A method for constructing an HPLC fingerprint of Qingrehuadu pills and its application

Abstract

The present invention relates to a fingerprint construction and detection method for Qingrehuadu Pills. The construction method comprises the following steps: taking a reference substance and a Qingrehuadu Pill sample to prepare a reference substance solution and a test solution, respectively; detecting the reference substance solution and the test solution by high performance liquid chromatography to obtain corresponding reference substance spectra and sample spectra; importing the sample spectra into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system for analysis to obtain a fingerprint of Qingrehuadu Pills with identification, obtaining 29 common peaks in total, identifying 7 components among them, and assigning the components to the 29 common peaks. The fingerprint of the Qingre Huadu Pills constructed by the present invention has rich chromatographic peaks and contains effective ingredients belonging to multiple medicinal materials such as Forsythia suspensa, Gentiana scabra, Scutellaria baicalensis, Coptis chinensis, Chrysanthemum flos, Rhubarb, Licorice, Indigo naturalis, etc., which can more comprehensively reflect the overall chemical composition of the Qingre Huadu Pills, provide an effective means for the overall quality control and evaluation of the Qingre Huadu Pills, overcome the singleness and one-sidedness of the original quality control method, better monitor and evaluate the quality of the product, guide standardized production, and have high application value.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and particularly relates to establishing a fingerprint of Qingre Huadu Pills by adopting a high performance liquid chromatography method for controlling the quality of the Qingre Huadu Pills. Background Art

[0002] Heat syndrome refers to symptoms caused by hyperactivity of the human body due to exposure to pathogenic heat or excess yang and deficiency of yin. The terms "heat," "fire," and "toxicity" in Traditional Chinese Medicine all refer to manifestations of heat syndrome. Fire is worse than heat, and toxicity is worse than fire, so heat toxicity is the most serious stage in the development of heat syndrome. Currently, the main drugs on the market for clearing heat and detoxifying, reducing swelling and relieving pain include Qingre Huadu Pills, Huadu Pills, Wufu Huadu Pills, Niuhuang Jiedu Tablets, Shangqing Pills, and Yinhuang Drops. Heat-clearing and detoxifying drugs not only have antibacterial and anti-inflammatory properties, but also regulate the internal organs and endocrine system, enhancing the body's immune function for rapid recovery. They have the advantages of rapid effectiveness, short treatment course, and no recurrence after recovery. Heat-clearing and detoxifying drugs are a Traditional Chinese Medicine prescription for treating excess heat syndrome.

[0003] Qingre Huadu Pills, included in Volume 2 of the Ministry's Standardized Formulas of Traditional Chinese Medicine, are effective in clearing heat and detoxifying, reducing swelling and relieving pain. They are used for pediatric fever and irritability, sore throat, mouth and tongue ulcers, skin boils, bad breath, constipation, and residual toxins after a rash. The ingredients in the formula, such as Forsythia suspensa, Coptis chinensis, Scutellaria baicalensis, and Scrophularia ningpoensis, clear heat and detoxify, reduce swelling and invigorate blood circulation; Indigo naturalis and Buffalo horn cool blood and calm convulsions, clear heat and remove dampness; Gentiana scabra clears excess liver and gallbladder fire and eliminates damp-heat in the lower abdomen; Radix Trichosanthis clears heat and clears heat, promotes fluid production and quenches thirst, drains pus and reduces swelling; Rhubarb is a laxative, clears heat and purges fire; Poria cocos promotes diuresis and eliminates dampness, invigorates the spleen and calms the mind; Chrysanthemum dispels wind and dampness; Platycodon grandiflorum relieves cough and eliminates phlegm; Borneol disperses stagnant heat; Cinnabar calms the mind and relieves convulsions; Licorice harmonizes the ingredients, protecting the stomach and detoxifying. Together, these ingredients achieve the effects of clearing heat and detoxifying, reducing swelling and relieving pain.

