A high-throughput mass detection method for Qingfei Paidu granules

Abstract

This invention discloses a high-throughput quality control method for Qingfei Paidu Granules. Based on liquid chromatography-mass spectrometry, this method establishes a comprehensive quality control method for Qingfei Paidu Granules. This method enables specific identification of the 19 ingredients in the Qingfei Paidu Granules, limit testing of the toxic component aristolochic acid I, fingerprint analysis, and content determination of four indicator components in a single analysis, significantly improving detection efficiency and reducing testing costs. This method enables rapid, comprehensive, and accurate quality assessment of Qingfei Paidu Granules, providing technical support for the development of new Qingfei Paidu Granules.

Description

Technical Field

[0001] The invention relates to a high-throughput quality detection method for Qingfei Paidu granules, and belongs to the field of quality analysis of traditional Chinese medicines. Background Art

[0002] Qingfei Paidu Granules originated from Zhang Zhongjing's Treatise on Febrile and Miscellaneous Diseases in the Han Dynasty. They are an optimized combination of four classic prescriptions: Ma Xing Shi Gan Tang, Wu Ling San, Xiao Chai Hu Tang, and She Gan Ma Huang Tang. They are mild in nature and have the effects of clearing the lungs, relieving asthma, and detoxifying and removing dampness. Clinically, they are mainly used for patients with pneumonia infected with the new coronavirus. A large amount of clinical data has shown that the total effective rate of Qingfei Paidu Granules is over 90%, effectively preventing patients from developing critical illness [Traditional Chinese Medicine Bulletin, 2020, 19(01):9]. Qingfei Paidu Granules are composed of 21 Chinese medicinal herbs with a complex chemical composition, with flavonoids, alkaloids, triterpenoid saponins, and organic acids as the main components [Zhou Yanyan. Chinese Journal of Traditional Chinese Medicine, 2020, 45(13):3035-3044]. By predicting and collecting the targets of identified compounds, a target network model of Qingfei Paidu Granules was established. Studies have shown that Qingfei Paidu Granules are associated with anti-inflammatory and antiviral effects in the treatment of novel coronavirus pneumonia [Ruocong Yang. Pharmacological Research, 2020, 157: 1-13; Zhao Jing. Chinese Herbal Medicine, 2020, 51(4): 829-835]. Traditional Chinese medicine prescriptions are a complex system, and establishing scientific and reasonable quality control methods is key to ensuring their clinical effectiveness and safety.

[0003] At present, the 2020 edition of the Chinese Pharmacopoeia has not yet included Qingfei Paidu Granules. The literature reports a method for determining the content of two flavonoid components in Qingfei Paidu Granules [Wu Yuanbo. Chinese Ethno-Folk Medicine, 2021, 30(12):34-38]. However, only the content of the main components of the prescription, baicalin and hesperidin, was determined, and there was a lack of effective control of the active ingredients of the monarch drug. In addition, Chen Yijun et al. used liquid chromatography-mass spectrometry to detect and quantitatively analyze the toxic component aristolochic acid I in Qingfei Paidu Granules [Chen Yijun. Acta Pharmaceutica Sinica, 2020, 55(8):1903-1907], which provided a certain guarantee for the safety of the drug. However, the existing quality control is limited to the determination of the content of the main components and the inspection of the toxic component aristolochic acid I, which cannot systematically and comprehensively reflect the intrinsic quality of Qingfei Paidu Granules. In addition, the analysis method is cumbersome, time-consuming, and the detection cost is high. Therefore, it is particularly important to establish a rapid, comprehensive, and accurate quality detection method for Qingfei Paidu Granules. Summary of the Invention

[0004] The present invention aims to provide a high-throughput quality control method for Qingfei Paidu Granules and its application. Based on liquid chromatography-mass spectrometry, a comprehensive quality control method for Qingfei Paidu Granules has been established. This single analysis enables specific identification of 19 medicinal ingredients in Qingfei Paidu Granules, limit testing and fingerprint analysis of the toxic component aristolochic acid I, and content determination of four indicator components, significantly improving detection efficiency and reducing testing costs.

[0005] The specific technical solutions of the present invention are:

[0006] 1. A high-throughput mass detection method for Qingfei Paidu granules, comprising the following steps:

[0007] a) Preparation of reference solution: respectively take ephedrine hydrochloride, pseudoephedrine hydrochloride, baicalin, and glycyrrhizic acid reference substances, add methanol or acetonitrile solvent to prepare a mixed reference solution containing ephedrine hydrochloride, pseudoephedrine hydrochloride, baicalin, and glycyrrhizic acid;

[0008] b) Preparation of test solution: Take Qingfei Paidu Granules and add 50-100% (preferably 75%-100%) methanol or 50-100% (preferably 75%-100%) ethanol at a solid-liquid ratio (sample to methanol or ethanol) of 1:10-1:300 g / mL (preferably 1:100-1:250 g / mL). Reflux or sonicate for 15-60 min, cool to room temperature, make up the weight loss with solvent, shake well, filter, and collect the filtrate to obtain the Qingfei Paidu Granule test solution.

