Establishment method of Chinese olive pill HPLC (High Performance Liquid Chromatography) fingerprint spectrum and fingerprint spectrum thereof

Abstract

The invention relates to the technical field of medicine analysis, in particular to a method for establishing a Chinese olive pill HPLC fingerprint spectrum and the fingerprint spectrum of the Chinese olive pill. According to the method, time and reagent cost are saved through repeated exploration, and component extraction efficiency, chromatographic peak separation degree, characteristic peak information amount, chromatographic peak shape symmetry and method environmental protection performance are comprehensively considered; finally, 20 mL of 70% ethyl alcohol is selected, heating reflux is conducted for 2 h to serve as the optimal extraction condition, acetonitrile-water (containing 0.1% formic acid) serves as a mobile phase, gradient elution is conducted, the detection wavelength is 280 nm, the column temperature is 28 DEG C, and the flow speed is 0.8 mL.min <-1 >, the method combining the Chinese olive pill HPLC fingerprint spectrum, content determination and the chemometrics technology is established, the method has the advantages of being easy and convenient to operate, accurate in result and high in repeatability, and the method is suitable for industrial production. The method can be effectively applied to quality evaluation of Chinese olive pills.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis, and particularly relates to a method for establishing an HPLC fingerprint of Qingguo Pills and the fingerprint thereof. Background Art

[0002] The throat is the gateway of the lungs and is also known as the "fortress" of health. "Ling Shu Xuan Jie" also states: "The throat is the path for water and grains. The larynx is where qi goes up and down." As a traditional Chinese medicine compound preparation for clearing heat and relieving sore throat, Qingguo Pills are composed of eight Chinese herbs, namely, Chinese olive, honeysuckle flower, scutellaria root, rhizoma menispermi, dwarf lilyturf tuber, figwort root, white peony root, and platycodon root. In the formula, Chinese olive detoxifies and relieves sore throat, and promotes the production of body fluid to moisten the throat, serving as the monarch drug. Honeysuckle flower clears heat and detoxifies, and disperses wind-heat; scutellaria root clears the lungs and purges fire, and detoxifies and relieves sore throat; rhizoma menispermi clears heat and detoxifies, and relieves sore throat and pain. The combination of these three herbs serves as the ministerial drugs. Dwarf lilyturf tuber, figwort root, and white peony root can nourish yin and reduce fire, detoxify and disperse nodules, and moisten the throat and relieve sore throat; platycodon root disperses the lung qi and relieves sore throat, opens the voice, and also can carry the drugs upward to reach the affected area, serving as the adjuvant and guiding drugs. Their compatibility has the effects of clearing heat and relieving sore throat, and reducing swelling and pain. Clinically, it is commonly used to treat redness and swelling of the pharynx, sore throat, aphonia, dry cough with little sputum, and dry mouth and tongue caused by the accumulation of heat in the lungs and stomach, and has good anti-inflammatory, antibacterial, antiviral and other effects. Clinical practice in recent years has proved that this drug has good curative effects on acute pharyngitis, acute laryngitis, tonsillitis, bronchitis, etc.

[0003] As is well known to those skilled in the art, the effect of a traditional Chinese medicine compound preparation is related to the compatibility between drugs, and is more closely related to the pharmacodynamic effects of each drug itself. Each herb may determine the effect of the entire formula. However, the current quality standard of "Chinese Pharmacopoeia" for Qingguo Pills only uses baicalin as a single quality control index, which is difficult to comprehensively reflect the overall content of multiple active ingredients in the compound and the stability between batches, and may lead to insufficient correlation between quality evaluation and clinical efficacy.

[0004] The fingerprint of traditional Chinese medicine can reveal in detail the chemical composition information in the sample, and the chemical pattern recognition technology can be used to deeply analyze the differences of common peaks in the sample. By establishing a characteristic common peak map and measuring the content of key components, the integrity and uniformity of the chemical components of the preparation can be systematically evaluated; further combined with chemical pattern recognition, the quality difference markers of different production batches or manufacturers can be accurately identified, providing data support for the optimization of quality standards. Although there have been research reports on the single content determination methods of baicalin and gallic acid in Qingguo Pills, the systematic analysis of the multiple active ingredients in the whole formula and the overall quality evaluation system based on chemical pattern recognition have not been perfected.

[0005] In view of the above technical problems, the present invention uses HPLC chromatography technology to establish a characteristic fingerprint of Qingguo Pills, and determines the contents of isochlorogenic acid A, isochlorogenic acid C, baicalin, baicalein, dauricine, harpagide, paeoniflorin, gallic acid, luteoloside, chlorogenic acid, and wogonin. Combining chemical pattern recognition analysis, by systematically analyzing the characteristic distribution law of the chemical components of Qingguo Pills, the marker components causing the quality fluctuations of multiple batches of Qingguo Pills are accurately identified, providing reliable technical support for improving the quality standard of Qingguo Pills and the research on the material basis of drug efficacy. Summary of the Invention

[0006] The primary object of the present invention is to provide an HPLC method for detecting Qingguo Pills. The method is detected using a high-performance liquid chromatograph, and the chromatographic conditions are as follows: Chromatographic column: Eclipse Plus C 18 , 250 mm × 4.6 mm, 5μm; Mobile phase: acetonitrile - water containing 0.1% formic acid, methanol - water containing 0.1% formic acid, acetonitrile - water containing 0.1% phosphoric acid, or acetonitrile - water containing 0.2% formic acid; The gradient elution conditions are as follows: 0 - 20 min, 2% - 20% acetonitrile; 20 - 35 min, 20% - 30% acetonitrile; 35 - 40 min, 30% - 37% acetonitrile; 40 - 43 min, 37% - 40% acetonitrile; 43 - 46 min, 40% - 44% acetonitrile; 46 - 50 min, 44% - 60% acetonitrile; 50 - 60 min, 60% - 64% acetonitrile; 60 - 70 min, 64% - 2% acetonitrile; Flow rate: 0.8 mL·min -1 ~1.0 mL·min -1 ; Column temperature: 28°C - 30°C; Detection wavelength: 210 nm ~ 327 nm; Injection volume: 10 μl.

[0007] Preferably, the mobile phase: acetonitrile - water containing 0.1% formic acid.

[0008] Preferably, the flow rate is 0.8 mL·min -1 .

[0009] Preferably, the detection wavelength is 280 nm ~ 327 nm.

[0010] The second object of the present invention is to provide a method for establishing an HPLC fingerprint of Qingguo Pills. The method includes: (1) Preparation of reference solution: Weigh each reference substance precisely, place it in a volumetric flask, dissolve it with methanol, and make up the volume to obtain a reference solution containing standard components with a predetermined concentration, and store it for future use; (2) Preparation of test solution: Precisely measure a sample, mix it with 20 - 40 ml of extraction solvent, extract, let it stand and cool, filter, and take the subsequent filtrate to obtain the test solution; (3) HPLC detection: Detect the test solution and reference solution in the above steps (1) and (2) by the HPLC method for detecting Qingguo Pills to obtain the fingerprint chromatogram of the test solution and the fingerprint chromatogram of the reference solution; (4) Generating a standard fingerprint: Generate a standard fingerprint based on the fingerprint chromatogram of the test solution, select the chromatographic peaks of the main components in Qingguo Pills with good resolution as characteristic peaks and determine them as common peaks, and identify the common peaks.

