UPLC (Ultra Performance Liquid Chromatography) fingerprint spectrum detection method of Sangju toxin vanquishing drink

Abstract

The invention provides an UPLC fingerprint spectrum detection method of a Sangju toxin-vanquishing beverage, and belongs to the technical field of pharmaceutical preparation detection. The UPLC fingerprint spectrum detection method comprises the following steps: preparing a Sangju toxin-vanquishing drink reference sample solution; preparing a test solution; preparing a reference substance solution; determining by using high performance liquid chromatography; and constructing a fingerprint spectrum. The chromatographic conditions are as follows: a CAPCELL PAK C18 chromatographic column is adopted; a mobile phase is an acetonitrile-0. 2% phosphoric acid aqueous solution; performing gradient elution; the volume flow rate is 0.3 mL / min; the column temperature is 35 DEG C; the detection wavelength is 350 nm; the sample size is 2 microliters. The method has the advantages of good repeatability, high stability and precision, simplicity and convenience in operation and the like. According to the present invention, the Sangju toxin-vanquishing drink fingerprint established by using the method has 33 common peaks and identifies 19 common characteristic peaks, such that the Sangju toxin-vanquishing drink is researched so as to lay the foundation for the subsequent research and development of the Sangju toxin-vanquishing drink.

Description

Technical Field

[0001] The invention belongs to the technical field of drug preparation detection, and particularly relates to a UPLC fingerprint detection method for Sangju Baidu Decoction. Background Art

[0002] Sangju Baidu Yin (Sangju Baidu Yin) is composed of 16 Chinese herbs, including mulberry leaves, chrysanthemums, honeysuckle, forsythia, scutellaria, peucedanum, and bupleurum. It primarily relieves stagnation in the lung defense system, addressing both the lungs and stomach, and both the exterior and interior. It is used to treat colds, fevers, coughs, and lung infections. In recent years, the overall quality assessment of Chinese medicine preparations has garnered increasing attention. Traditional Chinese medicine fingerprint analysis is a widely used analytical method for evaluating the quality of traditional Chinese medicines and provides effective quality control.

[0003] For example, Chinese patent CN100573138A discloses the establishment and fingerprinting of a Xiasangju preparation. The method includes: (a) preparation of a reference solution; (b) preparation of a test solution; (c) chromatographic conditions: a chromatographic column packed with octadecylsilane bonded silica gel; gradient elution with a mobile phase consisting of 0.103% acetic acid and methanol; column temperature: 25-50°C; UV detection wavelength: 285-295nm; flow rate: 0.5-1.5mL / min; and time: 30-80min; (d) determination: fingerprinting by high-performance liquid chromatography. This invention can effectively characterize the quality of Xiasangju preparations and facilitate product quality monitoring. It offers stability, high precision, and good reproducibility, enabling rapid and accurate product authenticity identification.

[0004] For example, Chinese patent CN114609321A discloses a method for establishing a fingerprint of a moringa leaf and mulberry leaf plant beverage and its fingerprint. The invention adopts high-performance liquid fingerprint technology combined with fingerprint evaluation software. By screening and optimizing the measurement wavelength, mobile phase elution program, flow rate, column temperature, and injection volume, a sample preparation method and fingerprint chromatographic conditions with good separation, stable baseline, no negative absorption chromatographic peaks, rich variety of ingredients and high content are obtained. After comparative analysis of the similarity of fingerprint spectra of multiple batches of products, it is found that the similarity of each fingerprint spectra is above 0.97, indicating that the inventive method can accurately, stably and reliably reflect the various component information in the moringa leaf and mulberry leaf plant beverage product in the fingerprint spectrum, and can comprehensively, objectively and scientifically evaluate and control the quality of the product, thereby ensuring the stability and effectiveness of the product quality.

[0005] For example, Chinese patent CN114252518A discloses a method for establishing a substance benchmark fingerprint for the classic prescription Sangbaipi Decoction, and its fingerprint. This fingerprint establishment method comprises the following steps: preparation of a substance benchmark for Sangbaipi Decoction; preparation of a test solution; preparation of a reference solution; determination using a high-performance liquid chromatograph; and construction of a fingerprint for Sangbaipi Decoction. Chromatographic conditions include: octadecylsilane bonded silica gel as the packing material; detection wavelength of 250 nm; flow rate of 1.0 ml / min; column temperature of 25°C; injection volume of 10 μl; and gradient elution using methanol and aqueous phosphoric acid as the mobile phase. This method offers advantages such as good reproducibility, high stability, high precision, and ease of operation. The substance benchmark fingerprint for Sangbaipi Decoction established using this method revealed 23 common peaks, four of which were identified as characteristic peaks. Peak 7 was selected as the reference peak in the fingerprint. This study of the substance benchmark for Sangbaipi Decoction laid the foundation for its subsequent research and development.

[0006] However, there is no fingerprint detection method for Sangju Baidu Drink in the prior art. The present invention adopts the UPLC method to establish the fingerprint analysis of Sangju Baidu Drink based on the quantity transfer law of characteristic components, in order to provide a reference for the quality evaluation standard of the prescription and ensure the safety and effectiveness of the preparation. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention provides a UPLC fingerprint detection method for Sangju Baidu Yin. This method, through testing 20 batches of Sangju Baidu Yin, identified 33 common peaks. By comparing the method with a reference substance, 19 common peaks were identified. This method also demonstrates excellent repeatability, stability, sample recovery, and a rich chromatographic peak profile.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] A UPLC fingerprint detection method for Sangju Baidu Drink comprises the following steps:

[0010] (1) Preparation of Sangju Baiduyin reference sample solution: Decoction the raw materials of Sangju Baiduyin and concentrate them to obtain the reference sample solution;

