Construction method, application and detection method of fingerprint spectrum of compound qinlan oral liquid

Abstract

The application discloses a construction method, application and detection method of a fingerprint spectrum of compound Qinlan oral liquid, and belongs to the technical field of Chinese medicine preparation detection. The technical problem to be solved is that because the chemical components of the compound Qinlan oral liquid are relatively complex, quantitative analysis of single or several index components cannot comprehensively control and evaluate the quality of the medicine, so the fingerprint spectrum detection method is established to represent the overall quality of the medicine, and the safety, effectiveness and controllable quality of the medicine are ensured.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine preparation detection technology. Specifically, it relates to a method for constructing, applying, and detecting the fingerprint spectrum of Compound Scutellaria baicalensis and Scutellaria baicalensis oral liquid. Background Technology

[0002] The application of traditional Chinese medicine (TCM) is becoming increasingly widespread both domestically and internationally, making its quality control a key factor influencing the development of the TCM industry. TCM has complex components, and traditional quality control methods have limitations, failing to fully reflect its quality. TCM fingerprinting technology comprehensively characterizes the chemical features of TCM through various advanced analytical techniques, providing a new means for TCM quality control.

[0003] Traditional Chinese medicine (TCM) fingerprinting technology is a method for comprehensive chemical composition analysis of TCM materials using modern analytical techniques. It aims to create characteristic "fingerprints" by separating, detecting, and quantifying multiple chemical components in TCM materials to characterize their quality. As a precise quality control method that comprehensively covers the production process, TCM fingerprinting technology effectively overcomes the limitations of traditional testing methods that only target the final product. This technology combines multiple advanced analytical methods such as high-performance liquid chromatography (HPLC), liquid chromatography (LC), gas chromatography (GC), and mass spectrometry (MS) to comprehensively and accurately reflect the chemical composition and quality characteristics of TCM materials. Specifically, the core principle of TCM fingerprinting technology is to comprehensively analyze the different chemical components in TCM materials and obtain their fingerprints. These fingerprints are a comprehensive representation of the chemical composition of TCM materials, containing detailed information on the main components and their characteristic peaks. By analyzing the fingerprints, the overall quality of the medicine is characterized, ensuring its safety, efficacy, and quality control.

[0004] The principle of liquid chromatography (LC) is to inject a sample solution into a chromatographic column. Utilizing the partitioning behavior of different chemical components within the column, the various components in the sample are separated, and their characteristic peaks are recorded by a detector, thus constructing a fingerprint spectrum. Specifically, in LC analysis, the sample solution first passes through a chromatographic column packed with solid-phase material (usually reversed-phase or normal-phase packing). During this process, due to the different interaction forces between the packing material and the chemical components in the sample, the components in the sample separate from each other within the column. When the mobile phase passes through the column, the components in the sample separate according to their chemical properties (such as polarity, hydrophilicity, etc.) and their residence time within the column. The commonly used mobile phase in LC is a mixture of water and organic solvents. The ratio and gradient of the mobile phase can be optimized according to the characteristics of the sample to enhance the separation effect.

[0005] Mass spectrometry (MS) is an analytical method used to determine the mass and structure of chemical components in a sample. It ionizes molecules in a sample and measures the mass and relative abundance of these ions, thereby constructing a fingerprint spectrum of traditional Chinese medicine (TCM) materials to reveal the chemical components and structural characteristics of the sample. In MS analysis, the sample is first introduced into an ion source. Common ionization methods include electron bombardment, chemical ionization, and electrospray ionization. The ionized sample molecules form charged ions, which are then separated according to their mass-to-charge ratio in the mass spectrometer's mass analyzer. Common mass spectrometers include time-of-flight mass spectrometers, ion trap mass spectrometers, and quadrupole mass spectrometers, all of which provide high-resolution mass data. Subsequently, in the generated mass spectrum, each peak corresponds to one ion in the sample, and the peak's position (mass-to-charge ratio) and intensity reflect the mass and concentration of that component.

[0006] Mass spectrometry is often coupled with high performance liquid chromatography or gas chromatography to further improve the separation capability and identification accuracy of mass spectrometry.

[0007] Relevant patent documents retrieved: The document, published in China (CN121298929A) on January 9, 2026, discloses a method for constructing a fingerprint spectrum of a compound preparation for relieving sore throat and detoxifying. The method includes the following steps: (1) preparation of the test solution; (2) detection of the test solution by high performance liquid chromatography (HPLC), using octadecylsilane-bonded silica gel as the packing material, and the mobile phase including an acidic aqueous solution and acetonitrile. The gradient elution program includes: 0→8min→25min→35min→50min→58min→70min→80min→82min→95min. The volume percentage of acetonitrile in the mobile phase is: 2%→2%→15%→20%→20%→28%→33%→60%→76%→76%. This invention can achieve the separation of 12 common characteristic peaks, and the obtained fingerprint spectrum has a stable baseline and good peak shape.

[0008] The document, published in China (CN102590424A) on July 18, 2012, discloses a quality detection method for a compound Houttuynia cordata mixture. The method uses high-performance liquid chromatography (HPLC) with octadecylsilane-bonded silica gel as the packing material. Gradient elution is performed using 0.1% phosphoric acid solution as mobile phase A and methanol as mobile phase B. The characteristic components of the four traditional Chinese medicines in the compound Houttuynia cordata mixture—Houttuynia cordata, Scutellaria baicalensis, Forsythia suspensa, and Lonicera japonica—are all reflected in the fingerprint chromatogram.