[0004] Qingrehuadu Pills have good clinical efficacy and a large market demand, but the quality control standards are low. The ministerial standards only include two thin-layer identification items, and there is no content determination item. There are only literature reports on the content determination of a single ingredient. For example, Yu Yuanyang et al. studied the determination of the content of forsythiaside A and baicalin in Qingrehuadu Pills by HPLC (Yu Yuanyang, Study on Content Determination of Qingrehuadu Pills, Electronic Journal of Clinical Medical Literature, 2020, No. 45, pp. 178-179); Li Tiechun et al. studied the simultaneous determination of the content of four anthraquinone components, rhein, emodin, chrysophanol, and physophanol methyl ether, by HPLC (Li Tiechun, Simultaneous Determination of Qingrehuadu Pills by HPLC Contents of four anthraquinone compounds in Qingrehuadu pills, Journal of Anshan Normal University, 2017, No. 4, pp. 34-37); Zheng Yan et al. studied the determination of forsythin content in Qingrehuadu pills by HPLC (Zheng Yan, Determination of forsythin content in Qingrehuadu pills by HPLC, Contemporary Chinese Medicine, 2014, No. 12, pp. 61-62); Yuan Qihua studied the determination of baicalin content in Qingrehuadu pills by HPLC (Yuan Qihua, Determination of baicalin content in Qingrehuadu pills by HPLC, Clinical Journal of Rational Drug Use, 2013, No. 2, p. 66); Liu Hui et al. studied the determination of baicalin and wogonin in Qingrehuadu Pills by HPLC (Liu Hui, Simultaneous Determination of Baicalin and Wogonin in Qingrehuadu Pills by Reverse Phase High Performance Liquid Chromatography, China Pharmaceutical, 2013, No. 1, pp. 17-18); Zhao Fei studied the determination of baicalin in Qingrehuadu Pills by HPLC (Zhao Fei, Determination of Baicalin in Qingrehuadu Pills by HPLC, Chinese Ethnic and Folk Medicine, 2012 10, pp. 48-49); Zhao Fei studied the determination of berberine hydrochloride in Qingrehuadu Pills by HPLC (Zhao Fei, Determination of berberine hydrochloride in Qingrehuadu Pills by HPLC, Qilu Pharmacy, 2011, pp. 646-647); Wang Jingtao et al. studied the determination of arctiin in Qingrehuadu Pills by HPLC (Wang Jingtao, Determination of arctiin in Qingrehuadu Pills by HPLC, Liaoning Journal of Traditional Chinese Medicine, 2006, pp. 12, 1624). To date, no fingerprint method has been used to control the quality of Qingrehuadu Pills.

[0005] At present, the content determination method of Qingre Huadu Pills is all to control the intrinsic quality of Qingre Huadu Pills with one or two compositions, and has certain one-sidedness.To strictly control the quality of Qingre Huadu Pills, it is not enough to characterize and control only for one or two chemical components, and its substance group as a whole must be controlled.So, setting up the detection method of Qingre Huadu Pills fingerprint and application thereof just seem particularly important, need to control its substance group, effectively characterize quality standard.Before the present invention was completed, the method for setting up fingerprint with the chemical component containing of Qingre Huadu Pills as index had no report. Summary of the Invention

[0006] One of the purposes of the present invention is to provide a fingerprint spectrum detection method for Qingre Huadu Pills and its application. The fingerprint spectrum constructed by this method can comprehensively reflect the overall chemical composition of Qingre Huadu Pills, providing an effective means for the overall quality control and evaluation of Qingre Huadu Pills, thereby better ensuring the quality stability, consistency and controllability of Qingre Huadu Pills, thereby ensuring the safety and effectiveness of Qingre Huadu Pills.

[0007] The second purpose of the present invention is to comprehensively reflect the status of each component in Qingre Huadu Pills through systematic component identification and attribution of single medicinal materials with common peaks in different batches of medicines, so as to provide a reference basis for the quality of Qingre Huadu Pills.

[0008] The Qingre Huadu Pills of the present invention are composed of 300g of Forsythia suspensa, 150g of Indigo naturalis, 30g of Coptis chinensis, 150g of Scutellaria baicalensis, 75g of Rhubarb, 300g of Chrysanthemum, 150g of Gentiana scabra, 150g of Radix Trichosanthis, 150g of Scrophularia ningpoensis, and 300g of Poria.

[0009] The pill is prepared from 15 Chinese herbs, including 300g of Platycodon grandiflorum, 150g of Licorice root, 45g of Cinnabar, 4.5g of Borneol, and 9g of Buffalo Horn Concentrated Powder. The specific preparation method is as follows: with the above 15 herbs, excluding Buffalo Horn Concentrated Powder, pulverize Cinnabar into a very fine powder and grind Borneol into a fine powder; crush the remaining 12 herbs, including Forsythia suspensa, into a fine powder, sieve, and mix; then grind and sieve the fine powders of the three herbs, including the Buffalo Horn Concentrated Powder, and mix; add 130-140g of refined honey to every 100g of the powder to make large honey pills.