[0011] (2) Preparation of test solution: Take the Sangju Baidu Drink standard sample solution, place it in a volumetric flask, add anhydrous methanol to below the scale line, sonicate, cool, and then dilute to the scale line with anhydrous methanol, shake well, centrifuge, take the supernatant, and filter through a microporous filter membrane to obtain the product;

[0012] (3) Preparation of reference solution: accurately weigh the reference substances of neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, 1,3-dicaffeoylquinic acid, luteolin, scutellarin, forsythiaside A, isochlorogenic acid B, isochlorogenic acid A, 1,5-dicaffeoylquinic acid, apigenin-7-O-glucuronide, rosmarinic acid, isochlorogenic acid C, isoliquiritigenin, baicalin, luteolin, wogonin, and peucedanum praeruptoside A, respectively, and add methanol to prepare a solution.

[0013] (4) Determination: Accurately aspirate the test solution and reference solution, inject them into the high performance liquid chromatograph, perform the determination, and record the chromatogram;

[0014] (5) Construction of fingerprint: The fingerprints of different batches of test samples were imported into the Chinese medicine chromatographic fingerprint similarity evaluation system for analysis to obtain the fingerprint of Sangju Baidu Drink.

[0015] As a preferred method, the decoction in step (1) is performed twice, with 12 times the amount of water added for 2 hours for the first time and 10 times the amount of water added for 1 hour for the second time.

[0016] As a preferred method, the concentration in step (1) is concentration to 150 mL.

[0017] As a preferred embodiment, the preparation method of the Sangju Baidu Yin reference sample solution described in step (1) is as follows: take 10g of mulberry leaves, 10g of chrysanthemums, 10g of honeysuckle, 10g of forsythia, 10g of schizonepeta, 10g of perilla leaves, 10g of notopterygium root, 10g of roasted ephedra, 10g of apricot kernel, 30g of gypsum, 6g of roasted licorice, 10g of peucedanum, 12g of bupleurum, 12g of stir-fried scutellaria, 12g of adenophora australis and 6g of cooked rhubarb, decoct them twice, add 12 times the amount of water for the first time and decoct for 2 hours, add 10 times the amount of water for the second time and decoct for 1 hour, combine the two filtrates, and concentrate to 150mL to obtain the reference sample solution.

[0018] As a preferred solution, the preparation method of the test solution described in step (2) is as follows: take 2 mL of the Sangju Baidu Drink reference sample solution, put it into a 5 mL volumetric flask, add anhydrous methanol to below the scale line, ultrasonicate (400W, 40kHz) for 30 minutes, cool and dilute to the scale line with anhydrous methanol, shake well, centrifuge at 12000r / min for 10 minutes, take the supernatant, and filter through a 0.22μm microporous filter membrane to obtain the product.

[0019] As a preferred embodiment, the preparation method of the reference solution described in step (3) is as follows: accurately weigh neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, 1,3-dicaffeoylquinic acid, luteolin, scutellarin, forsythiaside A, isochlorogenic acid B, isochlorogenic acid A, 1,5-dicaffeoylquinic acid, apigenin-7-O-glucuronide, rosmarinic acid, isochlorogenic acid C, isoliquiritin, baicalin, luteolin, wogonin, and peucedanum praeruptoside A reference substances, respectively, and add methanol to prepare a concentration of 240 μg. / mL, 230μg / mL, 224μg / mL, 200μg / mL, 250μg / mL, 205μg / mL, 180μg / mL, 205μg / mL, 270μg / mL, 220μg / mL, 173.33μg / mL, 170μg / mL, 186μg / mL, 345μg / mL, 232μg / mL, 120μg / mL, 350μg / mL, 70μg / mL, and 280μg / mL of the reference solution are obtained.

[0020] As a preferred solution, the determination described in step (4) is to accurately aspirate 2 μL of the test solution and the reference solution, respectively, and inject them into a high performance liquid chromatograph for determination.

[0021] As a preferred solution, the chromatographic conditions for the determination in step (4) are:

[0022] Chromatographic column: CAPCELL PAK C 18 Chromatographic column (150 mm × 2.0 mm, 2 μm);

[0023] Mobile phase: acetonitrile-0.2% phosphoric acid aqueous solution;

[0024] Gradient elution: 0-15 min, 6%-10% acetonitrile; 15-18 min, 10%-12% acetonitrile; 18-48 min, 12%-14% acetonitrile; 48-68 min, 14%-30% acetonitrile; 68-85 min, 30%-95% acetonitrile;

[0025] Volume flow rate: 0.3 mL / min;

[0026] Column temperature: 35°C;

[0027] Detection wavelength: 350nm;

[0028] Injection volume: 2 μL.

[0029] As a preferred solution, after constructing the fingerprint spectrum in step (5), similarity evaluation is performed. The similarity evaluation method is: the fingerprint spectrum is imported into the similarity evaluation software for similarity evaluation analysis, and 33 common peaks are determined, which are numbered as peaks 1-33.

[0030] Among them, peak 1 is neochlorogenic acid, peak 2 is chlorogenic acid; peak 3 is caffeic acid, peak 4 is cryptochlorogenic acid, peak 5 is 1,3-dicaffeoylquinic acid; peak 11 is luteolin; peak 12 is scutellarin, peak 14 is forsythiaside A; peak 15 is isochlorogenic acid B, peak 16 is isochlorogenic acid A, peak 18 is 1,5-dicaffeoylquinic acid; peak 19 is apigenin-7-O-glucuronide; peak 20 is rosmarinic acid; peak 22 is isochlorogenic acid C; peak 25 is isoliquiritigenin; peak 26 is baicalin; peak 27 is luteolin; peak 31 is wogonin; peak 33 is peucedanum scabraein.