[0009] The publication country is China, publication number CN106109673A, publication date November 16, 2016. This document discloses a compound houttuynia cordata syrup and its preparation and detection methods. The syrup is made from the following raw materials in parts by weight: houttuynia cordata 100 parts by weight, scutellaria baicalensis 25 parts by weight, isatis indigotica 25 parts by weight, forsythia suspensa 10 parts by weight, and honeysuckle 10 parts by weight. High-performance liquid chromatography (HPLC) was used for fingerprint analysis of its quality. Chromatographic conditions: Waters Xbridge C100 column. 18 The column used acetonitrile as mobile phase A and water as mobile phase B, with gradient elution: from 0 min to 12 min, mobile phase A linearly increased from 15% to 30%, and mobile phase B linearly decreased from 85% to 70%; from 12 min to 45 min, mobile phase A linearly increased from 30% to 80%, and mobile phase B linearly decreased from 70% to 20%; from 45 min to 55 min, mobile phase A remained at 80%, and mobile phase B remained at 20%. The detection wavelength was 203 nm, and the flow rate was 1.0 mL / min. -1 Column temperature 30℃, injection volume 10μL.

[0010] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: 1. The detection method in CN121298929A has limited effectiveness in detecting trace amounts of active ingredients, with evidence showing that only 12 characteristic peaks can be detected.

[0011] 2. The quality testing method in CN102590424A has limited ability to detect active pharmaceutical ingredients. The relevant evidence is that it can only detect the characteristic components of the four Chinese herbs in Compound Houttuynia Cordata Mixture: Houttuynia cordata, Scutellaria baicalensis, Forsythia suspensa, and Lonicera japonica, but cannot detect the characteristic components of Isatis indigotica.

[0012] 3. The detection method in CN106109673A has limited effectiveness in detecting trace amounts of active ingredients, with evidence showing that only 10 characteristic peaks can be detected.

[0013] Compound Scutellaria and Scutellaria Oral Solution contains four medicinal herbs: honeysuckle, scutellaria, forsythia, and isatis root. These herbs possess the effects of relieving exterior syndromes with pungent and cooling properties, and clearing heat and detoxifying. Due to the complexity of its chemical composition, quantitative analysis of single or a few indicator components is insufficient to comprehensively control and evaluate the quality of the drug. Therefore, it is urgent to establish a method for constructing a fingerprint spectrum for Compound Scutellaria and Scutellaria Oral Solution to characterize the overall quality of the drug and ensure its safety, efficacy, and quality control. Summary of the Invention

[0014] The purpose of this invention is to provide: A method for constructing, applying, and detecting the fingerprint spectrum of Compound Scutellaria and Ligusticum Oral Solution, and related technologies, to solve the technical problems, such as the difficulty in comprehensively controlling and evaluating the quality of the drug due to the complex chemical composition of Compound Scutellaria and Ligusticum Oral Solution and the difficulty in quantitative analysis of single or several indicator components, or a combination thereof.

[0015] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0016] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0017] The definition of the standard term can be found in the reference Luo Guoan, Liang Qionglin, Wang Yiming. Fingerprint Spectroscopy of Traditional Chinese Medicine: Quality Evaluation, Quality Control and New Drug Development [M]. Beijing: Chemical Industry Press, 2009.

[0018] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0019] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0020] The term "fingerprint spectrum" used in this article refers to a spectrum obtained by using analytical techniques such as chromatography and mass spectrometry, which can comprehensively characterize the overall chemical composition of Chinese medicinal materials or Chinese medicine preparations, and is used for the overall quality control of Chinese medicine.

[0021] The term "mobile phase" as used in this article refers to the liquid solvent system that carries the sample components through the chromatographic column in liquid chromatography. It can be prepared by mixing one or more solvents in proportion to achieve the separation and elution of the analyte.

[0022] The term "chromatographic conditions" as used in this article refers to all the experimental parameters set when performing liquid chromatography detection, including key parameters such as column type, mobile phase ratio and elution program, flow rate, column temperature, and detection wavelength.

[0023] The term "common peak" used in this article refers to the chromatographic peak that appears in all samples from multiple qualified test samples from the same batch, and is a chromatographic characterization of the inherent common chemical components of medicinal materials.

[0024] The term "characteristic peak" used in this article refers to a specific chromatographic peak that can specifically identify the target medicinal material or preparation and distinguish it from other medicinal materials, and can be used as a basis for qualitative identification.

[0025] The term "high performance liquid chromatography" as used in this article refers to an analytical technique that uses a liquid as the mobile phase, relies on the differences in the distribution and adsorption of analytes between the stationary and mobile phases to achieve component separation, and is equipped with a detector for quantitative and qualitative analysis. It is the main detection method for traditional Chinese medicine components in this study.

[0026] The term "mass spectrometry" as used in this article refers to a precision analytical instrument that ionizes the analyte into charged ions and uses the ion mass-to-charge ratio to achieve component identification, molecular weight determination, and content auxiliary determination. It is often used in conjunction with liquid chromatography.

[0027] The term "reference fingerprint spectrum" used in this article refers to a standard reference spectrum established by averaging multiple batches of qualified medicinal materials from authentic regions, which serves as the benchmark for comparing the fingerprint spectrum of the sample to be tested.

[0028] The term "similarity evaluation" used in this article refers to an evaluation method that uses mathematical algorithms to calculate the similarity coefficient between the fingerprint spectrum of the sample to be tested and the control fingerprint spectrum, and quantitatively evaluates the consistency of the overall chemical composition of the samples.

[0029] The term "specificity" used in this article refers to the methodological indicators that allow an analytical method to accurately detect the target analyte without interference, even in the presence of coexisting impurities, excipients, and interfering components.