[0011] Preferably, the extraction solvent in step (2) is water, 70% ethanol, 95% ethanol, 50% methanol or absolute methanol, and the volume is 20 - 40 ml.

[0012] Preferably, the extraction method in step (2) is heating under reflux extraction or ultrasonic extraction, and the extraction time is 30 min - 2 h.

[0013] The third object of the present invention is to provide a method for detecting the quality of Qingguo Pills products, including: obtaining the fingerprint chromatogram of Qingguo Pills products by using the method for establishing the HPLC fingerprint chromatogram of Qingguo Pills described above; comparing the obtained fingerprint chromatogram of Qingguo Pills products with the standard fingerprint chromatogram, if the similarity is greater than 0.9, it indicates that the quality of the Qingguo Pills products is qualified.

[0014] The fourth object of the present invention is to provide the application of the method for establishing the HPLC fingerprint chromatogram of Qingguo Pills in detecting and identifying Qingguo Pills.

[0015] The fifth object of the present invention is to provide the fingerprint chromatogram obtained by the method for establishing the HPLC fingerprint chromatogram of Qingguo Pills.

[0016] The beneficial effects of the present invention are: (1) The present invention provides a method for HPLC detection and fingerprint chromatogram of Qingguo Pills. The method is obtained through repeated exploration, combined with saving time and reagent costs, and comprehensively considering the extraction efficiency of components, chromatographic peak resolution, information content of characteristic peaks, symmetry of chromatographic peak shapes, and environmental protection performance of the method. Finally, 20 mL of 70% ethanol and heating under reflux for 2 h are selected as the optimal extraction conditions. The chromatographic conditions are determined as acetonitrile - 0.1% formic acid water as the mobile phase, gradient elution, detection wavelength of 280 nm, column temperature of 28 °C, and flow rate of 0.8 mL·min -1 Flow rate.

[0017] (2) In the fingerprint spectrum method described above, the relative standard deviation (RSD) of the peak areas of all common peaks is less than 2.89%, and the RSD of the relative retention times is lower than 0.075%, indicating that the instrument has good precision. The RSD of the peak areas is less than 2.96%, and the RSD of the relative retention times is lower than 0.087%, indicating that the method has good repeatability. The RSD of the peak areas is less than 2.84%, and the RSD of the relative retention times is lower than 0.076%, indicating that the sample solution has good stability within 24 h and good batch-to-batch consistency.

[0018] (3) The chromatographic peaks of each component are well separated and symmetrical, and match the reference substance, proving that the method has strong specificity and can accurately and efficiently analyze and identify components; the linear relationships of 11 components are good. The RSDs of the peak areas of gallic acid, luteoloside, isochlorogenic acid A, isochlorogenic acid C, baicalin, baicalein, dauricine, harpagoside, paeoniflorin, chlorogenic acid, and wogonin are 2.41%, 2.68%, 0.13%, 2.84%, 1.44%, 1.80%, 1.45%, 2.79%, 0.61%, 0.34%, and 0.27% respectively, indicating that the instrument has good precision.

[0019] The RSDs of the peak areas of gallic acid, luteoloside, isochlorogenic acid A, isochlorogenic acid C, baicalin, baicalein, dauricine, harpagoside, paeoniflorin, chlorogenic acid, and wogonin are 1.75%, 2.74%, 0.16%, 2.56%, 1.68%, 2.34%, 2.76%, 2.83%, 1.32%, 0.36%, and 0.34% respectively. The results show that the method has good repeatability.

[0020] The RSDs of the peak areas of gallic acid, luteoloside, isochlorogenic acid A, isochlorogenic acid C, baicalin, baicalein, dauricine, harpagoside, paeoniflorin, chlorogenic acid, and wogonin are 2.14%, 2.68%, 1.56%, 2.81%, 2.30%, 1.39%, 1.92%, 2.87%, 2.58%, 1.67%, and 0.93% respectively. The results show that the method has good stability within 24 h.

[0021] (4) Based on the similarity analysis of the HPLC fingerprints of 22 batches of Qingguo Pills, the overall similarity range among batches was 0.949 - 1.000, indicating a high quality consistency among production batches. Among them, the similarity between batches from manufacturers Q1 and Q3 was higher than 0.990, approaching 1.000, indicating high product quality stability; the similarity of batches from manufacturers Q2 and Q4 was between 0.970 - 0.995, showing good overall consistency but slightly lower than the first category; the similarity of batches from manufacturer Q5 was slightly lower than the previous two categories, which might be related to factors such as differences in the origin of raw medicinal materials used, changes in harvesting time, differences in storage conditions, fluctuations in extraction process parameters of the production process, differences in processing methods, and deviations in the implementation of quality control standards. Using chemometric methods for quality analysis of 22 batches of Qingguo Pills, the results showed that the samples could be divided into 4 clusters, and the classification results were correlated with the production manufacturers. OPLS-DA analysis identified 16 key differential markers with VIP > 1.0, mainly flavonoids, organic acids, and alkaloids. The content distribution results showed that the main active ingredients in Qingguo Pills from 5 manufacturers were paeoniflorin and baicalin, which were higher than other components, probably related to their high proportion of raw medicinal materials and high solubility characteristics in the water extraction process.

[0022] (5) The present invention established an HPLC fingerprint of Qingguo Pills, which has strong specificity, is accurate and reliable, and the pretreatment of the test solution is simple. According to the principle of Q-Marker "component measurability", it provides a reference basis for comprehensively evaluating the effective quality control of Qingguo Pills and can be used to control the quality of Qingguo Pills. Description of the Drawings