[0031] Twenty batches of Sangju Baidu Yin reference samples were measured using the aforementioned method, resulting in 15 batches of Sangju Baidu Yin chromatograms S1-S20. The similarity of the HPLC chromatograms of each batch of Sangju Baidu Yin substance reference sample was evaluated using the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" and the similarity results were 0.935, 0.977, 0.998, 0.997, 0.996, 0.983, 0.966, 0.979, 0.991, 0.987, 0.983, 0.983, 0.986, 0.974, 0.945, 0.954, 0.985, 0.980, 0.994, and 0.993, respectively, with similarities all greater than 0.93. This indicates that the 20 batches of Sangju Baidu Yin UPLC reference samples have good overall similarity, and the established fingerprints are stable and can reflect the fingerprint characteristics of the substance reference.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The present invention established fingerprints for 20 batches of Sangju Baidu Yin, with similarities greater than or equal to 0.93. This method can more comprehensively reflect the component information contained in Sangju Baidu Yin and better characterize the quality of Sangju Baidu Yin. To obtain rich chromatographic information, fingerprints are usually tested over a long period of time to obtain more common peaks and improve the separation of common peaks. However, the present invention obtained 33 common peaks in only 85 minutes, and the separation between the peaks was good.

[0034] (2) The present invention calibrated 33 common peaks and identified 19 common characteristic peaks, among which Peak 1 is neochlorogenic acid, Peak 2 is chlorogenic acid; Peak 3 is caffeic acid, Peak 4 is cryptochlorogenic acid, Peak 5 is 1,3-dicaffeoylquinic acid; Peak 11 is luteolin; Peak 12 is scutellarin, Peak 14 is forsythiaside A; Peak 15 is isochlorogenic acid B, Peak 16 is isochlorogenic acid A, Peak 18 is 1,5-dicaffeoylquinic acid; Peak 19 is apigenin-7-O-glucuronide; Peak 20 is rosmarinic acid; Peak 22 is isochlorogenic acid C; Peak 25 is isoliquiritigenin; Peak 26 is baicalin; Peak 27 is luteolin; Peak 31 is wogonin; Peak 33 is peucedanin. The fingerprint obtained by the detection method provided by the present invention has a moderate retention time, a separation degree between the previous and next chromatographic peaks greater than 1.5, high accuracy and good repeatability, can accurately reflect the component information contained in the fingerprint, and can effectively ensure the stability and effectiveness of product quality.

[0035] (3) Sangju Baidu Yin is mainly made from 16 raw materials, including mulberry leaves, chrysanthemums, honeysuckle, forsythia, scutellaria, peucedanum, and bupleurum, and other auxiliary materials through a complex process. Its chemical composition is relatively complex, and the content determination of a single component is often one-sided and cannot reflect the overall composition information of the product. The high-performance liquid chromatography fingerprint method established in this invention has achieved the first quality control of the entire Sangju Baidu Yin formula. Instead of identifying a single compound or medicinal material, it can more effectively guide the feeding of materials, strictly regulate production operations, and ensure the safety and effectiveness of clinical medication.

[0036] (4) The fingerprint method established in the present invention is used for the quality monitoring of the material standard of Sangju Baidu Drink, which can comprehensively and effectively control the quality of Sangju Baidu Drink as a whole, thereby ensuring the stability, reliability and safety of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The HPLC fingerprints of the substances of 20 batches of Sangju Baidu Yin and the reference fingerprints (R);

[0038] Figure 2 UPLC spectra of the reference substance and Sangju Baidu Drink benchmark sample;

[0039] In the figure: 1. Neochlorogenic acid; 2. Chlorogenic acid; 3. Caffeic acid; 4. Cryptochlorogenic acid; 5. 1,3-dicaffeoylquinic acid; 11. Luteolin; 12. Scutellaria baicalensis; 14. Forsythiaside A; 15. Isochlorogenic acid B; 16. Isochlorogenic acid A; 18. 1,5-dicaffeoylquinic acid; 19. Apigenin-7-O-glucuronide; 20. Rosmarinic acid; 22 Isochlorogenic acid C; 25 Isoliquiritin; 26. Baicalin; 27. Luteolin; 31. Wogonin; 33 Peucedanum scutellariae A.

[0040] Figure 3 This is the UPLC spectrum of the Sangju Baidu Decoction reference sample and single herbal medicine slices;

[0041] Figure 4 This is the chromatogram of the characteristic peaks of Forsythia suspensa in Sangju Baidu Decoction;

[0042] Figure 5 This is the chromatogram of the characteristic peaks of Peucedanum chinense in Sangju Baidu Decoction;

[0043] Figure 6 This is the chromatogram of the characteristic peaks of licorice in Sangju Baidu Drink;

[0044] Figure 7 This is the chromatogram of the characteristic peaks of perilla leaves in Sangju Baidu Drink;

[0045] Figure 8 This is the chromatogram of the characteristic peaks of Scutellaria baicalensis in Sangju Baidu Yin.

[0046] Figure 9 This is the chromatogram of the characteristic peaks of honeysuckle and chrysanthemum in Sangju Baidu Drink. DETAILED DESCRIPTION

[0047] The following is a clear and complete description of the technical solutions in the embodiments of the present invention, in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts are within the scope of protection of the present invention.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0049] "Chromatography" refers to a physical separation method in which the components (i.e., chemical constituents) to be separated are distributed between two phases, one of which is stationary (the stationary phase) and the other (the mobile phase) moves in a well-defined direction. The mobile phase can be a gas ("gas chromatography," "GC") or a liquid ("liquid chromatography," "LC"). Chromatographic output data can be used in embodiments of the methods described herein.