[0030] The term "system suitability" used in this article refers to the verification items used to evaluate whether the separation performance of a chromatographic system meets the experimental requirements by means of parameters such as tailing factor, theoretical plate number, and resolution.

[0031] The term "reproducibility" as used in this article refers to the ability of the same personnel, instruments, and chromatographic conditions to prepare and measure multiple test solutions consecutively within a short period of time, with the deviation of the test results meeting the limit requirements, reflecting the intra-day stability of the method.

[0032] The term "solution stability" used in this article refers to the stability of a sample solution during storage, where the content and chromatogram of the analyte do not change significantly when the sample solution is sampled and measured at different time points within a specified environment and storage period.

[0033] The term "intermediate precision" used in this article refers to the precision of the test results obtained when different testers, different test dates, or different instruments of the same model conduct the tests, and the results meet the standard, examining the impact of routine laboratory test fluctuations on the test results.

[0034] The term "robustness" as used in this article refers to the ability of an analytical method to still meet the separation and determination requirements after minor adjustments to chromatographic conditions (flow rate, column temperature, mobile phase ratio, etc.), reflecting the ability of the detection method to withstand minor changes in test conditions.

[0035] In a first aspect, the present invention provides: a method for constructing the fingerprint spectrum of Compound Scutellaria and Scutellaria Oral Liquid, wherein the raw materials for preparing Compound Scutellaria and Scutellaria Oral Liquid include honeysuckle, scutellaria, forsythia and isatis root.

[0036] Preferably, the method for constructing the fingerprint map includes the following steps: A. Preparation of the test solution: Dilute Compound Scutellaria and Ligusticum Oral Solution with methanol, filter, and obtain the test solution; B. Preparation of the reference solution: Dissolve and dilute the reference substance in methanol, shake well, and obtain the reference substance solution; C. High-performance liquid chromatography (HPLC) determination: Inject the test solution and reference solution into the HPLC instrument separately and collect the data.

[0037] Preferably, the high-performance liquid chromatography (HPLC) conditions are as follows: octadecylsilane-bonded silica gel as the packing material, methanol as mobile phase A, phosphoric acid solution as mobile phase B, ZORBAX Eclipse plus column, and detection wavelength of 245±2 nm.

[0038] Preferably, the detection wavelength is selected from any value or range between 243-247nm, specifically from: 243nm, 244nm, 245nm, 246nm, 247nm or a range between two of them.

[0039] More preferably, the detection wavelength is 245nm.

[0040] Preferably, the flow rate of the high-performance liquid chromatography is 1 mL / min.

[0041] Preferably, the injection volume under the high performance liquid chromatography conditions is 10 µl.

[0042] Preferably, the preparation of the test solution in step A is specifically performed as follows: accurately measure 1 mL of Compound Scutellaria baicalensis oral liquid, dilute it with methanol to 10 mL, shake well, filter, and take the filtrate to obtain the test solution.

[0043] Preferably, the concentration of methanol in both steps A and B is 50%.

[0044] Preferably, the reference substance in step B is chlorogenic acid reference standard.

[0045] Preferably, the preparation of the reference solution in step B is specifically performed as follows: accurately weigh 2 mg of reference substance, dissolve and dilute it with 50% methanol to prepare a solution containing 40 μg of reference substance per 1 mL.

[0046] Preferably, the concentration of the phosphoric acid solution in step C is selected from any value or range between 0.09% and 0.11%, specifically from 0.09%, 0.10%, 0.11%, or a range between the two.

[0047] More preferably, the concentration of the phosphoric acid solution in step C is 0.10%.

[0048] Preferably, the chromatographic conditions in step C of the high-performance liquid chromatography determination further include gradient elution, with the following conditions: From 0 to 10 min, the volume percentage of mobile phase A increased from 15% to 25%, while the volume percentage of mobile phase B decreased from 85% to 75%. From 10 min to 15 min, the volume percentage of mobile phase A increased from 25% to 30%, while the volume percentage of mobile phase B decreased from 75% to 70%. From 15 min to 20 min, the volume percentage of mobile phase A increased from 30% to 33%, while the volume percentage of mobile phase B decreased from 70% to 67%. From 20 min to 35 min, the volume percentage of mobile phase A was 33%, and the volume percentage of mobile phase B was 67%. From 35 min to 50 min, the volume percentage of mobile phase A increased from 33% to 65%, while the volume percentage of mobile phase B decreased from 67% to 35%. From 50 min to 60 min, the volume percentage of mobile phase A increased from 65% to 100%, while the volume percentage of mobile phase B decreased from 35% to 0%.

[0049] Preferably, the column temperature of the chromatographic conditions in step C is selected from any value or range between 23-27℃, specifically from: 23℃, 24℃, 25℃, 26℃, 27℃ or a range between the two.

[0050] More preferably, the column temperature of the chromatographic conditions in step C is 25°C.

[0051] Preferably, the data acquisition time for the chromatographic conditions in step C is 60 minutes.

[0052] Preferably, the fingerprint spectrum of the compound Scutellaria baicalensis and Scutellaria baicalensis oral liquid has 23 common characteristic peaks, with peak 6 serving as the reference peak (chlorogenic acid peak), and the corresponding peak being peak S. The relative retention times are: 0.267 (peak 1), 0.530 (peak 2), 0.624 (peak 3), 0.669 (peak 4), 0.955 (peak 5), 1.000 (peak 6), 1.073 (peak 7), 1.114 (peak 8), 1.198 (peak 9), and 1.3... 28 (peak #10), 1.514 (peak #11), 1.649 (peak #12), 1.824 (peak #13), 1.951 (peak #14), 2.330 (peak #15), 2.569 (peak #16), 2.740 (peak #17), 2.902 (peak #18), 3.042 (peak #19), 3.131 (peak #20), 3.240 (peak #21), 3.404 (peak #22), 3.443 (peak #23).