[0010] The present invention adopts the following technical solution: a method for detecting the fingerprint of Qingre Huadu Pills, which uses high performance liquid chromatography to determine its fingerprint. Specifically, the fingerprint detection method of Qingre Huadu Pills includes the following steps:

[0011] a. Preparation of test solution: Take Qingre Huadu pills, chop them, and take approximately 0.5-2.0 g. Accurately weigh the mixture and place it in a stoppered conical flask. Accurately add 25-100 mL of methanol and weigh the mixture. Reflux and extract for 30-90 minutes. Cool, weigh again, and make up the loss with methanol. Shake well, filter, and collect the filtrate.

[0012] b. Preparation of reference solution: Accurately weigh appropriate amounts of chlorogenic acid, gentiopicroside, liquiritin, forsythiaside A, baicalin, berberine hydrochloride, baicalein, and wogonin reference substances, and prepare mixed reference solutions with concentrations of 0.006, 0.02, 0.02, 0.04, 0.04, 0.01, 0.01, and 0.005 mg / mL, respectively, using methanol as the solvent. These solutions serve as reference solutions.

[0013] c. Determination: Accurately pipette 5-15 μL of the reference solution and the test solution respectively, inject them into the high performance liquid chromatograph, record the chromatogram within 125 minutes, and process the chromatogram using fingerprint software to obtain the fingerprint of Qingre Huadu Pills.

[0014] In the above method, the chromatographic conditions for the high performance liquid chromatography determination are: octadecylsilane bonded silica gel as the filler, acetonitrile as the mobile phase A, 0.02-0.3% formic acid solution as the mobile phase B, gradient elution, a flow rate of 0.5-1.5 mL / min, a column temperature of 25-45°C, and a detection wavelength of 210-360 nm; the theoretical plate number calculated based on the baicalin peak should be no less than 5000.

[0015] Preferably, the fingerprint detection method of the Qingrehuadu Pills comprises the following steps:

[0016] a. Preparation of test solution: Take Qingre Huadu Pills, cut into pieces, take 1.0 g, accurately weigh, place in a stoppered conical flask, accurately add 50 mL of methanol, weigh, reflux extract for 60 minutes, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate.

[0017] b. Preparation of reference solution: Accurately weigh appropriate amounts of chlorogenic acid, gentiopicroside, liquiritin, forsythiaside A, baicalin, berberine hydrochloride, baicalein, and wogonin reference substances, place them in a 10 mL volumetric flask, and use methanol as the solvent to prepare reference substance stock solutions with mass concentrations of 0.5980, 0.4228, 0.4056, 0.4001, 0.4141, 0.5025, 0.5003, and 0.5060 mg / mL, respectively; then accurately pipette the reference substances into the 0.1, 0.5, 0.5, 1, 1, 0.2, 0.2, and 0.1 mL of the stock solution were placed in the same 10 mL volumetric flask, dissolved with methanol and diluted to the scale, shaken to prepare mixed reference solutions with mass concentrations of 0.00598, 0.02114, 0.02028, 0.004001, 0.04141, 0.01005, 0.01001, and 0.00506 mg / mL, respectively. Pass through a 0.45 μm microporous filter membrane and take the filtrate as the mixed reference solution.

[0018] c. Determination: Accurately pipette 10 μl of each reference solution and test solution, inject them into high performance liquid chromatograph, record the chromatogram within 125 minutes, and process the chromatogram using fingerprint software to obtain the fingerprint of Qingre Huadu Pills.

[0019] The chromatographic conditions for the high performance liquid chromatography determination are as follows: octadecylsilane bonded silica gel as the filler, the chromatographic column is: Intersil C18 250×4.60 mm; acetonitrile is used as the mobile phase A, 0.1% formic acid solution is used as the mobile phase B, gradient elution, flow rate 1.0 ml / min, column temperature 30°C, detection wavelength 280 nm, and the theoretical plate number calculated based on the baicalin peak should be no less than 5000.