[0050] A "chromatogram" is a representation of mass spectrometry data as a chromatogram, where the x-axis represents time and the y-axis represents total ion intensity. This data represents mass spectrometry data that can be obtained using a liquid chromatography-mass spectrometer (LC-MS) apparatus. Liquid chromatography separates mixtures of organic molecules, while mass spectrometry provides molecular formula assignments for individual organic molecules. Thus, the x-axis represents retention time on the LC column of an LC-MS apparatus.

[0051] "Separation" refers to the process of separating a complex mixture into its component molecules or metabolites. Common exemplary laboratory separation techniques include electrophoresis and chromatography.

[0052] "Retention time" refers to the time that elapses during a chromatography process since the sample was introduced into a separation device. The retention time of a sample component refers to the time that elapses during a chromatography process between the time the sample is injected into the separation device and the time the sample component elutes (e.g., leaves) from the portion of the separation device containing the stationary phase.

[0053] 1. Experimental Materials

[0054] instrument:

[0055] Waters ACQUITY H-CLASS ultra-high performance liquid chromatograph and PDA detector (Waters Corporation, USA); KQ-500DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); refrigerated high-speed centrifuge (Thermo Fisher Scientific Technology Co., Ltd.); EYELAN-1200B rotary evaporator (Shanghai Ailan Instrument Co., Ltd.); FW80 high-speed universal grinder (Tianjin Test Instrument Co., Ltd.); SQP 1 / 10,000 balance (Sartorius Scientific Instruments Beijing Co., Ltd.); Milli-Q ultrapure water preparation system (Merck, USA).

[0056] Reagents and test drugs:

[0057] Reference substances: neochlorogenic acid (MUST-23120510), chlorogenic acid (MUST-20032310), caffeic acid (MUST-23061118), cryptochlorogenic acid (MUST-24011920), 1,3-dicaffeoylquinic acid (MUST-24022817), luteolin (MUST-22082211), forsythiaside A (MUST-22033105), isochlorogenic acid A (MUST-23042610), isochlorogenic acid B (MUST-22010705), isochlorogenic acid C (MUST-24032023), 1,5-dicaffeoylquinic acid (MUST-23060110), isoliquiritin (MUST-22050507 ), baicalin (MUST-22040419), and luteolin (MUST-19102418) were purchased from Chengdu Mansite Biotechnology Co., Ltd.; apigenin-7-O-glucuronide (Z11F8Z29057), wogonin (JB315037), scutellarin (J31GB156318), rosmarinic acid (JB241724), and praeruptorin A (Z1O10S100111) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; anhydrous methanol, ethanol, and chloroform were of analytical grade and were purchased from Jiangsu Qiangsheng Functional Chemical Co., Ltd.; methanol and acetonitrile were of chromatographic grade and were purchased from Merck KGaA; phosphoric acid was of chromatographic grade and was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; and water was ultrapure water prepared in the laboratory.

[0058] Example 1 A UPLC fingerprint detection method for Sangju Baidu Drink

[0059] (1) The preparation method of the Sangju Baiduyin reference sample solution is as follows: take 10g of mulberry leaves, 10g of chrysanthemum, 10g of honeysuckle, 10g of forsythia, 10g of schizonepeta, 10g of perilla leaves, 10g of notopterygium root, 10g of roasted ephedra, 10g of apricot kernel, 30g of gypsum, 6g of roasted liquorice, 10g of peucedanum, 12g of bupleurum, 12g of stir-fried scutellaria, 12g of adenophora australis and 6g of cooked rhubarb, decoct them twice, add 12 times the amount of water for the first time and decoct for 2 hours, add 10 times the amount of water for the second time and decoct for 1 hour, combine the two filtrates, and concentrate to 150mL to obtain the reference sample solution; the source information of Sangju Baiduyin decoction pieces is shown in Table 1, and the decoction pieces from different origins and batches are randomly combined into 20 batches of Sangju Baiduyin reference samples using a random number table method, numbered S1-S20, see Table 2;

[0060] (2) The test solution was prepared as follows: 2 mL of the Sangju Baidu Drink standard sample solution was placed in a 5 mL volumetric flask, anhydrous methanol was added to the mark, and ultrasonication (400 W, 40 kHz) was performed for 30 min. After cooling, the solution was diluted to the mark with anhydrous methanol, shaken, and centrifuged at 12,000 rpm for 10 min. The supernatant was collected and filtered through a 0.22 μm microporous filter membrane.

[0061] (3) The preparation method of reference solution is as follows: accurately weigh neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, 1,3-dicaffeoylquinic acid, luteolin, scutellarin, forsythiaside A, isochlorogenic acid B, isochlorogenic acid A, 1,5-dicaffeoylquinic acid, apigenin-7-O-glucuronide, rosmarinic acid, isochlorogenic acid C, isoliquiritin, baicalin, luteolin, wogonin, and peucedanum praeruptoside A, respectively, and add methanol to prepare the reference solutions at the concentrations of 240 μg / mL, 230 μg / mL, and 1,50 μg / mL, respectively. μg / mL, 224μg / mL, 200μg / mL, 250μg / mL, 205μg / mL, 180μg / mL, 205μg / mL, 270μg / mL, 220μg / mL, 173.33μ g / mL, 170μg / mL, 186μg / mL, 345μg / mL, 232μg / mL, 120μg / mL, 350μg / mL, 70μg / mL, 280μg / mL reference solution, you can get;

[0062] (4) Determination: Accurately pipette 2 μL of the test solution and reference solution respectively, inject them into the high performance liquid chromatograph for determination.