[0053] Secondly, the present invention provides the application of the above-mentioned construction method in the quality testing of Compound Scutellaria baicalensis and Scutellaria baicalensis oral liquid.

[0054] Thirdly, the present invention provides: a quality testing method for Compound Scutellaria baicalensis and Scutellaria baicalensis oral liquid, comprising the step of comparing the fingerprint spectrum of the Compound Scutellaria baicalensis and Scutellaria baicalensis oral liquid to be tested with a control fingerprint spectrum of the Compound Scutellaria baicalensis and Scutellaria baicalensis oral liquid; wherein the fingerprint spectrum of the Compound Scutellaria baicalensis and Scutellaria baicalensis oral liquid to be tested is constructed using the Compound Scutellaria baicalensis and Scutellaria baicalensis oral liquid to be tested according to the above-described construction method.

[0055] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: The first priority solution is a method for constructing a fingerprint spectrum of Compound Scutellaria and Ligusticum Oral Solution. This solution not only addresses the technical problem that "the chemical composition of Compound Scutellaria and Ligusticum Oral Solution is relatively complex, and quantitative analysis of single or a few indicator components is difficult to comprehensively control and evaluate the quality of the drug," but also further solves the technical problem that "the detection method is accurate, highly sensitive, has good repeatability, and stable detection results."

[0056] The beneficial effects of this invention are as follows: The present invention has at least the following beneficial effects: 1. The fingerprint spectrum construction method of Compound Scutellaria and Ligusticum Oral Solution provided by the present invention uses octadecylsilane-bonded silica gel as filler, methanol as mobile phase A, and phosphoric acid solution as mobile phase B. Through a specific elution procedure, 23 common characteristic peaks are obtained. The obtained fingerprint spectrum has good peak shape, which provides a basis for the quality detection and control of Compound Scutellaria and Ligusticum Oral Solution. It can also accurately locate the positions of stigmosiderin, (R,S)-gawisin, neochlorogenic acid, strophanthidin, chlorogenic acid, cryptochlorogenic acid, swertiamarin, caffeic acid, suspensaside, forsythoside I, strychnoside, salicylic acid, isostemioside, forsythoside A, luteolin, rutin, isochlorogenic acid C, forsythoside, baicalin, baicalin-7-O-glucuronide, and strychnoside A-7-O-β-D-glucuronide, fully reflecting the integrity and characteristics of Compound Scutellaria and Ligusticum Oral Solution.

[0057] 2. The method for constructing the fingerprint spectrum of Compound Scutellaria baicalensis and Scutellaria baicalensis oral liquid provided by this invention optimizes the mobile phase and flow gradient, detection wavelength, extraction solvent, injection volume, and other conditions to determine the optimal extraction process and chromatographic conditions. This results in a high number of chromatographic peaks with good resolution, excellent peak shape, and stable baseline, enabling more comprehensive quality monitoring of the fingerprint spectrum of Compound Scutellaria baicalensis and Scutellaria baicalensis oral liquid. Furthermore, the sample preparation of this method is simple, the chromatographic conditions are easy to achieve, and the method exhibits good reproducibility and accurate results.

[0058] 3. The quality testing method for Compound Scutellaria and Ligusticum Oral Solution provided by the present invention compares the fingerprint spectrum of the Compound Scutellaria and Ligusticum Oral Solution to be tested with the control fingerprint spectrum of Compound Scutellaria and Ligusticum Oral Solution, which can comprehensively, clearly and effectively test the quality of Compound Scutellaria and Ligusticum Oral Solution. Attached Figure Description

[0059] Figure 1 The chromatogram is of the compound Scutellaria baicalensis oral liquid test sample (B36250701) obtained in Example 1 of the present invention.

[0060] Figure 2 This is a chromatogram of the compound Scutellaria baicalensis oral liquid test sample prepared in Comparative Example 1 of the present invention.

[0061] Figure 3 This is a chromatogram of the compound Scutellaria baicalensis oral liquid test sample prepared in Comparative Example 2 of the present invention.

[0062] Figure 4 The image shows the chromatogram of the compound Scutellaria baicalensis oral liquid test sample prepared in Comparative Example 3 of this invention. The upper image is the chromatogram with a detection wavelength of 350 nm, and the lower image is the chromatogram with a detection wavelength of 245 nm.

[0063] Figure 5 This is a chromatogram of the compound Scutellaria baicalensis oral liquid test sample prepared in Comparative Example 4 of this invention.

[0064] Figure 6 This is a chromatogram of the compound Scutellaria baicalensis oral liquid test sample prepared in Comparative Example 5 of this invention.

[0065] Figure 7 This is a chromatogram of the compound Scutellaria baicalensis oral liquid test sample prepared in Comparative Example 6 of this invention.

[0066] Figure 8 The chromatograms are of 13 batches of samples from the construction method in Experimental Example 1 of this invention.

[0067] Figure 9 This is a positive mode TIC diagram of the compound Scutellaria baicalensis oral liquid in the construction method of Experimental Example 1 of the present invention.

[0068] Figure 10 This is a negative mode TIC diagram of Compound Scutellaria baicalensis Oral Solution in the construction method of Experimental Example 1 of the present invention.

[0069] Figure 11 This is a control fingerprint spectrum fitted to 13 batches of samples in the construction method of Experimental Example 1 of the present invention.

