuzhi Ping'an Pills, the method includes: (1) Preparation of the test solution: Take about 3 g of the traditional Chinese medicine powder of Yuzhi Ping'an Pills, weigh accurately, accurately add 50 ml of 50% methanol aqueous solution, stopper tightly, weigh, shake well, let stand overnight, ultrasonically treat for 60 minutes (ultrasonic power 500 W, frequency 40 kHz), cool, weigh again, make up the lost weight with 50% methanol aqueous solution, shake well, filter, take the continuous filtrate to obtain the test solution; (2) HPLC detection: Precisely pipette 10 μl of the test solutions of n batches respectively, and detect them by the HPLC method described in Claim 1 to obtain the test solution fingerprint spectra; (3) Generate a reference fingerprint spectrum: Generate a reference fingerprint spectrum based on the common peaks in the fingerprint spectra of n batches of test solutions. Among them, select the chromatographic peaks with good separation degree of the main components in the fingerprint spectrum of the traditional Chinese medicine powder of Yuzhi Ping'an Pills as characteristic peaks to determine the common peaks, where n≥10.

9. The method according to Claim 8, wherein, 23 common peaks are determined in step (3); and / or The reference fingerprint spectrum of the traditional Chinese medicine powder of Yuzhi Ping'an Pills is generally as shown in Figure 16.

10. A quality control method for the traditional Chinese medicine powder of Yuzhi Ping'an Pills, the method comprising the following steps: (1) Preparation of test solution: Take about 3 g of the traditional Chinese medicine powder of Yuzhi Ping'an Pills, weigh accurately, accurately add 50 ml of 50% methanol aqueous solution, stopper tightly, weigh, shake well, let stand overnight, ultrasonically treat for 60 minutes (ultrasonic power 500 W, frequency 40 kHz), cool, weigh again, make up the lost weight with 50% methanol aqueous solution, shake well, filter, take the continuous filtrate to obtain the test solution; (2) Determination of fingerprint spectrum: Precisely pipette 10 μL of the test solution, and determine it by the HPLC method described in Claim 1 to obtain the fingerprint spectrum of the test traditional Chinese medicine powder of Yuzhi Ping'an Pills to be inspected; (3) Pass / fail determination: Calculate the similarity between the fingerprint spectrum of the test traditional Chinese medicine powder of Yuzhi Ping'an Pills to be inspected and the reference fingerprint spectrum. If the similarity is greater than or equal to 0.9, it is a qualified product.

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