[0063] As a preferred solution, the chromatographic conditions for the determination in step (4) are:

[0064] Chromatographic column: CAPCELL PAK C 18 Chromatographic column (150 mm × 2.0 mm, 2 μm);

[0065] Mobile phase: acetonitrile-0.2% phosphoric acid aqueous solution;

[0066] Gradient elution: 0-15 min, 6%-10% acetonitrile; 15-18 min, 10%-12% acetonitrile; 18-48 min, 12%-14% acetonitrile; 48-68 min, 14%-30% acetonitrile; 68-85 min, 30%-95% acetonitrile;

[0067] Volume flow rate: 0.3 mL / min;

[0068] Column temperature: 35°C;

[0069] Detection wavelength: 350nm;

[0070] Injection volume: 2 μL.

[0071] Table 1 Source information of Sangju Baiduyin decoction pieces

[0072]

[0073]

[0074]

[0075] Table 2 Combination of 20 batches of Sangju Baiduyin decoction pieces

[0076]

[0077]

[0078] Example 2 Establishment of fingerprint and identification of common peaks

[0079] The chromatograms of the 20 batches of Sangju Baidu Drink benchmark test samples recorded in Example 1 were introduced into the Chinese medicine chromatographic fingerprint similarity evaluation system for analysis, and the common chromatographic peaks in each batch of Sangju Baidu Drink benchmark were selected to generate the Sangju Baidu Drink benchmark control fingerprint, and the peak area and retention time of each common peak were obtained. The results are shown in Table 3.

[0080] Table 3 Common peak areas and retention times of 20 batches of samples

[0081]

[0082]

[0083] The fingerprints of the samples were analyzed using the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 edition) software of the Chinese Pharmacopoeia Committee. The fingerprint of sample S4 was used as the reference spectrum for Mark peak matching. The time window width was 0.10 min. The median method was used to identify 33 common peaks, and an overlay of UPLC fingerprints of 20 batches of Sangju Baidu Yin benchmark samples was generated. Figure 1 .

[0084] By comparing with the reference substance, 19 common characteristic peaks were identified. Figure 2 Among them: Peak 1 is neochlorogenic acid, Peak 2 is chlorogenic acid; Peak 3 is caffeic acid, Peak 4 is cryptochlorogenic acid, Peak 5 is 1,3-dicaffeoylquinic acid; Peak 11 is luteolin; Peak 12 is scutellarin, Peak 14 is forsythiaside A; Peak 15 is isochlorogenic acid B, Peak 16 is isochlorogenic acid A, Peak 18 is 1,5-dicaffeoylquinic acid; Peak 19 is apigenin-7-O-glucuronide; Peak 20 is rosmarinic acid; Peak 22 is isochlorogenic acid C; Peak 25 is isoliquiritigenin; Peak 26 is baicalin; Peak 27 is luteolin; Peak 31 is wogonin; Peak 33 is peucedanin.

[0085] The common peaks were assigned by comparing the whole prescription and single herb spectra. Figure 3Among them, peaks 1, 2, and 4 are common peaks of chrysanthemum, honeysuckle, and mulberry leaves; peak 3 is a common peak of perilla leaves, chrysanthemum, honeysuckle, mulberry leaves, and forsythia; peak 8 is a common peak of perilla leaves and scutellaria; peak 10 is a common peak of peucedanum and notopterygium; peaks 11, 15, 16, 22, and 27 are common peaks of honeysuckle and chrysanthemum; peak 12 is a common peak of perilla leaves and scutellaria; peak 13 is a common peak of perilla leaves, scutellaria, and chrysanthemum. Peak 19 is a common peak of chrysanthemum, perilla leaf, and schizonepeta; peaks 7 and 14 belong to forsythia; peak 9 belongs to mulberry leaf; peaks 6 and 20 belong to perilla leaf; peak 30 belongs to chrysanthemum and schizonepeta; peaks 5, 17, 21, and 23 belong to chrysanthemum; peak 25 belongs to liquorice; peak 33 belongs to peucedanum; and peaks 26, 28, 29, 31, and 32 belong to scutellaria. The fingerprint of Sangju Baidu Decoction can reflect the information of most individual medicinal material pieces.

[0086] Then the classification was performed by comparing the UPLC characteristic spectra of the whole prescription, single herb and negative control samples. Figure 4 It was found that peak 14 belongs to the characteristic peak of Forsythia suspensa; peaks 26 and 31 belong to the characteristic peaks of Scutellaria baicalensis; peak 20 belongs to the characteristic peak of Perilla frutescens; peak 25 belongs to the characteristic peak of Licorice; peak 33 belongs to the characteristic peak of Peucedanum peucedanum; peaks 16 and 15 belong to the common characteristic peaks of Chrysanthemum morifolium and Lonicera japonica.

[0087] The similarity between the fingerprint and the reference was calculated. The similarity results of the 20 batches of Sangju Baiduyin reference samples S1-S20 were 0.935, 0.977, 0.998, 0.997, 0.996, 0.983, 0.966, 0.979, 0.991, 0.987, 0.983, 0.983, 0.986, 0.974, 0.945, 0.954, 0.985, 0.980, 0.994, and 0.993, respectively. The similarity was greater than 0.93. The results showed that the 20 batches of Sangju Baiduyin UPLC reference samples had good overall similarity, and the established fingerprints were stable and could reflect the fingerprint characteristics of the substance reference.