[0070] Figure 12 This is a chromatogram of the specificity detection in the analytical method of Experimental Example 2 of the present invention. Detailed Implementation

[0071] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0072] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0073] Table 1 Instruments and Equipment

[0074] Table 2. Reagents and their manufacturers and models

[0075] Table 3 Sample batch number, manufacturer, and specifications

[0076] Example 1: Method for constructing the fingerprint spectrum of Compound Scutellaria and Oryzae Oral Solution A. Preparation of the test solution Accurately measure 1 mL of Compound Scutellaria and Ligusticum Oral Solution (B36250701), place it in a 10 mL volumetric flask, dilute it to 10 mL with 50% methanol, shake well, filter, and take the filtrate to obtain the test solution. B. Preparation of reference solution Accurately weigh 2 mg of chlorogenic acid, dissolve and dilute it with 50% methanol to prepare a solution containing 40 μg of chlorogenic acid per 1 mL.

[0077] C. High-performance liquid chromatography (HPLC) determination: Inject the test solution and reference solution into the HPLC instrument separately and collect data; The high performance liquid chromatography (HPLC) conditions were as follows: octadecylsilane-bonded silica gel was used as the packing material, methanol was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, the chromatographic column was a ZORBAX Eclipse plus (250nm×4.6nm, 5μm), and the detection wavelength was 245nm. The theoretical plate number, calculated based on the chlorogenic acid peak, should be no less than 14,000; the resolution between adjacent peaks should be greater than 1.0. The gradient elution conditions are shown in Table 4 below: Table 4 Gradient elution table

[0078] Flow rate: 1 mL / min; Column temperature: 25℃; Detection wavelength: 245nm; Injection volume: 10 µl; Data collection time: 60 minutes.

[0079] Measurement results: Compared with the control fingerprint, the similarity was not less than 0.9, and the peak shape was complete, indicating good separation.

[0080] Comparative Example 1 The difference from Example 1 is that only the mobile phase B is changed to 0.25% glacial acetic acid, while the other conditions and steps are the same as in Example 1. The results showed that the number of chromatographic peaks was less than that of the phosphate system, and the asymmetry of the baicalin peak was lower.

[0081] Comparative Example 2 The difference from Example 1 is that only the column temperature is changed to 35°C, while all other conditions and steps are the same as in Example 1. The results showed that at a column temperature of 35℃, the chromatographic peaks shifted forward, and peaks 3, 4 and 5, 6 overlapped, affecting the resolution.

[0082] Comparative Example 3 The difference from Example 1 is that only the wavelength is changed to 350nm, while the other conditions and steps are the same as in Example 1. The results showed that the number of chromatographic peaks was significantly reduced.

[0083] Comparative Example 4 The difference from Example 1 is that only the chromatographic column was changed to ZORBAX C18, while all other conditions and steps were the same as in Example 1. The results showed that some chromatographic peaks could not be separated, and the resolution did not meet the requirements.

[0084] Comparative Example 5 The difference from Example 1 is that only the chromatographic column is changed to YMC-Pack ODS-A, while all other conditions and steps are the same as in Example 1. The results showed that some chromatographic peaks could not be separated, and the resolution did not meet the requirements.

[0085] Comparative Example 6 The difference from Example 1 is that only the gradient elution procedure is changed, as shown in Table 5 below: Table 5

[0086] All other conditions and steps are the same as in Example 1; The results showed that the separation between the S peak (chlorogenic acid) and the subsequent peaks was poor and the peak purity was insufficient in the first 15 minutes, and almost no chromatographic peaks appeared after 40 minutes.

[0087] Experiment Example 1: Construction Method, Identification of Characteristic Peaks, and Prescribed Values ​​of Fingerprint Spectra (1) Construction method Thirteen batches of Compound Scutellaria and Ligusticum Oral Solution (B14231001, A14231101, A14231201, B14240301, B14240601, B14240602, B36250101, B36250102, B14250202, B36241001, B36241002, B36241003, B36250701) were used as test samples. Test solutions were prepared according to the method in Example 1. The chromatographic conditions of Example 1 were used to detect the above test solutions. The results are shown in the figure. Figure 8 .

[0088] (2) Calibration of common fingerprint peaks By comparing and analyzing the fingerprint spectra of multiple batches of representative samples, chromatographic peaks appearing in all batches were selected as common peaks. High-content, highly specific chromatographic peaks were chosen as characteristic fingerprint peaks (with a single peak area not less than 5% of the reference peak area), and their relative retention times with the reference peak were used for calibration.

[0089] From Table 6 and Figure 8 The data shows that the proportion of non-common peaks in the total peak area of ​​each batch of samples ranged from 2.77% to 4.50%. Using the method that "the area of ​​a single peak is not less than 5% of the area of ​​the reference peak (6# chlorogenic acid peak)," a total of 23 characteristic peaks were identified, with the characteristic fingerprint peak area accounting for 85.82% to 91.42% of the total peak area. Therefore, the relative retention times were determined as follows: 0.267 (peak 1), 0.530 (peak 2), 0.624 (peak 3), 0.669 (peak 4), 0.955 (peak 5), 1.000 (peak 6), 1.073 (peak 7), 1.114 (peak 8), 1.198 (peak 9), 1.328 (peak 10), 1.514 (peak 11), 1.649 (peak 12), 1.82... Peaks 4 (13#), 1.951 (14#), 2.330 (15#), 2.569 (16#), 2.740 (17#), 2.902 (18#), 3.042 (19#), 3.131 (20#), 3.240 (21#), 3.404 (22#), and 3.443 (23#) are characteristic fingerprint peaks, with peak 6 being the reference peak, chlorogenic acid.

[0090] Table 6. Peak area data of fingerprint spectrum overlay for 13 batches of samples

[0091] Note: "-" represents blank content.