[0023] Figure 1 Chromatogram of the screening of gradient elution methods Note: From top to bottom are the chromatograms of gradient elution methods (1), (2), and (3) Figure 2 Chromatograms of different extraction solvents Note: From top to bottom are the chromatograms of extraction with 95% ethanol, 70% ethanol, water extraction, 50% methanol extraction, and 100% methanol extraction Figure 3 Chromatograms of different extraction methods Note: From top to bottom are the chromatograms of reflux extraction and ultrasonic extraction Figure 4 Chromatograms of extraction with different reflux times Note: From top to bottom are the chromatograms of reflux for 30 min, 1 h, and 2 h Figure 5 Chromatograms of extraction with different volumes Note: From top to bottom are the chromatograms with extraction volumes of 20 mL, 30 mL, and 40 mL Figure 6Chromatograms of different mobile phase systems Note: From top to bottom are chromatograms of methanol - water (containing 0.1% formic acid), acetonitrile - water (containing 0.1% phosphoric acid), acetonitrile - water (containing 0.2% formic acid), and acetonitrile - water (containing 0.1% formic acid). Figure 7 Chromatograms at different column temperatures Note: The upper figure is the chromatogram at a column temperature of 28 °C, and the lower figure is the chromatogram at a column temperature of 30 °C. Figure 8 Chromatograms at different flow rates Note: The upper figure is the chromatogram at a flow rate of 0.8 mL·min -1 chromatogram, and the lower figure is the chromatogram at a flow rate of 1 mL·min -1 chromatogram Figure 9 Chromatograms at different wavelengths Note: From top to bottom are chromatograms at wavelengths of 210 nm, 230 nm, 280 nm, and 327 nm. Figure 10 HPLC fingerprint chromatogram (A) and reference fingerprint chromatogram (B) of Qingguo Pills Note: 2 - gallic acid; 4 - chlorogenic acid; 8 - menisperine; 9 - paeoniflorin; 12 - luteoloside; 15 - isochlorogenic acid A; 16 - isochlorogenic acid C; 18 - baicalin; 20 - harpagoside; 27 - baicalein; 28 - wogonin Figure 11 Cluster analysis (CA) diagram of Qingguo Pills samples Figure 12 Principal component score diagram of 22 batches of Qingguo Pills samples Figure 13 Orthogonal partial least squares discriminant analysis (OPLS - DA) score diagram of Qingguo Pills samples Figure 14 OPLS - DA permutation test diagram of Qingguo Pills samples Note: The number of permutations is 200 times Figure 15 OPLS - DA VIP diagram of Qingguo Pills Figure 16 HPLC chromatogram of Qingguo Pills samples and mixed reference solution Note: A - mixed reference; B - sample; 2 - gallic acid; 4 - chlorogenic acid; 8 - menisperine; 9 - paeoniflorin; 12 - luteoloside; 15 - isochlorogenic acid A; 16 - isochlorogenic acid C; 18 - baicalin; 20 - harpagoside; 27 - baicalein; 28 - wogonin Figure 17 HPLC chromatogram of negative sample solution of Qingguo Pills Note: A - lack of Menispermi Rhizoma; B - lack of Scutellariae Radix; C - lack of Lonicerae Japonicae Flos; D - lack of Canarii Fructus; E - lack of Scrophulariae Radix; F - lack of Paeoniae Radix Alba; 2 - gallic acid; 4 - chlorogenic acid; 8 - dauricine; 9 - paeoniflorin; 12 - luteoloside; 15 - isochlorogenic acid A; 16 - isochlorogenic acid C; 18 - baicalin; 20 - harpagoside; 27 - baicalein; 28 - wogonin Figure 18 Radar chart of the contents of 11 components in Qingguo Pills samples Specific embodiments

[0024] The present invention will be further described in detail below in conjunction with test examples and specific embodiments. However, it should not be understood that the scope of the above-mentioned subject matter of the present invention is limited to the following embodiments. Any technology implemented based on the content of the present invention belongs to the scope of the present invention.

[0025] It should be noted that in the following embodiments, unless otherwise specified, the methods described are conventional methods, and the reagents can be obtained from commercial sources.

[0026] Based on the 2025 edition of the Chinese Pharmacopoeia and related research, Qingguo Pills have the effects of clearing heat and relieving sore throat, and reducing swelling and alleviating pain. Canarii Fructus contains various organic acids and flavonoid compounds, which have anti-inflammatory and antibacterial effects; Lonicerae Japonicae Flos mainly contains components such as chlorogenic acid, luteoloside, isochlorogenic acid A, and isochlorogenic acid C, which have broad-spectrum antibacterial, antiviral, and antipyretic effects; Baicalin, baicalein, and wogonin are the main active components of Scutellariae Radix, which have anti-inflammatory, antioxidant, and antipyretic effects; Menispermi Rhizoma contains various alkaloids, which have antibacterial, antiviral, antitumor, and anti-inflammatory effects; iridoid glycosides (harpagoside) in Scrophulariae Radix have the effects of clearing heat and cooling blood, and detoxifying; Paeoniae Radix Alba is rich in paeoniflorin, which has anti-inflammatory, analgesic, and antispasmodic effects. Based on the "component - efficacy" correlation principle, the present invention selects the above-mentioned index components for content determination. This multi-index component quantitative model can objectively reflect the chemical substance basis of Qingguo Pills and ensure that the quality control method is highly consistent with its traditional efficacy.

[0027] Example 1. Optimization of the method for detecting the HPLC fingerprint of Qingguo Pills 1 Instruments and reagents 1.1 Instruments Waters e2695 high performance liquid chromatograph (Waters Corporation, USA); temperature-controlled electric heating mantle (Beijing Kewi Yongxing Instrument Co., Ltd.); XS205DU electronic balance (Mettler-Toledo AG, Switzerland); Centrifuge5810R centrifuge (Eppendorf AG); energy-saving intelligent constant temperature bath (Ningbo Xinzhi Biotechnology Co., Ltd.); QE-100 high-speed crusher (Zhejiang Yili Industry and Trade Co., Ltd.); TYXH-I vortex oscillator (Shanghai Hanno Instrument Co., Ltd.).

[0028] 1.2 Drugs and Reagents

[0029] Reference substance isochlorogenic acid A (batch number: 17474, 94.6%); reference substance isochlorogenic acid C (batch number: 16660-S240901, 90.5%); reference substance baicalin (batch number: 12676-S241001, 97.1%); reference substance baicalein (batch number: 16645, 97.6%); reference substance dauricine (batch number: 14721, 99.7%); reference substance harpagoside (batch number: 15144, 96.2%); reference substance paeoniflorin (batch number: 18889-S240701, 96.8%); reference substance gallic acid (batch number: 16715, 98%); reference substance luteoloside (batch number: 111720-201810, 93.5%); reference substance chlorogenic acid (batch number: 170753-202018, 98%); reference substance wogonin (batch number: 15304, 99.1%), all purchased from Shanghai Standard Technology Service Co., Ltd. The sample information of Qingguo Pills is shown in Table 1 (specification: 8 g per bag). Acetonitrile (chromatographic pure), purified water is from Watson, and the rest of the reagents are of analytical grade.

[0030] Table 1 Sample Information of Qingguo Pills

[0031] 2. Preparation of Solutions 2.1 Reference Substance Solution Accurately weigh appropriate amounts of reference substances isochlorogenic acid A, isochlorogenic acid C, baicalin, baicalein, dauricine, harpagoside, paeoniflorin, gallic acid, luteoloside, chlorogenic acid, and wogonin, respectively, in 10 mL volumetric flasks, dissolve with methanol and dilute to the mark to prepare single reference substance solutions with mass concentrations of 0.9432 mg·mL -1 、0.9150 mg·mL -1 、0.9671mg·mL -1 、1.0221 mg·mL -1 、0.986 mg·mL -1 、0.9328 mg·mL -1 、1.4065 mg·mL -1 、1.0486mg·mL -1 、0.0906 mg·mL -1 、0.1333 mg·mL -1 、0.9583 mg·mL -1 respectively.

[0032] 2.2 Test sample solution Take an appropriate amount of Qingguo Pills sample, crush it, take about 1 g, weigh accurately, add 20 ml of 70% ethanol solution, weigh, heat under reflux for 2 h, let it stand at room temperature and weigh again, make up the weight with 70% ethanol solution, centrifuge (8000 r·min -1 , 10 min), then take an appropriate amount of the supernatant, filter it through a 0.45 μm microporous membrane filter, discard the initial filtrate, and collect the subsequent filtrate for standby.

[0033] 2.3 Negative sample solution According to the method under "2.2", and in accordance with the prescription process and proportion requirements, prepare 8 negative samples lacking Fructus Canarii, Flos Lonicerae, Rhizoma Menispermi, Radix Scutellariae, Radix Ophiopogonis, Radix Paeoniae Alba, Radix Platycodonis, and Radix Scrophulariae respectively, and that's it.