[0009] c) High Performance Liquid Chromatography-Tandem Mass Spectrometry: The reference solution prepared in step a) and the test solution prepared in step b) were accurately aspirated and injected into a high performance liquid chromatography-tandem mass spectrometer, and the chromatogram and mass spectrum were recorded; the chromatographic column was filled with octadecylsilane bonded silica gel, the mobile phase was methanol or acetonitrile (mobile phase A) and an aqueous phase containing formic acid or acetic acid as an additive (mobile phase B), the flow rate was 0.2 mL / min-0.5 mL / min, the injection volume was 0.5-2 μL, and the detection wavelength was 210-330 nm; the ionization modes used for mass spectrometry were electrospray positive ion ionization and negative ion ionization, the curtain gas was 25-45 psi; Gas 1: 30-70 psi; Gas 2: 30-70 psi; temperature: 300-500°C; ionization pressure: 4500-5500 V (positive), -4500--5500 V (negative); declustering voltage: 60-80 V; full scan range: m / z 50-1700; MS / MS scan range: m / z 50-1700; fragmentor voltage: 30-40 V (positive and negative);

[0010] d) Identification: Take 19 single-ingredient negative prescription samples (including, for example, ephedra single-ingredient negative prescription (Qingfei Paidu Granule prescription sample without ephedra medicinal taste): according to the Qingfei Paidu Granule prescription composition, remove ephedra medicinal taste, take the remaining 20 medicinal tastes such as gypsum and licorice, and prepare according to the Qingfei Paidu Granule preparation method, that is, the other 18 single-ingredient negative prescriptions such as licorice, apricot kernel, cinnamon twig, oriental rhizome, umbellate, white atractylodes, poria, bupleurum, scutellaria, ginger pinellia, ginger, aster, winter flower, beam dry, asarum, immature bitter orange, tangerine peel, patchouli, etc. are prepared respectively by the preparation method of ephedra single-ingredient negative prescription, and are respectively the Qingfei Paidu Granule prescription samples with the corresponding single medicinal taste removed;) and the Qingfei Paidu Granule samples are respectively prepared by the method of step b) Method, preparing test solutions of 19 single-ingredient negative prescriptions and a test solution of Qingfei Paidu Granules, determining according to step c), recording compound information, using it to identify the compounds in Qingfei Paidu Granules, then using the primary parent ions of the compounds to extract the primary mass spectra of Qingfei Paidu Granules and the 19 single-ingredient negative prescriptions, recording the compound mass spectrum peak areas, calculating negative interference (negative interference = the ratio of the mass spectrum peak area of ​​the target component in the single-ingredient negative prescription to the mass spectrum peak area of ​​the target component in Qingfei Paidu Granules), and using compounds with negative interference less than 5% (preferably less than 1%, more preferably 0%) as the specific components of the medicinal flavor, to establish a specific identification method for the 19 medicinal flavors in Qingfei Paidu Granules;

[0011] e) Content determination: Prepare a mixed reference solution of ephedrine hydrochloride, pseudoephedrine hydrochloride, baicalin, and glycyrrhizic acid according to the method of step a); prepare more than 3 batches (preferably more than 10 batches) of Qingfei Paidu Granule test solution according to the method of step b); determine them in sequence according to the method of step c); record the peak areas of ephedrine hydrochloride, pseudoephedrine hydrochloride, baicalin, and glycyrrhizic acid in the test solution and the reference solution; and use the external standard method ( Among them A x represents the peak area of ​​the component to be measured in the sample, Rf represents the correction factor, and m represents the sample mass. ), calculate the mass contents of ephedrine hydrochloride, pseudoephedrine hydrochloride, baicalin, and glycyrrhizic acid in the Qingfei Paidu granules sample.

[0012] In a specific embodiment of the present invention, an appropriate amount of aristolochic acid I reference substance is taken, accurately weighed, and methanol is added to prepare a reference solution containing 0.2-2 μg of aristolochic acid I per 1 mL (preferably 0.2-1 μg of aristolochic acid I per 1 mL), which is diluted 50 to 200 times (v / v) with methanol or acetonitrile solution (preferably 50 to 100 times (v / v) with methanol or acetonitrile solution). Qingfei Paidu granules are taken and prepared according to step b) to prepare a test solution, which is measured according to step c), and the primary ion map is recorded to calculate the signal-to-noise ratio (S / N, where S is the known concentration of aristolochic acid I). The signal measured by the aristolochic acid I reference solution, N is the signal measured by the methanol or acetonitrile solution); by comparing the primary ion patterns of the aristolochic acid I reference solution and the Qingfei Paidu Granule test solution when the signal-to-noise ratio is 3-10, the mass spectrum peak area of ​​the Qingfei Paidu Granule test solution at the position corresponding to the retention time of the aristolochic acid I reference solution should be 0 (i.e., there is no corresponding aristolochic acid I mass spectrum peak); if it is greater than 0, it means that it contains the toxic component aristolochic acid I (i.e., there is a corresponding aristolochic acid I mass spectrum peak); establish a limit inspection method for the toxic component aristolochic acid I in Qingfei Paidu Granules.