[0092] (3) Characteristic peak identification and assignment Preparation of single-herb medicinal slices, negative samples, and extract sample solutions: Both single-herb decoction pieces and negative samples were prepared into oral solutions using the corresponding amount of decoction pieces prescribed according to the preparation method in the pharmacopoeia standard of Compound Scutellaria and Ligusticum Oral Solution. For sample processing before testing, 1 ml of the corresponding sample was placed in a 10 ml volumetric flask, diluted to the mark with 50% methanol, shaken well, filtered, and the filtrate was collected.

[0093] The sample preparation method for the extract is as follows: accurately measure 4.950g of compound Scutellaria baicalensis extract, place it in a 100ml volumetric flask, dilute to the mark with 50% methanol, shake well, filter, and take the filtrate to obtain the extract.

[0094] Chromatographic peaks were identified using high-performance liquid chromatography-mass spectrometry (HPLC-MS) on single-herb medicinal slices, negative samples, extract samples, and the test solution prepared in Example 1. HPLC conditions are shown in Table 7, and ion source and scanning parameters for mass spectrometry are shown in Tables 8-9. Results are presented below. Figures 9-10 : Table 7 High Performance Liquid Chromatography Methods

[0095] Table 8 Ion source parameters

[0096] Table 9 Scanning Parameters

[0097] Data analysis methods: The precise relative molecular masses and secondary mass spectrometry fragments of each component were compared with the chemical components reported in the literature to identify the chemical components in Compound Scutellaria baicalensis and Scutellaria baicalensis oral liquid. The identification of some compounds was further confirmed by comparing the mass spectrometry data with the reference standard.

[0098] Based on the chemical composition and preparation process of each herb in the prescription, correlation analysis was conducted using fingerprint spectra of individual herbal pieces, negative samples, extracts, and test samples. The 23 characteristic peaks were assigned to specific herbs, clarifying their origins. The results are shown in Table 10.

[0099] Table 10. Analysis of the attribution of characteristic fingerprint peaks in fingerprint spectroscopy.

[0100] Note: "-" indicates that it was not detected.

[0101] (4) Selection of reference point Based on the correlation between medicinal slices, extracts, and preparations, and combined with a systematic study of pharmacodynamic characteristics, quality control standards, and preparation processes, this study selected peak 6 (chlorogenic acid peak) as the reference peak for the fingerprint spectrum of this product, and marked it with "S" in the spectrum. This peak has a moderate retention time, appropriate peak height, and stable elution in the fingerprint spectrum. At the same time, its components are derived from honeysuckle medicinal slices, the principal drug in the prescription, and are legally controlled indicators in the current quality standards. Therefore, the selection of the reference peak is reasonable and representative.

[0102] (5) Establishment of fingerprint profile In this study, based on the overall analysis of fingerprint chromatograms and related parameters of multiple batches of representative samples, the common pattern chromatogram generated by the "average method" in the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System was used as the control fingerprint chromatogram. (See attached image.) Figure 11 .

[0103] (6) Similarity evaluation The similarity of fingerprint spectra of different batches of samples was calculated and evaluated using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software from the National Pharmacopoeia Commission.

[0104] Results: The similarity between each batch and the control fingerprint was greater than 0.947. Detailed data are shown in Table 11.

[0105] Table 11. Similarity results of fingerprint spectra of 13 batches of samples

[0106] Experiment Example 2: Validation of Analytical Methods 1. Experimental Methods (1) Specificity detection Preparation of negative samples and blank excipient solutions: The negative sample was prepared into an oral solution using the corresponding amount of herbal pieces prescribed according to the preparation method in the pharmacopoeia standard of Compound Scutellaria and Ligusticum Oral Solution. Before testing, the sample was treated by taking 1 ml of the corresponding sample, placing it in a 10 ml volumetric flask, adding 50% methanol to dilute to the mark, shaking well, filtering, and taking the filtrate.

[0107] Preparation of blank excipient solution: Take 400ml of purified water, add 5.00g of steviol glycosides, boil for 15 minutes, cool to below 80℃, make up to 500ml, adjust the pH to 7.0 with 5% sodium hydroxide solution, stir for 30 minutes, coarsely filter with five filter papers (below 40℃); finely filter with five filter papers and a 0.8um filter membrane, fill, and sterilize at 105℃ for 40 minutes.

[0108] The test solution (B36250701) prepared in Example 1, the negative sample solution of Compound Scutellaria and Ligusticum Oral Solution (without Forsythia), the negative sample solution of Compound Scutellaria and Ligusticum Oral Solution (without Honeysuckle), the negative sample solution of Compound Scutellaria and Ligusticum Oral Solution (without Scutellaria), the negative sample solution of Compound Scutellaria and Ligusticum Oral Solution (without Isatis Root), the reference solution, and the blank excipient solution were tested according to the chromatographic conditions of Example 1.