[0034] 3. Optimization of chromatographic conditions 3.1 Selection of gradient elution method (1) Chromatographic conditions: Eclipse Plus C 18 Chromatographic column (4.6 mm×250 mm, 5 μm); mobile phase is acetonitrile - 0.1% formic acid water, gradient elution: 0 - 5 min, 2% - 12% acetonitrile; 5 - 15 min, 12% - 20% acetonitrile; 15 - 24 min, 20% - 28% acetonitrile; 24 - 34 min, 28% - 35% acetonitrile; 34 - 45 min, 35% - 47% acetonitrile; 45 - 52 min, 47% - 58% acetonitrile; 52 - 58 min, 58% - 4% acetonitrile; 58 - 60 min, 4% - 2% acetonitrile; flow rate 0.8 mL·min -1 , column temperature 28°C, injection volume 10 μL, detection wavelength 280 nm.

[0035] (2) Chromatographic conditions: Eclipse Plus C18 chromatographic column (4.6 mm×250 mm, 5 μm); mobile phase is acetonitrile - 0.1% formic acid water, gradient elution: 0 - 10 min, 2% - 12% acetonitrile; 10 - 30 min, 12% - 20% acetonitrile; 30 - 38 min, 20% - 28% acetonitrile; 38 - 50 min, 28% - 35% acetonitrile; 50 - 53 min, 35% - 55% acetonitrile; 53 - 58 min, 55% - 58% acetonitrile; 58 - 65 min, 58% - 60% acetonitrile; 65 - 70 min, 60% - 2% acetonitrile; flow rate 0.8 mL·min -1 , column temperature 28°C, injection volume 10 μL, detection wavelength 280 nm.

[0036] (3) Chromatographic conditions: Eclipse Plus C 18Chromatographic column (4.6 mm×250 mm, 5 μm); the mobile phase was acetonitrile - 0.1% formic acid in water, gradient elution: 0 - 20 min, 2% - 20% acetonitrile; 20 - 35 min, 20% - 30% acetonitrile; 35 - 40 min, 30% - 37% acetonitrile; 40 - 43 min, 37% - 40% acetonitrile; 43 - 46 min, 40% - 44% acetonitrile; 46 - 50 min, 44% - 60% acetonitrile; 50 - 60 min, 60% - 64% acetonitrile; 60 - 70 min, 64% - 2% acetonitrile; flow rate 0.8 mL·min -1 , column temperature 28°C, injection volume 10 μL, detection wavelength 280 nm.

[0037] The chromatogram of the gradient elution method screening was as shown in Figure 1 . The method of (1) was affected by the composition of the mobile phase, gradient elution, flow rate, change of mobile phase polarity, etc., and failed to effectively separate the components in the test sample. The baseline of the chromatogram fluctuated greatly, and the peak shape was abnormal, so the gradient elution conditions needed to be readjusted. The method of (2) had insufficient resolution for the components of the test sample, the proportion of the mobile phase was not good, the chromatographic peaks were dense in the latter 20 min, and the number of chromatographic peaks was small in the first 50 min, so the elution conditions needed to be further adjusted. The method of (3) effectively improved the baseline stability and peak resolution of the chromatogram by adjusting the elution gradient. Therefore, the method of (3) was selected as the final gradient elution scheme.

[0038] 3.2 Screening of different extraction solvents Chromatographic conditions: Eclipse Plus C 18 Chromatographic column (4.6 mm×250 mm, 5 μm); the mobile phase was acetonitrile - 0.1% formic acid in water, gradient elution: 0 - 20 min, 2% - 20% acetonitrile; 20 - 35 min, 20% - 30% acetonitrile; 35 - 40 min, 30% - 37% acetonitrile; 40 - 43 min, 37% - 40% acetonitrile; 43 - 46 min, 40% - 44% acetonitrile; 46 - 50 min, 44% - 60% acetonitrile; 50 - 60 min, 60% - 64% acetonitrile; 60 - 70 min, 64% - 2% acetonitrile; flow rate 0.8 mL·min -1 , column temperature 28°C, injection volume 10 μL, detection wavelength 280 nm.

[0039] Preparation of test sample solution: Take an appropriate amount of Qingguo Pills sample, grind it, take about 1 g, accurately weigh 5 portions, add 20 ml of 95% ethanol, 70% ethanol, water, 50% methanol or 100% methanol solution respectively, weigh, heat under reflux for 2 h, let it stand at room temperature and weigh again, make up with 70% ethanol solution, centrifuge (8000 r·min -1, after 10 min), an appropriate amount of the supernatant was aspirated, filtered through a 0.45 μm microporous membrane filter, the initial filtrate was discarded, and the subsequent filtrate was collected for standby.

[0040] As Figure 2 can be seen, when extracting with 95% ethanol, 50% methanol, and 100% methanol, the content of harpagoside is relatively low. When extracting with 50% methanol, the solvent peak is too high, interfering with the chromatographic peak. When extracting with water, the content of other components is low. Considering the above and combining with the reagent cost, 70% ethanol was finally selected as the best extraction solvent.

[0041] 3.3 Screening of different extraction methods Chromatographic conditions: Eclipse Plus C 18 Chromatographic column (4.6 mm × 250 mm, 5 μm); the mobile phase was acetonitrile - 0.1% formic acid water, gradient elution: 0 - 20 min, 2% - 20% acetonitrile; 20 - 35 min, 20% - 30% acetonitrile; 35 - 40 min, 30% - 37% acetonitrile; 40 - 43 min, 37% - 40% acetonitrile; 43 - 46 min, 40% - 44% acetonitrile; 46 - 50 min, 44% - 60% acetonitrile; 50 - 60 min, 60% - 64% acetonitrile; 60 - 70 min, 64% - 2% acetonitrile; flow rate 0.8 mL·min -1 , column temperature 28 °C, injection volume 10 μL, detection wavelength 280 nm.

[0042] Preparation of the test solution: Take an appropriate amount of Qingguo Pills sample, grind it, take about 1 g, accurately weigh it, add 20 ml of 70% ethanol solution, weigh it, heat under reflux for 2 h or extract by ultrasonic, let it stand at room temperature and weigh again, make up with 70% ethanol solution, centrifuge (8000 r·min -1 , after 10 min), an appropriate amount of the supernatant was aspirated, filtered through a 0.45 μm microporous membrane filter, the initial filtrate was discarded, and the subsequent filtrate was collected for standby.

[0043] The chromatograms of different extraction methods are as Figure 3 shown. When extracting by reflux, the contents of most components such as harpagoside, baicalein, and chlorogenic acid are higher than those by ultrasonic extraction, and there is a situation where the solvent peak is too high and interferes with the chromatographic peak during ultrasonic extraction. Therefore, the reflux extraction method was selected.

[0044] 3.4 Screening of extraction with different reflux times Chromatographic conditions: Eclipse Plus C 18Chromatographic column (4.6 mm × 250 mm, 5 μm); the mobile phase was acetonitrile - 0.1% formic acid water, gradient elution: 0 - 20 min, 2% - 20% acetonitrile; 20 - 35 min, 20% - 30% acetonitrile; 35 - 40 min, 30% - 37% acetonitrile; 40 - 43 min, 37% - 40% acetonitrile; 43 - 46 min, 40% - 44% acetonitrile; 46 - 50 min, 44% - 60% acetonitrile; 50 - 60 min, 60% - 64% acetonitrile; 60 - 70 min, 64% - 2% acetonitrile; flow rate 0.8 mL·min -1 , column temperature 28°C, injection volume 10 μL, detection wavelength 280 nm.