[0013] In a specific embodiment of the present invention, more than 3 batches (preferably more than 10 batches) of Qingfei Paidu Granule test solution are prepared according to the method of step b), and the results are measured in sequence according to step c), and the chromatograms are recorded. All the chromatograms are imported into the "Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System Software (2012 Edition)" software of the Chinese Pharmacopoeia Commission, and the common peaks (chromatographic peaks with a peak area accounting for more than 5% (preferably a peak area accounting for 10% to 50%)) are matched to obtain a control fingerprint, and a fingerprint method for Qingfei Paidu Decoction is established.

[0014] In a specific embodiment of the present invention, the reference substance solution in step a) is: appropriate amounts of ephedrine hydrochloride, pseudoephedrine hydrochloride, baicalin, and glycyrrhizic acid reference substances are accurately weighed, and methanol is added to prepare a mixed reference substance solution containing 20-100 μg of ephedrine hydrochloride per 1 mL (preferably 20-50 μg of ephedrine hydrochloride per 1 mL), 20-100 μg of pseudoephedrine hydrochloride (preferably 20-50 μg of pseudoephedrine hydrochloride), 20-100 μg of baicalin (preferably 40-80 μg of baicalin), and 20-100 μg of glycyrrhizic acid (preferably 20-50 μg of glycyrrhizic acid).

[0015] In a specific embodiment of the present invention, the chromatographic conditions of step c) are: the chromatographic column length is 50-250 mm, the diameter is 2.1-4.6 mm, the particle size is 1.7-5 μm, the mobile phase A is acetonitrile, the mobile phase B is an aqueous formic acid solution containing an additive with a volume concentration of 0.1-0.5%, and a gradient elution is adopted: 0-8 min, 2-5% A; 8-11 min, 5-13% A; 11-24 min, 13-20% A; 24-37 min, 20-42% A; 37-38 min, 42-90% A; 38-43 min, 90% A.

[0016] In a specific embodiment of the present invention, the identification in step d) is as follows: identifying 37 chemical components in Qingfei Paidu Granules, wherein the ephedra-specific components are ephedrine and pseudoephedrine, the licorice-specific components are liquiritin, apigenin, and glycyrrhizic acid, the apricot-specific components are amygdalin and prunin, the cassia twig-specific components are protocatechuic aldehyde / parahydroxybenzoic acid, the oriental-specific component is alismatol A, the polyporus-specific component is polyporus ketone A, the Atractylodes-specific component is atractylodesin A, the Poria-specific component is pachymic acid A, the bupleurum-specific component is saikosaponin A / B2, the scutellaria-specific components are baicalin, baicalin, wogonin, baicalein, and scutellaria flavonoid II, the ginger-specific component is sericinine, the ginger-specific component is 4-shogaol, and the aster-specific component is asterin A / asterinin. A. The specific components of coltsfoot flower are chlorogenic acid, cryptochlorogenic acid, kelp-like alkaloids, and isochlorogenic acid C; the specific components of belamcanda ternata are belamcandaside, irisin, irisin, and irisin; the specific component of asarum is 3,4-dimethoxycinnamyl alcohol; the specific component of immature bitter orange is citric acid; the specific component of dried tangerine peel is synephrine, naringin, hesperidin, and neohesperidin; the specific component of patchouli is bigninoside II. The specific qualitative identification of the 19 medicinal flavors in Qingfei Paidu Granules can be achieved by determining whether they contain mass spectrometry peaks of specific components (when a medicinal flavor contains multiple specific components, it must contain multiple specific components at the same time).

[0017] In a specific embodiment of the present invention, the Qingfei Paidu Granules described in step e) contain 0.24% to 0.45% by mass of ephedrine hydrochloride, 0.13% to 0.23% by mass of pseudoephedrine hydrochloride, 1.09% to 2.02% by mass of baicalin, and 0.14% to 0.26% by mass of glycyrrhizic acid, to ensure that the contents of the active ingredients and main components in the Qingfei Paidu Granules are uniform and stable.

[0018] In a specific embodiment of the present invention, in the limit test method for the toxic component aristolochic acid I in the Qingfei Paidu Granules, when the signal-to-noise ratio is equal to 3-10, the concentration of the aristolochic acid I reference solution is 1-20 ng / mL (preferably the concentration of the aristolochic acid I reference solution is 1-10 ng / mL), and the mass spectrum peak area of ​​the primary ion map of the test sample at the position corresponding to the retention time of the primary ion map of the aristolochic acid I reference should be 0, ensuring that the Qingfei Paidu Granules do not contain the toxic component aristolochic acid I.

[0019] In a specific embodiment of the present invention, in the fingerprint method of the Qingfei Paidu Decoction, similarity is calculated based on 10 common peaks with a peak area accounting for more than 5% (preferably a peak area accounting for 10% to 50%), and the similarity between the test sample chromatogram and the control chromatogram is not less than 0.95, thereby achieving overall quality control of the fingerprint of the Qingfei Paidu granules.