[0020] In the aforementioned fingerprint detection method for Qingre Huadu Pills, during the gradient elution process, the ratio of mobile phases A and B changes as follows: 0-10 min, 8% A; 10-20 min, 8-15% A; 20-40 min, 15% A; 40-55 min, 15-20% A; 55-70 min, 20-25% A; 70-80 min, 25% A; 80-100 min, 25-45% A; 100-110 min, 45-58% A; 110-125 min, 58-70% A.

[0021] The fingerprint has 29 common peaks. The chromatograms of the reference substances were compared with the fingerprints of the samples. The chromatogram peaks at the same retention time as the reference substances were identified. Figure 1 The results showed that chromatographic peaks 2, 4, 6, 8, 19, 21, 26, and 27 were identified as chlorogenic acid, gentiopicroside, liquiritin, forsythiaside A, baicalin, berberine hydrochloride, baicalein, and wogonin, respectively.

[0022] The beneficial effects of the present invention are:

[0023] 1. The present invention established HPLC fingerprints of 10 batches of Qingrehuadu Pills, and the similarities were all greater than 0.90, which can more comprehensively reflect the chemical information contained in Qingrehuadu Pills and better characterize their quality.

[0024] 2. 29 common peaks were identified, among which peaks 2, 4, 6, 8, 19, 21, 26 and 27 were identified as chlorogenic acid, gentiopicroside, liquiritin, forsythiaside A, baicalin, berberine hydrochloride, baicalein and wogonin, respectively.

[0025] 3. Since the fingerprint spectrum is not intended to determine the precise content of a certain component, but to fully reflect the information of the chemical composition, the present invention chooses to perform the measurement at a wavelength of 280nm, which has more peaks and reflects more complete information; the absorption value of each peak is good, the baseline is stable, and the large absorption of the near-ultraviolet impurity peak is avoided.

[0026] 4. The high-performance liquid chromatography fingerprint method established in the present invention has realized the quality control of the whole prescription of Qingre Huadu Pills for the first time. It does not identify a single compound or medicinal material. It can more effectively guide the feeding of materials, strictly regulate the production operation, and ensure the safety and effectiveness of clinical medication.

[0027] 5. According to the standard fingerprint conditions, the chromatographic peaks were attributed to eight medicinal materials, namely, rhubarb, chrysanthemum, gentian, forsythia, liquorice, scutellaria, coptis, and indigo. Peak 1 was attributed to rhubarb, peak 2 to chrysanthemum, peak 3 to gentian, peak 4 to gentian, peak 5 to forsythia, peak 6 to liquorice, peak 7 to scutellaria, peak 8 to forsythia, peak 9 to chrysanthemum, peak 10 to scutellaria, peak 11 to scutellaria and forsythia, peak 12 to chrysanthemum and scutellaria. Forsythia suspensa, peak 13 belongs to chrysanthemum, peak 14 belongs to chrysanthemum, peak 15 belongs to coptis chinensis, peak 16 belongs to chrysanthemum, peak 17 belongs to chrysanthemum, peak 18 belongs to scutellaria baicalensis, peak 19 belongs to scutellaria baicalensis, peak 20 belongs to scutellaria baicalensis and forsythia suspensa, peak 21 belongs to coptis chinensis, peak 22 belongs to liquorice, peak 23 belongs to scutellaria baicalensis, peak 24 belongs to rhubarb and chrysanthemum, peak 25 belongs to scutellaria baicalensis, peak 26 belongs to scutellaria baicalensis, peak 27 belongs to scutellaria baicalensis, peak 28 belongs to indigo naturalis, and peak 29 belongs to indigo naturalis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 HPLC chromatogram of mixed reference solution

[0029] Figure 2 Common pattern map of Qingre Huadu Pills

[0030] Figure 3 Overlay of fingerprint spectra of 10 batches of Qingre Huadu Pills

[0031] Figure 4 Comparison of sample fingerprints and chromatograms of various medicinal materials

[0032] Figure 5 Spectrum of Qingrehuadu Pills under 360nm detection wavelength

[0033] Figure 6 Spectrum of Qingrehuadu Pills under 320nm detection wavelength

[0034] Figure 7Spectrum of Qingrehuadu Pills under 280nm detection wavelength

[0035] Figure 8 Spectrum of Qingrehuadu Pills under 230nm detection wavelength

[0036] Figure 9 Spectrum of Qingrehuadu Pills under 210nm detection wavelength

[0037] Figure 10 Spectrum of Qingrehuadu Pills under 203nm detection wavelength DETAILED DESCRIPTION

[0038] In order to facilitate the understanding of the technical solution of the present invention, the establishment of the HPLC fingerprint of the Qingre Huadu Pill of the present invention and its fingerprint are further explained below in combination with specific test examples.