[0109] (2) Precision testing Take 10 μL of the test solution (batch number: B36250701) prepared in Example 1 and the reference solution, and inject them continuously 6 times under the chromatographic conditions of Example 1. Record the chromatographic peaks within 60 min. Calculate the RSD values ​​of the relative retention time and relative peak area ratio of each characteristic fingerprint peak using the reference peak. Compare the similarity results to examine the instrument precision. The injection operation is shown in Table 12 below: Table 12 Precision Test Injection Sequence Table

[0110] (3) Repeatability test Take 10 μL of the test solution prepared in Example 1 (batch number: B36250701), prepare 6 parallel test solutions, and determine them according to the chromatographic conditions of Example 1. Record the chromatographic peaks within 60 min; calculate the RSD value of the relative retention time and relative peak area ratio of each characteristic fingerprint peak with reference peak, compare the similarity results, and examine the repeatability of the method. The injection operation is shown in Table 13 below: Table 13 Sample Injection Sequence List for Repeatability Tests

[0111] (4) Stability testing Take 10 μL of the test solution prepared in Example 1 (batch number: B36250701), place it at room temperature, and inject it for analysis at 0 h, 2 h, 4 h, 6 h, 8 h, and 12 h, respectively. Record the chromatographic peaks within 60 min. Calculate the RSD value of the relative retention time and relative peak area ratio of each characteristic fingerprint peak using the reference peak, compare the similarity results, and examine the stability of the solution. The injection operation is shown in Table 14 below: Table 14 Stability Test Injection Sequence Table

[0112] (5) Intermediate precision testing Different analysts prepared the test solutions on different dates using different instruments, and determined the fingerprints according to the chromatographic conditions specified in Example 1; similarity was used as the evaluation index to examine the effect of random events on the precision of the analytical method. Different analysts (Analyst 1 and Analyst 2) injected 10 μL of the test solution (batch number: B36250701) and reference solution prepared in Example 1 into the liquid chromatograph (Agilent, Waters, Thermo) according to the chromatographic conditions of Example 1, and recorded the chromatographic peaks within 60 min to examine the instrument precision. The injection operation is shown in Table 15 below: Table 15 Injection sequence for intermediate precision test

[0113] (6) Durability testing The study investigated varying factors including column temperature, flow rate, different batches of chromatographic columns, mobile phase phosphoric acid concentration, and detection wavelength, and determined fingerprints under the chromatographic conditions specified in Example 1; it also examined the ability of the analytical method to remain unaffected by minor but deliberate changes. Take 10 μL of the test solution prepared in Example 1 (batch number: B36250701) and inject it into the liquid chromatograph according to the chromatographic conditions of Example 1 for determination. Record the chromatographic peaks within 60 min. The injection operation is shown in Table 16 below: Table 16. Sample Injection Sequence for Durability Test

[0114] Effect of column temperature fluctuation: Keeping other chromatographic conditions constant, fingerprint chromatograms were determined by injection at column temperatures of 23℃, 25℃, and 27℃ respectively. The effect of column temperature fluctuation was examined using similarity as the evaluation index. That is, under different column temperatures, other conditions remained constant, with Method 1 at 23℃ and Method 2 at 27℃.

[0115] Effect of flow rate fluctuation: Keeping other chromatographic conditions constant, fingerprint chromatograms were determined by injection at column flow rates of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min, respectively. The effect of flow rate fluctuation was examined using similarity as the evaluation index. That is, under different flow rate conditions, other conditions remained constant, with 0.8 mL / min for Method 1 and 1.2 mL / min for Method 2.

[0116] Keeping other chromatographic conditions unchanged, 0.09%, 0.10%, and 0.11% phosphoric acid solutions were prepared as mobile phase B components, and fingerprint chromatograms were determined by injection. The effect of phosphoric acid concentration fluctuations was investigated using similarity as the evaluation index.

[0117] The effect of fluctuations in mobile phase phosphoric acid concentration: The effect of column batches: Keeping other chromatographic conditions constant, using the same packing material, different batches of chromatographic columns were injected and fingerprint chromatograms were determined; the effect of different batches of chromatographic columns was investigated using similarity as the evaluation index.

[0118] The effect of detection wavelength fluctuations: Keeping other chromatographic conditions unchanged, the detection wavelengths were set to 243 nm, 245 nm, and 247 nm, respectively, and the fingerprint chromatograms were determined by injection. The effect of detection fluctuations was examined using similarity as the evaluation index.

[0119] 2. Results Evaluation (1) Specificity detection results The fingerprinting method is not affected by solvents and excipients, and all 23 characteristic peaks are derived from the four medicinal slices in the prescription, indicating that the method has strong specificity.

[0120] (2) Precision test results When the same test solution was injected six times consecutively, the relative retention time RSD values ​​of the 23 common peaks and the reference peak S peak in the obtained fingerprint spectra were between 0.008% and 0.088%, all less than 3%. The RSD values ​​of the ratio of the area of ​​the common peak to the area of ​​the S peak were between 0.08% and 1.1%, all less than 3%. The similarity was 1, indicating good precision.

[0121] (3) Repeatability test results For 6 parallel samples, the relative retention time RSD values ​​of the 23 common peaks and the reference peak S peak in each fingerprint spectrum were between 0.017% and 0.097%, all less than 3%. The RSD values ​​of the ratio of the common peak area to the S peak area were between 0.16% and 1.88%, all less than 3%. The similarity was 1 for all samples, indicating good repeatability.

[0122] (4) Stability test results For the same test solution, after being placed at room temperature for 12 hours and then injected, the relative retention time RSD values ​​of the 23 common peaks and the S peak in the obtained fingerprint spectra were between 0 and 0.24%, all less than 3%, and the RSD values ​​of the ratio of the peak area of ​​the common peak to the peak area of ​​the S peak were between 0.06 and 1.66%, all less than 3%, with a similarity of 1. This indicates that the prepared test solution has good stability after being placed at room temperature for 12 hours.

[0123] (5) Intermediate precision test results The similarity between the 23 common peaks and the S peak in the fingerprint spectra obtained by different analysts (Analyst 1 and Analyst 2) on different dates and using different instruments was not less than 0.90, indicating that the intermediate precision of the method was good.

[0124] Table 17 Intermediate Precision Similarity Data Table

[0125] (6) Durability test results The experiment was conducted at a column temperature of 25±2℃. The similarity between the samples was greater than 0.90, indicating that the samples can withstand the influence of column temperature fluctuations.