[0020] This method enables specific identification of the 19 ingredients in the Qingfei Paidu Granule formula, limit testing of the toxic component aristolochic acid I, fingerprint analysis, and content determination of four index components in a single analysis, significantly improving detection efficiency and reducing testing costs. This method enables rapid, comprehensive, and accurate quality assessment of Qingfei Paidu Granules, providing technical support for the development of new Qingfei Paidu Granules.

[0021] The beneficial effects of the present invention are as follows: the present invention establishes a high-throughput quality detection method for Qingfei Paidu Granules, which can realize the identification, inspection, fingerprint spectrum and content determination detection of Qingfei Paidu Granules through one analysis, and can quickly, comprehensively and accurately evaluate the quality of Qingfei Paidu Granules. The detection time is short, the efficiency is high, the specificity is good, and the accuracy is high, which greatly reduces the detection cost and provides technical support for the research and development of new drugs for Qingfei Paidu Granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The total ion current (TIC) diagrams of Qingfei Paidu Granules in positive and negative ion modes: A. TIC diagram of Qingfei Paidu Granules in positive ion mode; B. TIC diagram of Qingfei Paidu Granules in negative ion mode;

[0023] Figure 2 Positive ion extracted ion chromatograms (EICs) of ephedrine and pseudoephedrine, the specific components of ephedra: A. Positive ion EIC of Qingfei Paidu Granules; B. Negative positive ion EIC of ephedra alone;

[0024] Figure 3 Determination of the content of four index components of Qingfei Paidu Granules;

[0025] Figure 4 Positive ion extracted ion chromatogram (EIC) of the toxic component aristolochic acid I: A. Positive ion EIC of the reference sample; B. Positive ion EIC of the test sample

[0026] Figure 5 These are the UPLC fingerprints of 10 batches of Qingfei Paidu Granules. DETAILED DESCRIPTION

[0027] In order to provide a more detailed understanding of the features and technical content of the present invention, the implementation of the present invention is described in detail below. A high-throughput mass detection method for Qingfei Paidu granules of the present invention comprises the following steps:

[0028] Example 1: Specific identification of 19 medicinal ingredients

[0029] 1. Instruments and test drugs

[0030] 1.1 Instrument

[0031] An Agilent ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometer Q-TOF 6545 was used, an ACQUITY UPLC BEHC18 column (2.1×100 mm, 1.7 μm, Waters Corporation, USA), and an XSR105 electronic analytical balance (Mettler-Toledo Co., Ltd.) were used.

[0032] 1.2 Drug testing

[0033] Reference substances: aristolochic acid I (batch number: 110746-201912, specification: 20 mg / tube, purity: 99.20%), ephedrine hydrochloride (batch number: 171241-201508, specification: 100 mg / tube, purity: 99.80%), pseudoephedrine hydrochloride (batch number: 171237-201208, specification: 100 mg / tube, purity: 99.90%), baicalin (batch number: 110715-201821, specification: 40 mg / tube, purity: 95.40%), ammonium glycyrrhizate (batch number 110731-202021, specification: 20 mg / tube, purity: 96.20%) were purchased from China Food and Drug Inspection Institutes.

[0034] Reagents: Acetonitrile (mass spectrometry grade), methanol (chromatographic grade), and ethanol (chromatographic grade) were purchased from Sigma-Aldrich, and formic acid (mass spectrometry grade) was purchased from Aladdin. Laboratory water was obtained from a Milli-Q ultrapure water purification system (Milli-Q IQ 7000).

[0035] 1.3 Sample

[0036] Eleven batches of Qingfei Paidu Granule samples were prepared in the laboratory, batches QFP200701 to QFP200711 (numbers S1 to S11). The preparation method is as follows:

[0037]

[0038]

[0039] Preparation method: The above 21 ingredients, Asarum sieboldii, were decocted twice, the first time with 5L of water and decocted for 20 minutes, the second time with 4L of water and decocted for 15 minutes, the decoctions were combined, filtered, and the filtrate was passed through a polar modified octadecylsilane bonded silica gel column (name: C18YE, specification: 100×250mm, particle size: 30μm, packing weight: 1.2kg, column volume about 2L), and impurities were removed by eluting with pure water for 3 times the column volume, and eluted with 60% (v / v) ethanol for 3 times the column volume, and collected. Collect the 60% (v / v) ethanol eluate, pass the eluate through an anion exchange macroporous resin column (name: PA408 resin, specification: 100×250 mm, particle size: 400 μm, filler weight: 1.8 kg, column volume is about 2 L), collect the sample flow-through, then elute with 60% (v / v) ethanol for 3 times the column volume, collect the 60% (v / v) ethanol eluate, combine the sample flow-through and 60% (v / v) ethanol eluate as the asarum preparation solution for later use. Take gypsum and add 12L of water to decoct for 15 minutes, then add the remaining 19 ingredients including ephedra, and decoct twice, the first time adding 145L of water and decocting for 20 minutes, the second time adding 126L of water and decocting for 15 minutes, the decoctions are combined, filtered, and the filtrate is passed through polar-modified octadecylsilane bonded silica gel (name: C18YE, specification: 500×350mm, particle size: 30μm, filler weight: 40kg, column volume of approximately 66L), washed with pure water for 3 times the column volume to remove impurities, eluted with 60% (v / v) ethanol for 3 times the column volume, and the 60% (v / v) ethanol eluate is combined with the asarum preparation solution and concentrated into a clear paste with a relative density of 1.10-1.15 (60°C). The mixture is spray-dried to obtain a dry paste powder, and maltodextrin is added at a ratio of dry paste powder: excipients = 1.1:1, and dry granulation is performed to obtain the product.