[0088] Example 3 Methodological Investigation of the Fingerprint of Sangju Baidu Drink

[0089] 1. Precision inspection:

[0090] The test solution (S4) of Sangju Baidu Drink was taken and injected continuously 6 times according to the chromatographic conditions of Example 1. The chromatogram was recorded and the retention time and peak area of ​​each common peak were calculated. The results are shown in Tables 4-5 below. The RSDs of the retention times of the common peaks were all <0.29%, and the RSDs of the peak areas were all <4.87%, indicating that the instrument had good precision.

[0091] Table 4 Retention time of common peaks in precision test

[0092]

[0093]

[0094] Table 5 Peak areas of common peaks in precision test

[0095]

[0096]

[0097] 2. Repeatability inspection:

[0098] Take the Sangju Baidu Drink reference sample (S4), and prepare 6 test solutions in parallel according to the method of Example 1. The samples were injected separately according to the chromatographic conditions of Example 1, the chromatograms were recorded, and the retention time and peak area of ​​each common peak were calculated. The results are shown in Tables 6-7 below. The RSDs of the retention times of the common peaks were all <0.34%, and the RSDs of the peak areas were all <4.73%, indicating that the method had good repeatability.

[0099] Table 6 Relative retention time of common peaks in repeatability test

[0100]

[0101]

[0102] Table 7 Relative peak areas of common peaks in repeatability test

[0103]

[0104]

[0105] 3. Stability inspection:

[0106] The test solution (S4) of Sangju Baidu Drink was taken and sampled at 0, 2, 4, 6, 8, 10, 12, and 24 h according to the chromatographic conditions of Example 1. The chromatograms were recorded and the retention time and peak area of ​​each common peak were calculated. The results are shown in Tables 8-9 below. The RSDs of the retention times of the common peaks were all <0.30%, and the RSDs of the peak areas were all <4.78%, indicating that the test solution had good stability within 24 h.

[0107] Table 8 Relative retention time of each common peak in the stability test

[0108]

[0109]

[0110]

[0111] Table 9 Relative peak areas of common peaks in stability test

[0112]

[0113]

[0114] Example 4 Determination of the content of ingredients in Sangju Baidu Decoction

[0115] The chromatographic conditions were the same as in Example 1.

[0116] 1. Preparation of single herbal medicine sample solution:

[0117] The test sample solutions of single medicinal materials Forsythia suspensa, Scutellaria baicalensis, Licorice root, Peucedanum peucedanum, Honeysuckle and Chrysanthemum were prepared according to the method of Part I of the 2025 edition of the Pharmacopoeia of the People's Republic of China.

[0118] The preparation method of perilla leaf test solution is as follows: accurately weigh 0.5 g of perilla leaf powder (passed through No. 3 sieve), place it in a stoppered conical flask, accurately add 50 mL of 50% methanol, weigh the mass, seal it, and ultrasonically extract for 30 minutes (400W, 25Hz). After cooling to room temperature, make up for the lost mass, filter, filter through a 0.22μm microporous filter membrane, and take the filtrate to obtain the perilla leaf test solution.

[0119] 2. Preparation of mixed reference solution:

[0120] Appropriate amounts of forsythiaside A, baicalin, wogonin, rosmarinic acid, isoliquiritilicate, praeruptoside A, isochlorogenic acid A, and isochlorogenic acid B reference substances were accurately weighed, and methanol was added to prepare stock solutions with mass concentrations of 2193 μg / mL, 3264 μg / mL, 418 μg / mL, 250 μg / mL, 52 μg / mL, 1.610 mg / mL, 220 μg / mL, and 342 μg / mL, respectively.

[0121] 3. Methodological Investigation

[0122] 3.1 Linear relationship investigation:

[0123] Methanol was precisely added to the mixed reference substance stock solution and diluted stepwise to 5 concentrations. The peak area of ​​each component was measured according to the chromatographic conditions of Example 1. The standard curve was drawn with the reference substance mass concentration as the abscissa (X) and the peak area as the ordinate (Y). The regression equation and correlation coefficient were calculated, as shown in Table 10. The results showed that the above index components had a good linear relationship within their respective mass concentration ranges.

[0124] Table 10 Results of linear relationship investigation of 8 index components in Sangju Baidu Decoction

[0125] Element Regression equation <![CDATA[R 2 ]]> Linear range (μg / mL) Forsythiaside A Y=4838.8X+32086 0.9998 34.2656～2193 Baicalin Y=3757.8X+29733 0.9999 102～3264 Wogonin Y=5815.9X-48219 0.9993 13.0625～418 Rosmarinic acid Y=9808.6X-46110 0.9997 7.8125～250 Isoliquiritin Y=22939X+1939 0.9999 1.625～52 Praeruptorin A Y=2600.8X+2184 0.9999 10.0625～322 Isochlorogenic acid A Y=11692X-51425 0.9997 6.875～220 Isochlorogenic acid B Y=8803.7X-73047 0.9995 10.6875～342

[0126] 3.2 Precision test:

[0127] A reference sample numbered S5 was taken, and a test solution was prepared according to the method of Example 1. The sample was injected continuously six times according to the chromatographic conditions of Example 1, and the peak area of ​​each index component was recorded. As shown in Table 11 below, the RSD values ​​of the retention times of the index components forsythiaside A, baicalin, wogonin, rosmarinic acid, isoliquiritigenin, praeruptoside A, isochlorogenic acid A, and isochlorogenic acid B were 0.79%, 0.29%, 0.20%, 0.37%, 0.31%, 0.04%, 0.55%, and 0.63%, respectively, and the RSD values ​​of the peak areas were 0.50%, 0.73%, 0.40%, 0.74%, 4.47%, 1.23%, 0.82%, and 0.73%, respectively, indicating good instrument precision.