[0126] The experiment was conducted at a column flow rate of 1.0 ± 0.2 mL / min. The similarity between samples was greater than 0.90, indicating that the method can withstand the influence of column flow rate fluctuations.

[0127] Using the same chromatographic column packing material, three chromatographic columns from different batches were tested. The similarity between the samples was greater than 0.90, indicating that the influence of the batch of chromatographic columns was relatively small.

[0128] The experiment was conducted using a phosphoric acid solution with a concentration of 0.1 ± 0.01% as mobile phase B. The similarity between samples was greater than 0.90, indicating that the sample can withstand the influence of fluctuations in the column phosphoric acid concentration.

[0129] The experiment was conducted at a detection wavelength of 245±2nm, and the similarity between samples was greater than 0.90, indicating that the sample can withstand the influence of detection wavelength fluctuations.

[0130] Table 18. Durability Similarity Data at Different Column Temperatures

[0131] Table 19. Durability Similarity Data for Different Flow Rates

[0132] Table 20. Durability Similarity Data for Chromatographic Columns of the Same Type but Different Batch Numbers

[0133] Table 21. Durability Similarity Data of Phosphoric Acid at Different Concentrations

[0134] Table 22 Durability Similarity Data for Different Wavelengths

[0135] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for constructing the fingerprint spectrum of Compound Scutellaria and Oryzae Oral Solution, characterized in that, The raw materials for preparing the compound Scutellaria and Scutellaria oral liquid include honeysuckle, Scutellaria baicalensis, Forsythia suspensa and Isatis indigotica root; The method for constructing the fingerprint map includes the following steps: A. Preparation of the test solution: Dilute Compound Scutellaria and Ligusticum Oral Solution with methanol, filter, and obtain the test solution; B. Preparation of the reference solution: Dissolve and dilute the reference substance in methanol, shake well, and obtain the reference substance solution; C. High-performance liquid chromatography (HPLC) determination: Inject the test solution and reference solution into the HPLC instrument separately and collect data; The high-performance liquid chromatography (HPLC) conditions were as follows: octadecylsilane-bonded silica gel was used as the packing material, methanol was used as mobile phase A, phosphoric acid solution was used as mobile phase B, the chromatographic column was ZORBAX Eclipse plus, and the detection wavelength was 245±2nm.

2. The construction method according to claim 1, characterized in that, The concentration of methanol mentioned in both steps A and B is 50%; the reference substance mentioned in step B is chlorogenic acid standard.

3. The construction method according to claim 1, characterized in that, The chromatographic conditions in step C of the high-performance liquid chromatography determination also include gradient elution, with the following conditions: From 0 to 10 min, the volume percentage of mobile phase A increased from 15% to 25%, while the volume percentage of mobile phase B decreased from 85% to 75%. From 10 min to 15 min, the volume percentage of mobile phase A increased from 25% to 30%, while the volume percentage of mobile phase B decreased from 75% to 70%. From 15 min to 20 min, the volume percentage of mobile phase A increased from 30% to 33%, while the volume percentage of mobile phase B decreased from 70% to 67%. From 20 min to 35 min, the volume percentage of mobile phase A was 33%, and the volume percentage of mobile phase B was 67%. From 35 min to 50 min, the volume percentage of mobile phase A increased from 33% to 65%, while the volume percentage of mobile phase B decreased from 67% to 35%. From 50 min to 60 min, the volume percentage of mobile phase A increased from 65% to 100%, while the volume percentage of mobile phase B decreased from 35% to 0%.

4. The construction method according to claim 1, characterized in that, The column temperature for the high performance liquid chromatography conditions described in step C is 23-27℃.

5. The construction method according to claim 1, characterized in that, The data acquisition time for the chromatographic conditions described in step C is 60 minutes.

6. The construction method according to claim 1, characterized in that, The concentration of the phosphoric acid solution mentioned in step C is 0.1 ± 0.01%.

7. The construction method according to claim 6, characterized in that, The concentration of the phosphoric acid solution mentioned in step C is 0.1%.

8. The construction method according to claim 1, characterized in that, The fingerprint spectrum of the compound Scutellaria baicalensis and Scutellaria baicalensis oral liquid has 23 common characteristic peaks, with peak 6 being the reference peak (chlorogenic acid peak), and the corresponding peak being peak S. The relative retention times are as follows: 0.267 (peak 1), 0.530 (peak 2), 0.624 (peak 3), 0.669 (peak 4), 0.955 (peak 5), 1.000 (peak 6), 1.073 (peak 7), 1.114 (peak 8), 1.198 (peak 9), 1.328 (peak 10), 1.514 (peak 11), 1.649 (peak 12), 1.824 (peak 13), 1.951 (peak 14), 2.330 (peak 15), 2.569 (peak 16), 2.740 (peak 17), 2.902 (peak 18), 3.042 (peak 19), 3.131 (peak 20), 3.240 (peak 21), 3.404 (peak 22), 3.443 (peak 23).

9. The application of the construction method according to any one of claims 1-8 in the quality testing of Compound Scutellaria baicalensis and Scutellaria baicalensis oral liquid.

10. A quality testing method for Compound Scutellaria and Oryzae Oral Solution, characterized in that, The method includes the step of comparing the fingerprint spectrum of the Compound Scutellaria and Ligusticum Oral Solution to be tested with the control fingerprint spectrum of the Compound Scutellaria and Ligusticum Oral Solution; the fingerprint spectrum of the Compound Scutellaria and Ligusticum Oral Solution to be tested is constructed using the Compound Scutellaria and Ligusticum Oral Solution to be tested according to the construction method described in any one of claims 1-8.

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