[0040] Ephedra single-ingredient negative-yin prescription: Based on the formula composition of Qingfei Paidu Granules, remove the ephedra ingredient and take the remaining 20 ingredients, including gypsum and roasted licorice, and prepare according to the above preparation method. The other 18 single-ingredient negative-yin prescriptions, including roasted licorice, apricot kernel, cinnamon twig, oriental rhizome, polyporus, atractylodes, poria, bupleurum, scutellaria, pinellia, ginger, aster, winter flower, belamcanda, asarum, immature bitter orange, tangerine peel, and patchouli, are prepared using the same method as the ephedra single-ingredient negative-yin prescription (when there is no corresponding asarum or gypsum ingredient, the separate process for that ingredient can be omitted).

[0041] 2. Methods

[0042] 2.1 Chromatographic Conditions: Chromatographic column: ACQUITY UPLC BEH C18 column (2.1×100 mm, 1.7 μm, Waters Corporation, USA); mobile phase: acetonitrile (A)-0.1% formic acid solution (B); elution: 0–8 min, 2–5% A; 8–11 min, 5–13% A; 11–24 min, 13–20% A; 24–37 min, 20–42% A; 37–38 min, 42–90% A; 38–43 min, 90% A; flow rate: 0.4 mL / min; injection volume: 1 μL; column temperature: 30°C; detection wavelengths: 254 nm and 210 nm.

[0043] 2.2 Mass spectrometry conditions Ion source: ESI ion source; positive ion mode, negative ion mode; curtain gas: 35 psi; Gas 1: 55 psi; Gas 2: 55 psi; temperature: 350 °C; ionization pressure: 5500 V (positive), -5500 V (negative); declustering voltage: 75 V; full scan range: m / z 50-1700; MS / MS scan range: m / z 50-1700; fragmentation voltage: 40 V (positive, negative).

[0044] 2.3 Solution preparation

[0045] 2.3.1 Preparation of Reference Solution Take appropriate amounts of ephedrine hydrochloride reference substance, pseudoephedrine hydrochloride, baicalin, and ammonium glycyrrhizate reference substance, accurately weigh them, and add methanol to prepare a mixed solution containing 40 μg of each per 1 mL.

[0046] 2.3.2 Preparation of test solution: Take about 0.1 g of Qingfei Paidu Granules, weigh accurately, place in a stoppered conical flask, add 25 mL of 75% methanol accurately, weigh the weight, and ultrasonically treat (power 400 W, frequency 40 kHz) for 30 minutes. Let cool, weigh the mass again, make up the lost weight with 75% methanol, shake well, filter, and take the filtrate; use the same method to prepare a single-herb negative control solution.

[0047] 3. Results and Analysis

[0048] 3.1 Peak identification

[0049] The reference substance prepared according to the method "2.3.1" and the Qingfei Paidu Granule test solution prepared according to the method "2.3.2" were determined according to the chromatography method "2.1" and the mass spectrometry method "2.2", and the compound information was recorded. According to the retention time, precise molecular mass, fragment ion and other information of the compounds, combined with the literature ([1]Shen A,Zhou W,Xiong L,Jin H,Yu L,Wu H,Yu W,Yu D,Guo Z,Liu Y,Liang X.Chemical profiling of Qingfei Paidu Decoction bytriplex off-line two-dimensional liquid chromatography coupled withquadrupole time-of-flight mass spectrometry.J Sep Sci.2022Jan 9.doi:10.1002 / jssc.202100587.[2]Xu F,Hou T,Shen A,Jin H,Xiao Y,Yu W,Li X,Wang J,Liu Y,Liang X.Mechanism deconvolution of Qing Fei Pai Du decoction for treatment ofCoronavirus Disease 2019(COVID-19)by label-free integrative pharmacologyassays.J Ethnopharmacol. 2021 Nov 15; 280: 114488. doi: 10.1016 / j.jep.2021.114488.) were used for compound elucidation. The results are shown in Figure 1 , Table 1.