[0128] Table 11

[0129]

[0130]

[0131] 3.3 Stability test:

[0132] The reference sample solution numbered S5 was taken and the test solution was prepared according to the method of Example 1. The sample was injected and measured according to the chromatographic conditions of Example 1 at 0, 2, 4, 6, 8, 10, 12, and 24 hours, respectively. The peak area of ​​each index component was recorded and the RSD value was calculated. As shown in Table 12 below, the results show that the RSD values ​​of forsythiaside A, baicalin, wogonin, rosmarinic acid, isoliquiritin, praeruptorin A, and isochlorogenic acid A and isochlorogenic acid B were 0.88%, 0.54%, 0.38%, 0.74%, 0.65%, 0.07%, 0.68%, and 1.32% for retention time, and 1.02%, 0.84%, 0.46%, 0.97%, 4.80%, 1.16%, 0.89%, and 1.34% for peak area, respectively, indicating good stability of the solution within 24 hours.

[0133] Table 12

[0134]

[0135]

[0136] 3.4 Repeatability test:

[0137] The reference sample solution numbered S5 was taken, and six test solutions were prepared in parallel according to the method of Example 1. The samples were injected and measured according to the chromatographic conditions of Example 1, and the peak area of ​​each index component was recorded. As shown in Table 13 below, the results showed that the retention time RSD values ​​of forsythiaside A, baicalin, wogonin, rosmarinic acid, isoliquiritigenin, praeruptorin A, isochlorogenic acid A, and isochlorogenic acid B were 0.79%, 0.33%, 0.23%, 0.46%, 0.36%, 0.04%, 0.73%, and 0.87%, respectively, and the peak area RSD values ​​were 1.33%, 1.26%, 1.24%, 2.10%, 1.62%, 2.04%, 1.18%, and 1.55%, respectively, indicating good reproducibility of the method.

[0138] Table 13

[0139]

[0140] 3.5 Sample recovery test:

[0141] Accurately measure 2.5 mL of the Sangju Baidu Drink reference sample with the determined index component content, for a total of 6 portions. Add each reference substance at a 1:1 ratio of the sample content, and adjust the volume to 5 mL. Inject and measure the sample according to the chromatographic conditions of Example 1, record the peak area of ​​each index component, and calculate the sample recovery and RSD value. Results The average recoveries of forsythiaside A, baicalin, wogonin, rosmarinic acid, isoliquiritigenin, praeruptoside A, isochlorogenic acid A and isochlorogenic acid B were 102.87%, 101.37%, 102.83%, 99.31%, 96.63%, 101.33%, 100.59% and 99.31%, respectively, and the RSDs were 0.67%, 1.12%, 0.97%, 1.96%, 2.15%, 1.19%, 2.65% and 3.51%, respectively, indicating that the method has a good recovery rate. The results are shown in Table 14.

[0142] Table 14 Sample recovery test results

[0143]

[0144]

[0145] Example 5 Chromatographic Conditions

[0146] In the process of establishing the UPLC fingerprint of Sangju Baidu Drink, the effects of acetonitrile, methanol, different concentrations of phosphoric acid water, and different concentrations of formic acid water on the separation effect of the sample were investigated. It was found that when acetonitrile-0.2% phosphoric acid water was used as the mobile phase, the baseline was stable, the separation effect between the peaks was the best, there were fewer impurity peaks, and the analysis method was more accurate. Therefore, acetonitrile-0.2% phosphoric acid water was selected as the optimal fingerprint mobile phase condition.

[0147] At a wavelength of 350nm, the response value of the comprehensive chromatographic peak is obvious, the peak shape is good, and the peak information is comprehensive. Finally, a wavelength of 350nm was selected as the detection wavelength for the fingerprint spectrum and quantity transfer law of the Sangju Baidu Drink benchmark sample.

[0148] Twenty batches of Sangju Baidu Decoction reference samples were tested, and the resulting fingerprints showed good similarity, all greater than 0.92. Thirty-three common peaks were identified, and 19 common characteristic peaks were identified by comparison with the reference. Comparison of the UPLC chromatograms of the full formula and individual herbs revealed that some individual herbs did not contribute to the common peaks of the full formula. Possible reasons for this include: some ingredients have low or no response at a UV wavelength of 350 nm. For example, saikosaponin a and saikosaponin d, the main components of Bupleurum chinense, only exhibit terminal UV absorption within the UV wavelength range of 190-400 nm, making them suitable for detection using an evaporative light scattering detector. Therefore, Bupleurum chinense does not contribute to the characteristic peaks of the full formula at 350 nm. In contrast, the main components of rhubarb, such as rhein, emodin, and aloe-emodin, all respond best at 254 nm and have low responses at 350 nm. Amygdalin in bitter almonds, ephedrine hydrochloride in ephedra, and the main components of Adenophora australis, taraxacone and lupeol acetate, all absorb at the ultraviolet end of 210nm. Therefore, the above single medicinal materials have no contribution to the fingerprint spectrum.

[0149] Based on the UPLC fingerprint of the whole formula and relevant provisions of the pharmacopoeia, forsythiaside A, baicalin, wogonin, rosmarinic acid, isoliquiritigenin, praeruptoside A, isochlorogenic acid A and isochlorogenic acid B in Sangju Baidu Decoction with clear pharmacological activities were finally selected as indicator ingredients for investigation.