[0045] Preparation of test solution: Take an appropriate amount of Qingguo Pills sample, grind it, take about 1 g, accurately weigh 3 portions, add 20 ml of 70% ethanol solution, weigh, reflux for 30 min, 1 h or 2 h respectively by heating, let it stand at room temperature and weigh again, make up with 70% ethanol solution, centrifuge (8000 r·min -1 , 10 min), then take an appropriate amount of the supernatant, filter through a 0.45 μm microporous membrane, discard the initial filtrate, and collect the subsequent filtrate for standby.

[0046] Chromatograms extracted at different reflux times are as Figure 4 shown. When reflux extraction was carried out for 2 h, the contents of harpagide, baicalin, baicalein, chlorogenic acid, menisperine, isochlorogenic acid A, gallic acid, paeoniflorin, and luteoloside were all higher than those at reflux for 30 min and 1 h. Therefore, the reflux extraction time was selected as 2 h.

[0047] 3.5 Screening of different extraction volumes Chromatographic conditions: Eclipse Plus C 18 Chromatographic column (4.6 mm × 250 mm, 5 μm); the mobile phase was acetonitrile - 0.1% formic acid water, gradient elution: 0 - 20 min, 2% - 20% acetonitrile; 20 - 35 min, 20% - 30% acetonitrile; 35 - 40 min, 30% - 37% acetonitrile; 40 - 43 min, 37% - 40% acetonitrile; 43 - 46 min, 40% - 44% acetonitrile; 46 - 50 min, 44% - 60% acetonitrile; 50 - 60 min, 60% - 64% acetonitrile; 60 - 70 min, 64% - 2% acetonitrile; flow rate 0.8 mL·min -1 , column temperature 28°C, injection volume 10 μL, detection wavelength 280 nm.

[0048] Preparation of test solution: Take an appropriate amount of Qingguo Pills sample, grind it, take about 1 g, accurately weigh 3 portions, add 20 ml, 30 ml or 40 ml of 70% ethanol solution respectively, weigh, heat under reflux for 2 h, let it stand at room temperature and weigh again, make up with 70% ethanol solution, centrifuge (8000 r·min -1 , 10 min), then take an appropriate amount of the supernatant, filter through a 0.45 μm microporous membrane, discard the initial filtrate, and collect the subsequent filtrate for standby.

[0049] The chromatograms extracted with different extraction volumes are as Figure 5 shown. The extraction effects of 20 mL, 30 mL and 40 mL of solvents are not very different. To save actual costs, the extraction solvent volume of 20 mL is selected.

[0050] 3.6 Screening of different mobile phase systems Chromatographic conditions: Eclipse Plus C 18 chromatographic column (4.6 mm×250 mm, 5 μm); the mobile phase is selected from acetonitrile - 0.1% formic acid water, methanol - 0.1% formic acid water, acetonitrile - 0.1% phosphoric acid water or acetonitrile - 0.2% formic acid water, gradient elution: 0 - 20 min, 2% - 20% acetonitrile; 20 - 35 min, 20% - 30% acetonitrile; 35 - 40 min, 30% - 37% acetonitrile; 40 - 43 min, 37% - 40% acetonitrile; 43 - 46 min, 40% - 44% acetonitrile; 46 - 50 min, 44% - 60% acetonitrile; 50 - 60 min, 60% - 64% acetonitrile; 60 - 70 min, 64% - 2% acetonitrile; flow rate 0.8 mL·min -1 , column temperature 28℃, injection volume 10 μL, detection wavelength 280 nm.

[0051] Preparation of test solution: The same as "2".

[0052] The chromatograms of different mobile phase systems are as Figure 6 shown. When the mobile phase is acetonitrile - 0.1% formic acid water, the resolution of chromatographic peaks is good, the peak shape of chromatographic peaks is better, and the baseline is relatively straight; when the mobile phase is methanol - 0.1% formic acid water, the resolution of chromatographic peaks is poor, and the chromatographic peaks are dense in the latter 20 min; when the mobile phase is acetonitrile - 0.1% phosphoric acid water or acetonitrile - 0.2% formic acid water, the response value of the solvent peak is too large and there is a certain interference. Therefore, acetonitrile - 0.1% formic acid water is selected for subsequent experiments.

[0053] 3.7 Screening of different column temperatures Chromatographic conditions: Eclipse Plus C 18Chromatographic column (4.6 mm×250 mm, 5 μm); the mobile phase was selected as acetonitrile - 0.1% formic acid water, gradient elution: 0 - 20 min, 2% - 20% acetonitrile; 20 - 35 min, 20% - 30% acetonitrile; 35 - 40 min, 30% - 37% acetonitrile; 40 - 43 min, 37% - 40% acetonitrile; 43 - 46 min, 40% - 44% acetonitrile; 46 - 50 min, 44% - 60% acetonitrile; 50 - 60 min, 60% - 64% acetonitrile; 60 - 70 min, 64% - 2% acetonitrile; flow rate 0.8 mL·min -1 , the column temperature was selected as 28 °C or 30 °C, the injection volume was 10 μL, and the detection wavelength was 280 nm.

[0054] Preparation of the test solution: same as "2".

[0055] Chromatograms at different column temperatures are as Figure 7 shown. The separation effects at 28 °C and 30 °C were the same. From the perspective of the compatibility of the experimental equipment, 28 °C avoided adverse effects on the detector due to excessive temperature, such as affecting the sensitivity or stability of the detector. Therefore, the column temperature was selected as 28 °C.

[0056] 3.8 Screening of different flow rates Chromatographic conditions: Eclipse Plus C 18 Chromatographic column (4.6 mm×250 mm, 5 μm); the mobile phase was selected as acetonitrile - 0.1% formic acid water, gradient elution: 0 - 20 min, 2% - 20% acetonitrile; 20 - 35 min, 20% - 30% acetonitrile; 35 - 40 min, 30% - 37% acetonitrile; 40 - 43 min, 37% - 40% acetonitrile; 43 - 46 min, 40% - 44% acetonitrile; 46 - 50 min, 44% - 60% acetonitrile; 50 - 60 min, 60% - 64% acetonitrile; 60 - 70 min, 64% - 2% acetonitrile; flow rate 0.8 mL·min -1 or 1 mL·min -1 , the column temperature was selected as 28 °C, the injection volume was 10 μL, and the detection wavelength was 280 nm.

[0057] Preparation of the test solution: same as "2".

[0058] Chromatograms at different flow rates are as Figure 8 shown. It can be seen that by reducing the flow rate, the resolution is better, the baseline of the chromatogram is flatter, and the stability is better. Therefore, the flow rate was determined to be 0.8 mL·min -1 for the analysis of samples.