[0050] 3.2 Specificity Identification

[0051] Qingfei Paidu Granules, batch number QFP200701, were used to prepare Qingfei Paidu Granule test solutions according to method 2.3.2. Nineteen single-ingredient negative prescriptions were also prepared according to method 2.3.2. These solutions were then analyzed using the chromatographic method in 2.1 and the mass spectrometry method in 2.2. For Ephedra sinica, the primary mass spectrum of the Qingfei Paidu Granule test solution was extracted using the primary precursor ion m / z 166.1222 for ephedrine and pseudoephedrine described in 3.1 Peak Identification. Peak areas for ephedrine and pseudoephedrine were recorded as 47,281,381 and 37,825,118, respectively. Fragment ion information for ephedrine and pseudoephedrine was then extracted, and the results were consistent with the fragment ions in Table 1, confirming the presence of ephedrine and pseudoephedrine in Qingfei Paidu Granules. The primary parent ion m / z 166.1222 of ephedrine and pseudoephedrine in "3.1 Peak Identification" was used to extract the primary mass spectrum of the test solution of the single-flavor negative prescription of ephedra. The peak areas of ephedrine and pseudoephedrine were both recorded as 0. The negative interference was calculated (negative interference = the ratio of the mass spectrum peak area of ​​the target component in the single-flavor negative prescription to the mass spectrum peak area of ​​the target component in the Qingfei Paidu Granules). The results showed that the negative interference was less than 5%. Ephedrine (peak 3) and pseudoephedrine (peak 4) were used as the specific components of ephedra, and the specific identification of ephedra in Qingfei Paidu Granules was achieved. It was shown that the presence of both ephedrine (peak 3) and pseudoephedrine (peak 4) can be used to identify ephedra in Qingfei Paidu Granules. The results are shown in Figure 3. Figure 2By analogy, the specific components of licorice root were determined to be liquiritin (peak 10), apiosylliquiritin (peak 11), and glycyrrhizic acid (peak 33); the specific components of almond were amygdalin (peak 8) and prunin (peak 9); the specific components of cassia twig were protocatechuic aldehyde / parahydroxybenzoic acid (peak 2); the specific components of oriental rhizome were alisol A (peak 28); the specific components of polio were polioside A (peak 6); the specific components of atractylodes were atractylodesin A (peak 24); and the specific components of poria were schizonepeta tenuifolia. The specific component is tuckahoe acid A (peak 16), the specific component of bupleurum is saikosaponin A / B2 (peak 36), the specific components of scutellaria are baicalin (peak 14), baicalin (peak 23), wogonin (peak 27), baicalein (peak 31), scutellaria flavonoid II (peak 35), the specific component of pinellia is sericin (peak 37), the specific component of ginger is 4-shogaol (peak 34), and the specific component of aster is asterin A / asterinin The specific components of coltsfoot flower were chlorogenic acid (peak 5), cryptochlorogenic acid (peak 7), kelpine (peak 12), and isochlorogenic acid C (peak 21); the specific components of Belamcanda chinensis were belamcandaside (peak 13), iridin (peak 19), irisin (peak 30), and irisin (peak 32); the specific component of Asarum was 3,4-dimethoxycinnamyl alcohol (peak 29); the specific component of Citrus aurantium was citric acid (peak 26); the specific components of Citrus reticulatae were synephrine (peak 1), naringin (peak 17), hesperidin (peak 20), and neohesperidin (peak 22); the specific component of Patchouli was bigninoside II (peak 25). The specific identification of 19 medicinal flavors in Qingfei Paidu Granules was achieved with negative interference less than 5%. The method had strong specificity, indicating that the 37 chemical components can be used for the identification of the 19 medicinal flavors in Qingfei Paidu Granules.

[0052] Table 1 Specific identification of 19 medicinal flavors

[0053]

[0054]

[0055]

[0056]

[0057] *Confirmed with reference material

[0058] Example 2: Determination of the contents of 4 index components

[0059] 1. Instruments and reagents: Same as those in Example 1 "1"

[0060] 2. Methods

[0061] 2.1. Chromatographic conditions: Same as those in "2.1" of Example 1

[0062] 2.2.Solution conditions

[0063] 2.2.1. Preparation of reference solution: same as the preparation of reference solution in "2.3.1" of Example 1

[0064] 2.2.2. Preparation of test solution: Same as the preparation of test solution in "2.3.2" in Example 1

[0065] 3. Methodological Investigation

[0066] 3.1 Linear Relationship

[0067] The concentrations of ephedrine, pseudoephedrine hydrochloride, baicalin and glycyrrhizic acid were 2.2080, 2.0080, 11.3266 and 1.9394 μg / mL; 4.4160, 4.0160, 22.6532 and 3.8788 μg / mL; 8.8320, 8.0319, 45.3064 and 7.7576 μg / mL; 13.248 0, 12.0479, 67.9596, 11.6364 μg / mL; 17.6640, 16.0638, 90.6129, 15.5151 μg / mL; 22.0800, 20.0798, 113.2661, 19.3939 μg / mL of a series of mixed reference solutions were measured by high performance liquid chromatography and the peak areas were recorded. A standard curve was drawn with concentration (μg / mL) as the abscissa and peak area (mAU) as the ordinate. The results are shown in Table 2. The R 2 The peak areas were all greater than 0.999, and the peak areas were linearly correlated with the concentrations.