[0039] Example 1: Detection of fingerprints of different batches of Qingrehuadu pills

[0040] 1. Instruments and test drugs

[0041] 1.1Agilent 1220 high performance liquid chromatograph (Agilent, USA); KQ-250B ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); BSA 124S electronic balance (Sartorius); QUINTIX35-1CN (Sartorius).

[0042] 1.2 Purified water (Wahaha), acetonitrile (chromatographic grade), and all other reagents were of analytical grade. Chlorogenic acid, gentiopicroside, liquiritin, forsythiaside A, baicalin, berberine hydrochloride, baicalein, and wogonin reference standards (batch numbers 110753-202119, 110770-202219, 111610-202209, 111810-202209, 110715-202223, 110713-202015, 111595-201808, and 111514-202207, with purities of 96.3%, 98.1%, 95.2%, 96.4%, 97.2%, 85.9%, 97.9%, and 100.0%, respectively) were used. Qingrehuadu Pills (Batch numbers: 20220401, 20220402, 20220403, 20220601, 20220602, 20221101, 20221102, 20230101, 20230102, 20230201, Jilin Jichun Pharmaceutical Co., Ltd.

[0043] 2. Fingerprint determination

[0044] 2.1 Chromatographic conditions: Octadecylsilane bonded silica gel was used as the filler, acetonitrile was used as the mobile phase A, 0.1% formic acid solution was used as the mobile phase B, gradient elution was performed, the flow rate was 1.0 ml / min, the column temperature was 30°C, the detection wavelength was 280 nm, and the mobile phase gradient elution program was as follows: 0-10 min, 8% A; 10-20 min, 8-15% A; 20-40 min, 15% A; 40-55 min, 15-20% A; 55-70 min, 20-25% A; 70-80 min, 25% A; 80-100 min, 25-45% A; 100-110 min, 45-58% A; 110-125 min, 58-70% A.

[0045] 2.2 Preparation of test solution: Take Qingre Huadu Pills, cut into pieces, take 1.0 g, accurately weigh, place in a stoppered conical flask, accurately add 50 mL of methanol, weigh the weight, reflux extraction for 60 minutes, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate.

[0046] 2.3 Preparation of control medicinal materials and control extracts: According to the prescription ratio and preparation method, weigh the control extract or control medicinal material and prepare it according to the preparation method of the test sample.

[0047] 2.4 Preparation of reference solution: Accurately weigh appropriate amount of chlorogenic acid, gentiopicroside, liquiritin, forsythiaside A, baicalin, berberine hydrochloride, baicalein, and wogonin reference substances, accurately weigh them, place them in a 10 mL volumetric flask, and use methanol as solvent to prepare reference substance stock solutions with mass concentrations of 0.5980, 0.4228, 0.4056, 0.4001, 0.4141, 0.5025, 0.5003, and 0.5060 mg / mL, respectively; then accurately pipette reference solution into the 10 mL volumetric flask. 0.1, 0.5, 0.5, 1, 1, 0.2, 0.2, and 0.1 mL of the sample stock solution were placed in the same 10 mL volumetric flask, dissolved in methanol and diluted to the scale, shaken to prepare mixed reference solution with mass concentrations of 0.00598, 0.02114, 0.02028, 0.004001, 0.04141, 0.01005, 0.01001, and 0.00506 mg / mL, respectively. Pass through a 0.45 μm microporous filter membrane, and take the filtrate as the mixed reference solution;

[0048] 2.4 Determination: Accurately pipette 10 μL of reference solution and test solution respectively, inject into high performance liquid chromatograph, record the chromatogram within 125 minutes, see Figure 1 、 Figure 2 .