[0059] 3.9 Screening of different wavelengths Chromatographic conditions: Eclipse Plus C 18 Chromatographic column (4.6 mm×250 mm, 5 μm); the mobile phase was selected as acetonitrile - 0.1% formic acid water, gradient elution: 0 - 20 min, 2% - 20% acetonitrile; 20 - 35 min, 20% - 30% acetonitrile; 35 - 40 min, 30% - 37% acetonitrile; 40 - 43 min, 37% - 40% acetonitrile; 43 - 46 min, 40% - 44% acetonitrile; 46 - 50 min, 44% - 60% acetonitrile; 50 - 60 min, 60% - 64% acetonitrile; 60 - 70 min, 64% - 2% acetonitrile; flow rate 0.8 mL·min -1 , column temperature was selected as 28 °C, injection volume 10 μL, detection wavelengths were selected as 210 nm, 230 nm, 280 nm or 327 nm.

[0060] Preparation of test solution: same as "2".

[0061] Results at different wavelengths were as Figure 9 shown. At wavelengths of 210 nm and 230 nm, the chromatogram baseline was unstable. At a wavelength of 327 nm, the number of peaks was less than that at 280 nm. Considering the peak appearance effects of each component comprehensively, a wavelength of 280 nm was selected.

[0062] In summary, in this example, the extraction solvents (water, 70% ethanol, 95% ethanol, 50% methanol, absolute methanol), extraction methods (ultrasonic, reflux), extraction times (30 min, 1 h, 2 h), extraction volumes (20 mL, 30 mL, 40 mL), wavelengths (210 nm, 230 nm, 280 nm, 327 nm), mobile phase systems (acetonitrile - 0.1% formic acid water, methanol - 0.1% formic acid water, acetonitrile - 0.1% phosphoric acid water or acetonitrile - 0.2% formic acid water), column temperatures (28 °C, 30 °C) and flow rates (0.8 mL·min -1 、1.0 mL·min -1 ) were investigated. Considering the component extraction efficiency, chromatographic peak resolution, characteristic peak information content, chromatographic peak shape symmetry, and method environmental protection performance comprehensively, 20 mL of 70% ethanol, heating under reflux for 2 h were finally selected as the optimal extraction conditions. The chromatographic conditions were determined as acetonitrile - 0.1% formic acid water as the mobile phase, gradient elution, detection wavelength of 280 nm, column temperature of 28 °C and flow rate of 0.8 mL·min -1 flow rate.

[0063] Example 2. Study on HPLC fingerprint of Qingguo Pills 1. Preparation of solutions 1.1 Reference substance solution Accurately weigh appropriate amounts of reference substances of isochlorogenic acid A, isochlorogenic acid C, baicalin, baicalein, dauricine, harpagoside, paeoniflorin, gallic acid, luteoloside, chlorogenic acid, and wogonin. Respectively place them in 10 mL volumetric flasks, dissolve with methanol and dilute to the mark to prepare single reference substance solutions with mass concentrations of isochlorogenic acid A, isochlorogenic acid C, baicalin, baicalein, dauricine, harpagoside, paeoniflorin, gallic acid, luteoloside, chlorogenic acid, and wogonin being 0.9432 mg·mL -1 、0.9150 mg·mL -1 、0.9671mg·mL -1 、1.0221 mg·mL -1 、0.986 mg·mL -1 、0.9328 mg·mL -1 、1.4065 mg·mL -1 、1.0486mg·mL -1 、0.0906 mg·mL -1 、0.1333 mg·mL -1 、0.9583 mg·mL -1 respectively.

[0064] 1.2 Test solution Take an appropriate amount of Qingguo Pills sample, crush it, take about 1 g, accurately weigh it, add 20 ml of 70% ethanol solution, weigh it, heat under reflux for 2 h, let it stand at room temperature and then weigh again, make up the weight with 70% ethanol solution, centrifuge (8000 r·min -1 , 10 min), then take an appropriate amount of the supernatant, filter it through a 0.45 μm microporous membrane, discard the initial filtrate, and collect the subsequent filtrate for standby.

[0065] 1.3 Negative sample solution According to the method under "1.2", prepare 8 negative samples lacking Qingguo, Flos Lonicerae, Rhizoma Menispermi, Radix Scutellariae, Radix Ophiopogonis, Radix Paeoniae Alba, Radix Platycodonis, and Radix Scrophulariae respectively according to the prescription process and proportion requirements.

[0066] 2. Chromatographic conditions Eclipse Plus C 18Chromatographic column (4.6 mm×250 mm, 5 μm); the mobile phase was acetonitrile - 0.1% formic acid water, gradient elution: 0 - 20 min, 2% - 20% acetonitrile; 20 - 35 min, 20% - 30% acetonitrile; 35 - 40 min, 30% - 37% acetonitrile; 40 - 43 min, 37% - 40% acetonitrile; 43 - 46 min, 40% - 44% acetonitrile; 46 - 50 min, 44% - 60% acetonitrile; 50 - 60 min, 60% - 64% acetonitrile; 60 - 70 min, 64% - 2% acetonitrile; flow rate 0.8 mL·min -1 , column temperature 28 °C, injection volume 10 μL, detection wavelength 280 nm.

[0067] 3. Fingerprint study 3.1 Precision test Accurately weigh 1 g of the powder of this product (sample number S2), prepare the solution according to the method under item "1.2", and inject samples continuously 6 times according to the chromatographic conditions in item "2". Using peak 4 (chlorogenic acid) as the reference peak, calculate the peak areas and relative retention times of each common peak.

[0068] The results showed that the relative standard deviations (RSDs) of the peak areas of each common peak were all less than 2.89%, and the RSDs of the relative retention times were all lower than 0.075%, indicating that the instrument had good precision.

[0069] 3.2 Repeatability test Accurately weigh 6 portions of the same batch of powder (S2), prepare the test solution according to the method under item "1.2", and determine it under the optimal chromatographic conditions, and record the chromatogram.

[0070] The results showed that the RSDs of the peak areas were all less than 2.96%, and the RSDs of the relative retention times were all lower than 0.087%, indicating that the method had good repeatability.

[0071] 3.3 Stability test Accurately weigh 1 g of the powder of this product (S2), prepare the test solution according to the method described under item "1.2", and determine it according to the chromatographic conditions in item "2" at 0, 2, 4, 8, 12, and 24 h respectively, and record the chromatogram.

[0072] The results showed that the RSDs of the peak areas were all less than 2.84%, and the RSDs of the relative retention times were all lower than 0.076%, indicating that the sample solution had good stability within 24 h.

[0073] 3.4 Establishment and similarity evaluation of HPLC fingerprint Twenty-two batches of samples (S1 - S22) were used to construct HPLC chromatograms according to the method described under "1.2". The obtained HPLC chromatographic data were imported into the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints. Using the chromatogram of S21 as the reference chromatogram, 29 characteristic common peaks (with high response values, symmetric peak shapes, and good resolution) were screened, and the reference characteristic chromatogram and its HPLC fingerprint chromatogram of Qingguo Pills were constructed.

[0074] As Figure 10 shown, by comparing with reference substances, 11 chromatographic peaks were successfully identified, including peak 2 as gallic acid, peak 4 as chlorogenic acid, peak 8 as dauricine, peak 9 as paeoniflorin, peak 12 as luteoloside, peak 15 as isochlorogenic acid A, peak 16 as isochlorogenic acid C, peak 18 as baicalin, peak 20 as harpagoside, peak 27 as baicalein, and peak 28 as wogonin. The similarities of each sample to the reference characteristic chromatogram were calculated to be 0.997, 0.997, 0.996, 0.998, 0.997, 0.996, 0.985, 0.998, 0.998, 0.999, 0.999, 0.999, 0.996, 0.997, 0.997, 0.998, 0.996, 0.995, 0.998, 0.972, 0.971, and 0.997, all > 0.90, indicating that the preparation has good batch - to - batch consistency.