[0068] Table 2 Linear relationships and linear equations of the four indicator components

[0069]

[0070] 3.2 Precision test

[0071] Qingfei Paidu Granules (batch number QFP200701) were used to prepare the test solution according to method 2.2.2. Six consecutive injections were made. The retention time and peak area RSDs of ephedrine hydrochloride, pseudoephedrine hydrochloride, baicalin, and glycyrrhizic acid were calculated. The retention time RSDs were 0.12%, 0.22%, 0.19%, and 0.32%, respectively, and the peak area RSDs were 0.67%, 0.82%, 0.41%, and 0.72%, respectively. The results showed that the retention time and peak area RSDs of the four index components were all less than 1%, indicating good instrument precision.

[0072] 3.3 Stability test

[0073] Qingfei Paidu Granules (batch number QFP200701) were used to prepare test solutions according to method 2.2.2. The solutions were incubated at room temperature for 0, 6, 12, 18, and 24 hours before injection into a liquid chromatograph. The RSDs of the peak areas for ephedrine hydrochloride, pseudoephedrine hydrochloride, baicalin, and glycyrrhizic acid were calculated to be 0.45%, 0.68%, 0.23%, and 0.72%, respectively. The results showed that the RSDs of the peak areas for all four components were less than 1%, indicating that the test solutions were stable for 24 hours at room temperature.

[0074] 3.4 Repeatability test

[0075] Six parallel aliquots of Qingfei Paidu Granules (batch number QFP200701) were prepared according to the method described in section 2.2.2. The aliquots were injected into a liquid chromatograph and the RSDs for ephedrine hydrochloride, pseudoephedrine hydrochloride, baicalin, and glycyrrhizic acid were calculated to be 0.61%, 0.32%, 0.59%, and 0.88%, respectively. The results showed that the RSDs for the four index components were all less than 1%, demonstrating good reproducibility of the method.

[0076] 3.5 Sample recovery test

[0077] Take the Qingfei Paidu Granules with batch number QFP200701, accurately add known amounts of three reference substance mixed solutions at 50%, 100%, and 150% mass concentration levels of ephedrine hydrochloride (3220.57μg per gram of sample), pseudoephedrine hydrochloride (1718.56μg per gram of sample), baicalin (15303.20μg per gram of sample), and glycyrrhizic acid (2231.65μg per gram of sample), and prepare the test solution according to "2.2.2". Nine test sample solutions, each containing three samples at low, medium, and high concentrations, were prepared and analyzed separately. The recoveries of the four quantitative parameters were calculated (% recovery = (measured amount - content in sample) / amount of reference substance added * 100%). The average recoveries of ephedrine hydrochloride, pseudoephedrine hydrochloride, baicalin, and glycyrrhizic acid were 96.32%, 95.15%, 98.15%, and 94.74%, respectively, with RSDs of 1.78%, 1.45%, 1.27%, and 1.52%, respectively. The results showed that the recoveries of the four quantitative parameters were all within 90-108%, with RSDs less than 5%, indicating that the method has high accuracy.

[0078] 3.6 Calculation of correction factors

[0079] The reference sample mixed solution prepared according to the method of "2.2.1" was injected 5 times continuously and the correction factor was calculated. ) are shown in Table 3. The results show that the RSD values ​​of the correction factors of ephedrine hydrochloride, pseudoephedrine hydrochloride, baicalin and glycyrrhizic acid are all less than 2%, which meets the requirements.

[0080] Table 3 Correction factors for ephedrine hydrochloride, pseudoephedrine hydrochloride, baicalin, and glycyrrhizic acid

[0081]

[0082]

[0083] 4. Sample Measurement

[0084] Take 10 batches of Qingfei Paidu Granules and prepare the test solution according to the method of "2.2.2". Inject the sample for analysis and use the external standard method ( Among them A x Represents the peak area of ​​the component to be measured in the sample, Rf represents the correction factor, and m represents the sample weight) (the average correction factor of ephedrine hydrochloride is 21281.10, the average correction factor of pseudoephedrine hydrochloride is 21313.61, the average correction factor of baicalin is 2088.01, and the average correction factor of glycyrrhizic acid is 925.64) The mass contents of the four index components, including ephedrine hydrochloride, pseudoephedrine hydrochloride, baicalin, and glycyrrhizic acid, in Qingfei Paidu Granules were calculated. The results are shown in Figure 3 Table 4. Qingfei Paidu Granules should contain 0.24%-0.45% ephedrine hydrochloride, 0.13%-0.23% pseudoephedrine hydrochloride, 1.09%-2.02% baicalin, and 0.14%-0.26% glycyrrhizic acid to ensure uniform and stable contents of the active ingredients and main components.