[0150] Obviously, the described embodiments are only individual embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other implementations obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims

1. A UPLC fingerprint detection method for Sangju Baidu Yin, characterized by: The steps include: (1) Preparation of Sangju Baiduyin reference sample solution: Decoction the raw materials of Sangju Baiduyin and concentrate them to obtain the reference sample solution; (2) Preparation of test solution: Take the Sangju Baidu Drink standard sample solution, place it in a volumetric flask, add anhydrous methanol to below the scale line, sonicate, cool, and then dilute to the scale line with anhydrous methanol, shake well, centrifuge, take the supernatant, and filter through a microporous filter membrane to obtain the product; (3) Preparation of reference solution: accurately weigh the reference substances of neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, 1,3-dicaffeoylquinic acid, luteolin, scutellarin, forsythiaside A, isochlorogenic acid B, isochlorogenic acid A, 1,5-dicaffeoylquinic acid, apigenin-7-O-glucuronide, rosmarinic acid, isochlorogenic acid C, isoliquiritin, baicalin, luteolin, wogonin, and peucedanum praeruptoside A, respectively, and add methanol to prepare a solution. (4) Determination: Accurately aspirate the test solution and reference solution, inject them into the high performance liquid chromatograph, perform the determination, and record the chromatogram; (5) Construction of fingerprint: The fingerprints of different batches of test samples were imported into the Chinese medicine chromatographic fingerprint similarity evaluation system for analysis to obtain the fingerprint of Sangju Baidu Drink.

2. The UPLC fingerprint detection method according to claim 1, wherein: The preparation method of the Sangju Baidu Yin reference sample solution described in step (1) is as follows: take 10g of mulberry leaves, 10g of chrysanthemums, 10g of honeysuckle, 10g of forsythia, 10g of schizonepeta, 10g of perilla leaves, 10g of notopterygium, 10g of roasted ephedra, 10g of apricot kernel, 30g of gypsum, 6g of roasted licorice, 10g of peucedanum, 12g of bupleurum, 12g of stir-fried scutellaria, 12g of adenophora australis and 6g of cooked rhubarb, decoct them twice, add 12 times the amount of water for the first time and decoct for 2 hours, add 10 times the amount of water for the second time and decoct for 1 hour, combine the two filtrates, and concentrate to 150mL to obtain the reference sample solution.

3. The UPLC fingerprint detection method according to claim 1, wherein: The preparation method of the test solution described in step (2) is as follows: take 2 mL of the Sangju Baidu Drink reference sample solution, put it into a 5 mL volumetric flask, add anhydrous methanol to below the scale line, ultrasonicate at 400W, 40kHz, 30min, cool and dilute to the scale line with anhydrous methanol, shake well, centrifuge at 12000r / min for 10min, take the supernatant, and filter through a 0.22μm microporous filter membrane to obtain the product.

4. The UPLC fingerprint detection method according to claim 1, wherein: The preparation method of the reference solution described in step (3) is as follows: accurately weigh neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, 1,3-dicaffeoylquinic acid, luteolin, scutellarin, forsythiaside A, isochlorogenic acid B, isochlorogenic acid A, 1,5-dicaffeoylquinic acid, apigenin-7-O-glucuronide, rosmarinic acid, isochlorogenic acid C, isoliquiritin, baicalin, luteolin, wogonin, and peucedanum praeruptoside A reference standards, respectively, and add methanol to prepare a concentration of 240 μg / mL, The reference solutions of 230μg / mL, 224μg / mL, 200μg / mL, 250μg / mL, 205μg / mL, 180μg / mL, 205μg / mL, 270μg / mL, 220μg / mL, 173.33μg / mL, 170μg / mL, 186μg / mL, 345μg / mL, 232μg / mL, 120μg / mL, 350μg / mL, 70μg / mL, and 280μg / mL are obtained.

5. The UPLC fingerprint detection method according to claim 1, wherein: The mobile phase in the chromatographic conditions of the determination described in step (4) is a mixture of acetonitrile and phosphoric acid.

6. The UPLC fingerprint detection method according to claim 5, wherein: The phosphoric acid is a 0.2% phosphoric acid aqueous solution.

7. The UPLC fingerprint detection method according to claim 6, wherein: The chromatographic conditions for the determination described in step (4) are: Chromatographic column: CAPCELL PAK C 18 Chromatographic column (150 mm × 2.0 mm, 2 μm); Mobile phase: acetonitrile-0.2% phosphoric acid aqueous solution; Gradient elution: 0-15 min, 6%-10% acetonitrile; 15-18 min, 10%-12% acetonitrile; 18-48 min, 12%-14% acetonitrile; 48-68 min, 14%-30% acetonitrile; 68-85 min, 30%-95% acetonitrile; Volume flow rate: 0.3 mL / min; Column temperature: 35°C; Detection wavelength: 350nm; Injection volume: 2 μL.

8. The UPLC fingerprint detection method according to claim 1, wherein: After the fingerprint is constructed in step (5), similarity evaluation is performed. The similarity evaluation method is as follows: the fingerprint is imported into similarity evaluation software for similarity evaluation analysis. The similarities are all greater than 0.93, and 33 common peaks are determined, which are numbered as peaks 1-33.

9. The UPLC fingerprint detection method according to claim 8, wherein: Peak 1 is neochlorogenic acid, peak 2 is chlorogenic acid; peak 3 is caffeic acid, peak 4 is cryptochlorogenic acid, peak 5 is 1,3-dicaffeoylquinic acid; peak 11 is luteolin; peak 12 is scutellarin, peak 14 is forsythiaside A; peak 15 is isochlorogenic acid B, peak 16 is isochlorogenic acid A, peak 18 is 1,5-dicaffeoylquinic acid; peak 19 is apigenin-7-O-glucuronide; peak 20 is rosmarinic acid; peak 22 is isochlorogenic acid C; peak 25 is isoliquiritigenin; peak 26 is baicalin; peak 27 is luteolin; peak 31 is wogonin; peak 33 is peucedanum scabraein.

10. A fingerprint obtained by the UPLC fingerprint detection method of Sangju Baidu Drink according to claim 1.

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