[0049] Example 2: Fingerprint analysis of 10 batches of Qingrehuadu pills

[0050] 1. Fingerprint similarity analysis

[0051] Ten batches of Qingrehuadu pills were prepared according to the test sample preparation method, sampled and analyzed under chromatographic conditions, and the fingerprints of Qingrehuadu pills were recorded. The "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine 2012A Edition" issued by the State Pharmacopoeia Committee was used to perform similarity analysis on the fingerprints of the ten batches of Qingrehuadu pills. S1 was set as the reference spectrum, and the median method was used with a time window width of 0.1 min. A total of 29 common peaks were calibrated. The similarities of the ten batches of Qingrehuadu pills were all greater than 0.90. The similarity evaluation results are shown in Table 1. The relative retention times of the 29 common peaks were basically consistent, while the relative peak areas were quite different. The standard fingerprint data are shown in Table 2. The superposition diagram of the fingerprints of the ten batches of Qingrehuadu pills is shown in Figure 3 , Figure 3 In the list, S1-S10 correspond to batch numbers 20220401, 20220402, 20220403, 20220601, 20220602, 20221101, 20221102, 20230101, 20230102, and 20230201, respectively.

[0052] Table 1 Similarity evaluation results of 10 batches of Qingrehuadu pill samples

[0053]

[0054]

[0055] Table 2 Standard fingerprint data

[0056]

[0057]

[0058] 2. Common peak identification: The HPLC chromatogram of the reference substance ( Figure 1 , where peak 2 is chlorogenic acid, peak 4 is gentiopicroside, peak 6 is liquiritin, peak 8 is forsythiaside A, peak 19 is baicalin, peak 21 is berberine hydrochloride, peak 26 is baicalein, and peak 27 is wogonin)) and the sample fingerprint ( Figure 3 ) were compared with the reference samples, and the chromatographic peaks at the same retention time as the reference samples were identified. The results showed that among the 29 common peaks, peaks 2, 4, 6, 8, 19, 21, 26, and 27 were identified as chlorogenic acid, gentiopicroside, liquiritin, forsythiaside A, baicalin, berberine hydrochloride, baicalein, and wogonin, respectively.

[0059] 3. Medicinal material identification: According to the standard fingerprint conditions, the chromatographic peaks are identified. Figure 4 , Figure 4Among them, S1-S15 are respectively forsythia, scutellaria, gentian, poria, cinnabar, indigo, rhubarb, radix trichosanthis, platycodon, borneol, coptis, chrysanthemum, figwort, liquorice, buffalo horn concentrated powder medicinal materials, and S16 is a sample. The 29 common peaks are respectively attributed to eight medicinal materials, namely rhubarb, chrysanthemum, gentian, forsythia, liquorice, scutellaria, coptis, indigo; among them, peak 1 is attributed to rhubarb, peak 2 is attributed to chrysanthemum, peak 3 is attributed to gentian, peak 4 is attributed to gentian, peak 5 is attributed to forsythia, peak 6 is attributed to liquorice, peak 7 is attributed to scutellaria, peak 8 is attributed to forsythia, peak 9 is attributed to chrysanthemum, peak 10 is attributed to scutellaria, peak 11 is attributed to scutellaria and forsythia, peak 12 is attributed to chrysanthemum and forsythia, peak 13 is attributed to chrysanthemum. Flowers, peak No. 14 belongs to chrysanthemum, peak No. 15 belongs to coptis chinensis, peak No. 16 belongs to chrysanthemum, peak No. 17 belongs to chrysanthemum, peak No. 18 belongs to scutellaria baicalensis, peak No. 19 belongs to scutellaria baicalensis, peak No. 20 belongs to scutellaria baicalensis and forsythia suspensa, peak No. 21 belongs to coptis chinensis, peak No. 22 belongs to liquorice, peak No. 23 belongs to scutellaria baicalensis, peak No. 24 belongs to rhubarb and chrysanthemum, peak No. 25 belongs to scutellaria baicalensis, peak No. 26 belongs to scutellaria baicalensis, peak No. 27 belongs to scutellaria baicalensis, peak No. 28 belongs to indigo, and peak No. 29 belongs to indigo.