[0075] 3.5 Chemical Pattern Recognition 3.5.1 Cluster Analysis The areas of 29 common peaks of 22 batches of Qingguo Pills were imported into SIMCA 14.1, and the Ward system clustering method was used to calculate the areas of each chromatographic peak ( Figure 11 ). Through cluster analysis of the samples, it was found that when the distance was 3, the samples could be divided into 4 categories: S11 was the first category, S2 - S6 was the second category, S20 - S22 was the third category, and the remaining batches were grouped into the fourth category. Although the previous similarity analysis showed that the similarity of each batch of fingerprint chromatograms was > 0.90, the results of cluster analysis revealed that there were still distinguishable quality differences among different batches. Such differences may stem from factors such as fluctuations in the origin of raw medicinal materials, changes in the harvesting season, minor deviations in production process parameters, and differences in storage conditions.

[0076] 3.5.2 Principal Component Analysis (PCA) Principal component analysis (PCA) was performed on the areas of 29 common peaks of 22 batches of Qingguo Pills samples. Using SPSS25.0 software, with the eigenvalue > 1 as the condition for extracting principal components, 5 principal component factors were obtained, and the cumulative variance contribution rate reached 90.25%.

[0077] The main components and variance contribution rates are shown in Table 2. The results of the rotated component matrix show that the chromatographic peaks numbered 5, 8 (dauricine), 9 (paeoniflorin), 10, 12 (luteoloside), 18 (baicalin), 19, 21, 22, and 24 in principal component 1, the chromatographic peaks numbered 1, 3, 26, 27 (baicalein), 28 (wogonin), and 29 in principal component 2, the chromatographic peaks numbered 2 (gallic acid), 11, and 16 (isochlorogenic acid C) in principal component 3, the chromatographic peaks numbered 7 and 20 (harpagide) in principal component 4, and the chromatographic peak numbered 13 in principal component 5 all have relatively high loading values. The PCA score plot analysis of the common peak areas was performed using SIMCA 14.1 software ( Figure 12 ), and the results showed that the 22 batches of samples presented 4 clustering trends, which was consistent with the previous clustering analysis results, confirming the existence of a stable quality difference pattern among different batches.

[0078] Table 2 Eigenvalues and variance contribution rates of the main components of Qingguo Pills samples

[0079] 3.5.3 Orthogonal partial least squares discriminant analysis (OPLS-DA) Through chemical pattern recognition methods, key components that significantly contribute to sample differences can be effectively screened out, thereby more comprehensively revealing the chemical characteristic differences among samples. An OPLS-DA model was established using SIMCA 14.1 software for the 29 common peak area data of 22 batches of Qingguo Pills samples. The cumulative interpretation ability parameters of the model R 2 X , R 2 Y were 0.879 and 0.908 respectively, and the prediction ability parameter Q 2 = 0.688, indicating good goodness of fit and prediction reliability of the model.

[0080] The OPLS-DA scores ( Figure 13 ) showed that the samples within the same group had high similarity, indicating good production process stability; the samples from different manufacturers showed an obvious separation trend, indicating a high quality difference among manufacturers. The results of 200 permutation tests ( Figure 14 ) showed that the model did not show overfitting and had good prediction ability. According to the variable importance in projection (VIP) analysis ( Figure 15), 16 peaks with VIP > 1.0 were screened out, including peak 24, peak 18, peak 29, peak 26, peak 22, peak 21, peak 19, peak 11, peak 5, peak 1, peak 16, peak 14, peak 8, peak 28, peak 25, peak 27. Among them, peak 8 is dauricine, peak 16 is isochlorogenic acid C, peak 18 is baicalin, peak 27 is baicalein, and peak 28 is wogonin, indicating that these 16 components can be used as potential markers for quality control.

[0081] Example 3. Content determination 1. Specificity test The test solution, reference solution, and negative sample solution were prepared according to the method in Example 2 and determined under the chromatographic conditions described in Example 2. As Figure 16 and Figure 17 shown, the results showed that the chromatographic peaks of each component were well separated and symmetric, and matched with the reference substance, proving that the method has strong specificity and can accurately and efficiently analyze and identify the components.

[0082] 2. Investigation of linear relationship Appropriately precise amounts of the above reference substances were taken and fixed volume in a 10 mL volumetric flask with methanol to prepare a mixed reference solution containing isochlorogenic acid A, isochlorogenic acid C, baicalin, baicalein, dauricine, harpagoside, paeoniflorin, gallic acid, luteoloside, chlorogenic acid, and wogonin with mass concentrations of 18.863 2, 9.149 6, 241.779, 30.363 4, 29.581, 2.796 5, 232.073 2, 18.874 8, 18.12, 10.662 4, 19.165 9 μg·mL -1 respectively. It was serially diluted and determined under the chromatographic conditions described in Example 2, and the areas of each peak were recorded. The standard curve between the concentration ( X ) and the peak area ( Y ) was plotted. The results showed that the correlation coefficient r ≥ 0.9981. As can be seen from Table 3, the 11 components had good linear relationships.

[0083] Table 3 Linear regression equations and linear ranges of 11 components in Qingguo Pills

[0084] 3. Precision test Prepare the mixed reference substance solution according to the method of Example 2, inject the sample 6 times, and measure that the RSDs of the peak areas of gallic acid, luteoloside, isochlorogenic acid A, isochlorogenic acid C, baicalin, baicalein, dauricine, harpagoside, paeoniflorin, chlorogenic acid, and wogonin are 2.41%, 2.68%, 0.13%, 2.84%, 1.44%, 1.80%, 1.45%, 2.79%, 0.61%, 0.34%, and 0.27% respectively, indicating that the instrument precision is good.

[0085] 4. Repeatability test Accurately weigh 6 portions of 1 g of Qingguo Pills powder (S2), prepare the sample solution according to the method and chromatographic conditions of Example 2, and measure that the RSDs of the peak areas of gallic acid, luteoloside, isochlorogenic acid A, isochlorogenic acid C, baicalin, baicalein, dauricine, harpagoside, paeoniflorin, chlorogenic acid, and wogonin are 1.75%, 2.74%, 0.16%, 2.56%, 1.68%, 2.34%, 2.76%, 2.83%, 1.32%, 0.36%, and 0.34% respectively. The results show that the method has good repeatability.

[0086] 5. Stability test Accurately weigh 1 g of Qingguo Pills sample powder (S2), according to the preparation method of Example 2, at room temperature, measure the samples at 0, 2, 4, 8, 12, and 24 h respectively according to the chromatographic conditions of Example 2. Measure that the RSDs of the peak areas of gallic acid, luteoloside, isochlorogenic acid A, isochlorogenic acid C, baicalin, baicalein, dauricine, harpagoside, paeoniflorin, chlorogenic acid, and wogonin are 2.14%, 2.68%, 1.56%, 2.81%, 2.30%, 1.39%, 1.92%, 2.87%, 2.58%, 1.67%, and 0.93% respectively. The results show that the method has good stability within 24 h.