[0060] Example 3: Wavelength selection

[0061] Take 20220401 batches of samples for testing, and examine the overall effect of the chromatogram at different wavelengths such as 360, 320, 280, 230, 210, and 203 respectively. Other chromatographic conditions are the same as in Example 1. Figure 5 、 6 , 7, 8, 9, 10. From the measured chromatogram, we know that when the wavelength is 360nm, the peak is very small, the response value is low and the number of peaks is small ( Figure 5 ); When the wavelength is 320nm, the response value is low and the number of peaks is relatively small ( Figure 6 ); When the wavelength is 280nm, the chromatogram baseline is flat, the peak shape is better, the number of peaks is larger, and the response value is the highest, which is the best condition ( Figure 7 ); When the wavelength is 230nm, the chromatogram baseline is not flat, the chromatogram baseline is not in the standard position, and the number of peaks is small ( Figure 8 When the wavelength is 210, the chromatogram baseline is not flat, the chromatogram baseline is not in the standard position, and the chromatographic peak fluctuates greatly ( Figure 9 ); When the wavelength is 203nm, the chromatogram baseline is not at the standard position ( Figure 10 ). Comprehensively selected, the fingerprint spectrum with 280nm as the detection wavelength can most comprehensively reflect the composition of the preparation, and has the best peak shape, so 280nm is preferred as the optimal detection wavelength.

[0062] Example 4: Methodological Investigation

[0063] The 20220401 batch of samples was tested, and the chromatographic conditions and test sample preparation method were the same as in Example 1.

[0064] (1) Precision test

[0065] A test solution was prepared using Qingrehuadu Pills. Six consecutive injections were made, and the retention times and areas of the 29 major chromatographic peaks were recorded. Using baicalin as the reference peak, the relative peak area RSDs were <3.0% (n=6) and the relative retention time RSDs were <1.0% (n=6), demonstrating good instrument precision. The experimental results are shown in Table 3.

[0066] (2) Stability test

[0067] A test solution was prepared from Qingrehuadu Pills and tested at 0, 3, 6, 9, 12, and 24 hours. The retention times and areas of the 29 major chromatographic peaks were recorded. Using baicalin as the reference peak, the relative peak area RSD was <3.0% (n=6) and the relative retention time RSD was <1.0% (n=6), demonstrating good stability within 24 hours. The experimental results are shown in Table 3.

[0068] (3) Repeatability test

[0069] Six test solutions of Qingrehuadu Pills were prepared and tested, recording the retention times and areas of the 29 major chromatographic peaks. Using baicalin as the reference peak, the relative peak area RSDs were <3.0% (n=6) and the relative retention time RSDs were <1.0% (n=6), demonstrating good stability. The experimental results are shown in Table 3.

[0070] Table 3 Methodological investigation experimental results

[0071]

[0072]

Claims

1. A method for constructing an HPLC fingerprint of Qingrehuadu Pills, characterized by: The following steps are involved: a. Preparation of test solution: Take Qingre Huadu Pills, chop them, take 0.5-2.0g, accurately weigh, and place in a stoppered conical flask. Accurately add 25-100mL of methanol and weigh the solution. Reflux for 30-90 minutes. Cool, weigh again, make up the loss with methanol, shake well, filter, and collect the filtrate. b. Preparation of reference solution: Accurately weigh appropriate amounts of chlorogenic acid, gentiopicroside, liquiritin, forsythiaside A, baicalin, berberine hydrochloride, baicalein, and wogonin reference substances, and prepare mixed reference solutions with concentrations of 0.006, 0.02, 0.02, 0.04, 0.04, 0.01, 0.01, and 0.005 mg / mL, respectively, using methanol as the solvent. These solutions serve as reference solutions. c. Determination: Accurately pipette 5 to 15 μL of each reference solution and test solution, inject into high performance liquid chromatograph, record the chromatogram within 125 minutes, and process the map with fingerprint software to obtain the fingerprint of Qingre Huadu Pills; The chromatographic conditions of the high performance liquid chromatography determination are as follows: octadecylsilane bonded silica gel as the filler, acetonitrile as the mobile phase A, 0.02-0.3% formic acid solution as the mobile phase B, gradient elution, flow rate 0.5-1.5 mL / min, column temperature 25-45°C, detection wavelength 210-360 nm; the theoretical plate number calculated based on the baicalin peak should be no less than 5000; During the gradient elution process, the ratio of mobile phase A and B changes as follows: 0-10min, 8% A; 10-20min, 8~15%A; 20-40min, 15%A; 40-55min, 15~20%A; 55-70min, 20~25%A; 70-80min, 25%A; 80-100min, 25~45%A; 100-110min, 45~58%A; 110-125min, 58~70%A.

2. The method for constructing an HPLC fingerprint of Qingre Huadu Pill according to claim 1, characterized in that: The following steps are involved: a. Preparation of test solution: Take Qingrehuadu pills, chop them, take 1.0g, accurately weigh, place in a stoppered conical flask, accurately add 50mL of methanol, weigh, reflux for 60 minutes, cool, weigh again, make up the loss with methanol, shake well, filter, and collect the filtrate. b. Preparation of reference solution: Accurately weigh appropriate amounts of chlorogenic acid, gentiopicroside, liquiritin, forsythiaside A, baicalin, berberine hydrochloride, baicalein, and wogonin reference substances, place them in a 10 mL volumetric flask, and use methanol as the solvent to prepare reference substance stock solutions with mass concentrations of 0.5980, 0.4228, 0.4056, 0.4001, 0.4141, 0.5025, 0.5003, and 0.5060 mg / mL, respectively; then accurately pipette the reference substances into the Place 0.1, 0.5, 0.5, 1, 1, 0.2, 0.2, and 0.1 mL of the stock solution in the same 10 mL volumetric flask, dissolve with methanol and dilute to the mark, shake well, and prepare mixed reference substance solutions with mass concentrations of 0.00598, 0.02114, 0.02028, 0.004001, 0.04141, 0.01005, 0.01001, and 0.00506 mg / mL, respectively. Pass through a 0.45 μm microporous filter membrane, and take the filtrate as the mixed reference substance solution; c. Determination: Accurately pipette 10 μl of each reference solution and test solution, inject them into high performance liquid chromatograph, record the chromatogram within 125 minutes, and process the chromatogram using fingerprint software to obtain the fingerprint of Qingre Huadu Pills.

3. The method for constructing an HPLC fingerprint of Qingre Huadu Pills according to claim 2, characterized in that: The chromatographic conditions for the high performance liquid chromatography determination are as follows: octadecylsilane bonded silica gel as the filler, the chromatographic column is: Intersil C18 250×4.60 mm; acetonitrile is used as the mobile phase A, 0.1% formic acid solution is used as the mobile phase B, gradient elution, flow rate 1.0 ml / min, column temperature 30°C, detection wavelength 280 nm, and the theoretical plate number calculated based on the baicalin peak should be no less than 5000.

4. The method for constructing the HPLC fingerprint of Qingrehuadu Pills according to claim 1-3, characterized in that: The fingerprint has a total of 29 common peaks, of which chromatographic peaks 2, 4, 6, 8, 19, 21, 26, and 27 are respectively identified as chlorogenic acid, gentiopicroside, liquiritin, forsythiaside A, baicalin, berberine hydrochloride, baicalein, and wogonin; according to the standard fingerprint conditions, the chromatographic peaks are assigned to eight medicinal materials, namely, rhubarb, chrysanthemum, gentian, forsythia, liquorice, scutellaria, coptis, and indigo naturalis; Peak 1 belongs to Rhubarb, Peak 2 belongs to Chrysanthemum, Peak 3 belongs to Gentiana, Peak 4 belongs to Gentiana, Peak 5 belongs to Forsythia, Peak 6 belongs to Licorice, Peak 7 belongs to Scutellaria, Peak 8 belongs to Forsythia, Peak 9 belongs to Chrysanthemum, Peak 10 belongs to Scutellaria, Peak 11 belongs to Scutellaria and Forsythia, Peak 12 belongs to Chrysanthemum and Forsythia, Peak 13 belongs to Chrysanthemum, Peak 14 belongs to Chrysanthemum, Peak 15 belongs to Rhubarb. Peak 16 belongs to chrysanthemum, peak 17 belongs to chrysanthemum, peak 18 belongs to scutellaria baicalensis, peak 19 belongs to scutellaria baicalensis, peak 20 belongs to scutellaria baicalensis and forsythia suspensa, peak 21 belongs to coptis chinensis, peak 22 belongs to liquorice, peak 23 belongs to scutellaria baicalensis, peak 24 belongs to rhubarb and chrysanthemum, peak 25 belongs to scutellaria baicalensis, peak 26 belongs to scutellaria baicalensis, peak 27 belongs to scutellaria baicalensis, peak 28 belongs to indigo naturalis, and peak 29 belongs to indigo naturalis.

5. Use of the fingerprint of Qingre Huadu Pills obtained by the method for constructing the fingerprint of Qingre Huadu Pills according to any one of claims 1 to 4 in the quality control of Qingre Huadu Pills.

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