[0087] 6. Recovery test Weigh 6 portions of Qingguo Pills powder (S2), each about 0.5 g, and add the corresponding reference substances to each portion according to a ratio of 1:1, that is, the amount of the reference substance is equal to the expected content in the sample. According to the method and chromatographic conditions in Example 2, calculate the recovery of each component based on the measured peak area, and the results are shown in Table 4.

[0088] Table 4 Results of the recovery test for 11 components in Qingguo Pills

[0089]

[0090]

[0091] 2.5.7 Determination of Sample Content According to the solution preparation method and chromatographic conditions in Example 2, 22 batches of Qingguo Pills samples (S1 - S22) were injected, and the contents were calculated. The results are shown in Table 5. The results showed that the average content of gallic acid in all samples was 0.4810 mg·g -1 , the average content of luteoloside was 0.8793 mg·g -1 , the average content of isochlorogenic acid A was 0.6707 mg·g -1 , the average content of isochlorogenic acid C was 0.3479 mg·g -1 , the average content of baicalin was 10.1044 mg·g -1 , the average content of baicalein was 1.0248 mg·g -1 , the average content of menisperine was 0.5759 mg·g -1 , the average content of harpagoside was 0.1283 mg·g -1 , the average content of paeoniflorin was 14.3848 mg·g -1 , the average content of chlorogenic acid was 0.2750 mg·g -1 , the average content of wogonin was 0.3965 mg·g -1 . The content radar chart was made using Origin21, as shown in Figure 18 .

[0092] Table 5 Determination Results of Each Component Content in Qingguo Pills

[0093] Note: The unit is mg·g -1 , n = 3 In summary, the present invention established a fingerprint analysis method for Qingguo Pills by HPLC, and determined the contents of 11 components, including gallic acid, chlorogenic acid, dauricine, paeoniflorin, luteoloside, isochlorogenic acid A, isochlorogenic acid C, baicalin, harpagoside, baicalein, and wogonin. By analyzing 22 batches of samples through chemical pattern recognition technology, the key markers affecting the quality differences among batches were identified, which could comprehensively reflect the chemical characteristics of the preparation. The comprehensive evaluation model of "fingerprint - multi - component quantification - pattern recognition" established in this study provided a reliable technical means for the quality control of Qingguo Pills, which was helpful to ensure the safety and effectiveness of clinical medication. Based on the similarity analysis of the HPLC fingerprints of 22 batches of Qingguo Pills samples, the overall similarity range among batches was 0.949 - 1.000, indicating a high quality consistency among production batches. Among them, the similarity between batches of manufacturers Q1 and Q3 was higher than 0.990, approaching 1.000, indicating a high stability of product quality; the similarity of manufacturers Q2 and Q4 was between 0.970 - 0.995, with good overall consistency but slightly lower than the first category; the similarity of manufacturer Q5 was slightly lower than the previous two categories, which might be related to factors such as the differences in the origin of raw medicinal materials used, the changes in harvesting time, the differences in storage conditions, as well as the fluctuations in extraction process parameters of the production process, the differences in processing methods, and the deviations in the implementation of quality control standards. The quality analysis of 22 batches of Qingguo Pills using chemometric methods showed that the samples could be divided into 4 clusters, and the classification results were correlated with the manufacturers. The OPLS - DA analysis identified 16 key differential markers with VIP > 1.0, mainly including flavonoids, organic acids, and alkaloids. The content distribution results showed that the main active components in Qingguo Pills of the 5 manufacturers were paeoniflorin and baicalin, which were higher than other components, probably related to their high proportion of raw medicinal materials and high solubility characteristics in the water extraction process.

Claims

1. An HPLC method for detecting Qingguo Pills, characterized in that, The described method is detected by a high performance liquid chromatograph, and the chromatographic conditions are as follows: Chromatographic column: Eclipse Plus C 18 , 250 mm × 4.6 mm, 5μm; Mobile phase: acetonitrile - water containing 0.1% formic acid, methanol - water containing 0.1% formic acid, acetonitrile - water containing 0.1% phosphoric acid, or acetonitrile - water containing 0.2% formic acid; The gradient elution conditions are as follows: 0 - 20 min, 2% - 20% acetonitrile; 20 - 35 min, 20% - 30% acetonitrile; 35 - 40 min, 30% - 37% acetonitrile; 40 - 43 min, 37% - 40% acetonitrile; 43 - 46 min, 40% - 44% acetonitrile; 46 - 50 min, 44% - 60% acetonitrile; 50 - 60 min, 60% - 64% acetonitrile; 60 - 70 min, 64% - 2% acetonitrile; Flow rate: 0.8 mL·min -1 ~1.0 mL·min -1 ; Column temperature: 28°C - 30°C; Detection wavelength: 210 nm - 327 nm; Injection volume: 10 μl.

2. The method according to claim 1, characterized in that, The described mobile phase: acetonitrile - water containing 0.1% formic acid.

3. The method according to claim 1, wherein The flow rate is 0.8 mL·min -1 .

4. The method according to claim 1, wherein The described detection wavelength is 280 nm - 327 nm.

5. A method for establishing an HPLC fingerprint of Qingguo Pills, characterized in that, The described method includes: (1) Preparation of the reference solution: Weigh each reference substance precisely, place it in a volumetric flask, dissolve it with methanol, and make up the volume to obtain a reference solution containing the standard components with a predetermined concentration, and store it for later use; (2) Preparation of the test solution: Precisely measure the sample, mix it with 20 - 40 ml of extraction solvent, extract, let it stand and cool, filter, and take the subsequent filtrate to obtain it; (3) HPLC detection: Detect the test solution and the reference solution in (1) and (2) above by the HPLC method for detecting Qingguo Pills according to Claim 1 to obtain the test fingerprint chromatogram and the reference fingerprint chromatogram; (4) Generate the standard fingerprint chromatogram: Generate the standard fingerprint chromatogram based on the test fingerprint chromatogram, select the chromatographic peaks with good separation of the main components in Qingguo Pills as the characteristic peaks and determine them as the common peaks, and identify the common peaks.

6. The method for establishing the HPLC fingerprint of Qingguo Pills according to claim 5, characterized in that, The extraction solvent in step (2) is water, 70% ethanol, 95% ethanol, 50% methanol, or absolute methanol, and the volume is 20 - 40 ml.

7. The method for establishing the HPLC fingerprint of Qingguo Pills according to claim 5, characterized in that, The extraction method in step (2) is heating under reflux extraction or ultrasonic extraction, and the extraction time is 30 min - 2 h.

8. A method for detecting the quality of Qingguo Pills, characterized in that, Includes: Use the method for establishing the HPLC fingerprint chromatogram of Qingguo Pills according to any one of Claims 5 - 7 to obtain the fingerprint chromatogram of the Qingguo Pills product; Compare the obtained fingerprint chromatogram of the Qingguo Pills product with the standard fingerprint chromatogram. If the similarity is greater than 0.9, it indicates that the quality of the Qingguo Pills product is qualified.

9. The application of the method for establishing the HPLC fingerprint chromatogram of Qingguo Pills according to any one of Claims 5 - 7 in the detection and identification of Qingguo Pills.

10. The fingerprint chromatogram obtained by the method for establishing the HPLC fingerprint chromatogram of Qingguo Pills according to any one of Claims 5 - 7.

Images