[0085] Table 4 Determination of index component content

[0086]

[0087]

[0088] Example 3: Inspection of the toxic component aristolochic acid I

[0089] 1. Instruments and test drugs

[0090] Same as the instrument and reagent of Example 1 "1"

[0091] 2. Methods

[0092] 2.1 Chromatographic conditions: Same as those in "2.1" of Example 1

[0093] 2.2 Mass spectrometry conditions: Same as those in "2.2" of Example 1

[0094] 2.3 Solution conditions

[0095] 2.3.1 Preparation of reference solution: Take an appropriate amount of aristolochic acid I reference substance, weigh it accurately, and add methanol to make a reference solution containing 0.4 μg of aristolochic acid I per 1 mL.

[0096] 2.3.2 Preparation of test solution: Same as the preparation of test solution in "2.3.2" in Example 1 3. Detection limit

[0097] Take the aristolochic acid I reference solution and dilute it 10 times with methanol solution to obtain aristolochic acid I dilution solution (a); then take the aristolochic acid I dilution solution (a) and dilute it 10 times with methanol solution to obtain aristolochic acid I dilution solution (b); determine the aristolochic acid I dilution solutions (a) and (b) according to the chromatographic conditions of "2.1" and the mass spectrometry conditions of "2.2", and take the signal-to-noise ratio (S / N, where S is the signal measured from the aristolochic acid I reference solution of known concentration and N is the signal measured from the methanol or acetonitrile solution) equal to 3 as the detection limit. The determination results are 4.97 ng / mL, respectively, and the concentrations of the reference solutions for the limit test are determined.

[0098] 4. Limit inspection of aristolochic acid I

[0099] Take Qingfei Paidu Granules with batch number QFP200701 and prepare the test solution according to the method in "2.3.2". Determine according to the chromatographic conditions in "2.1" and the mass spectrometry conditions in "2.2". Use the electrospray ionization source (ESI) in positive ion mode to extract and compare the Qingfei Paidu Granules test solution with the aristolochic acid I reference solution with a concentration of 4.97 ng / mL at a signal-to-noise ratio of 3 [M+NH4] + The results showed that the mass spectrum peak area of ​​the primary ion map of the Qingfei Paidu granules test sample was 0 at the position corresponding to the retention time of the primary ion map of the aristolochic acid I reference substance, indicating that aristolochic acid I was not detected in the Qingfei Paidu granules. This method has high sensitivity and can be used for the limit test of aristolochic acid I. The results are shown in Figure 4 , Table 5.

[0100] Table 5 Aristolochic acid I limit inspection

[0101]

[0102] Example 4: Establishment of fingerprint

[0103] 1. Instruments and reagents: Same as those in Example 1 "1"

[0104] 2. Methods

[0105] 2.1 Chromatographic conditions: Same as those in "2.1" of Example 1

[0106] 2.2 Solution conditions

[0107] 2.2.1 Preparation of reference solution: same as the preparation of reference solution in "2.3.1" in Example 1

[0108] 2.2.2 Preparation of test solution: the same as the preparation of test solution in "2.3.2" in Example 1

[0109] 3. Methodological Investigation

[0110] 3.1 Precision test

[0111] Prepare the test solution using Qingfei Paidu Granules (batch number QFP200701) according to method 2.2.2. Six consecutive injections were made, and the fingerprint was recorded. The relative retention time RSD (relative standard deviation) of each characteristic peak was less than 1%, and the relative peak area RSD of the 10 common peaks (identified in "4") was less than 5%, indicating good instrument precision.

[0112] 3.2 Stability test

[0113] Qingfei Paidu Granules (batch number QFP200701) were used to prepare test solutions according to the method in section 2.2.2. The solutions were incubated at room temperature for 0, 6, 12, 18, and 24 hours before injection into a liquid chromatograph and the fingerprints were recorded. The relative retention time RSDs of each characteristic peak were less than 1%, and the relative peak area RSDs of the ten common peaks were less than 5%, indicating good stability of the test solution within 24 hours.

[0114] 3.3 Repeatability test

[0115] Six test solutions of Qingfei Paidu Granules (batch number QFP200701) were prepared according to the method described in section 2.2.2. The samples were injected and their fingerprints recorded. The relative retention time RSDs of each characteristic peak were less than 1%, and the relative peak area RSDs of the ten common peaks were less than 5%, demonstrating good reproducibility of the method.

[0116] 4. Establishment of fingerprint

[0117] Take 10 batches of Qingfei Paidu Granules, prepare the test solution according to the method of "2.2.2", inject the sample for analysis, record the chromatogram, and obtain the UPLC fingerprint of Qingfei Paidu Granules ( Figure 5 ), all of them were imported into the "Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System Software" (2012 version), with S1 as the reference spectrum, the median was used for automatic matching and multi-point correction, and combined with the results of "3.1" of Example 1, 10 common peaks (chromatographic peaks with a peak area of ​​more than 5%) were determined to obtain the control spectrum (R) of Qingfei Paidu Granules, as shown in FIG. Figure 5 As shown. Using the Qingfei Paidu Granule control spectrum (R) as a reference, the similarities between the 10 batches of Qingfei Paidu Granules and the control spectrum were calculated, and all were greater than 0.95, achieving overall quality control of the Qingfei Paidu Granule fingerprint. The results are shown in Table 6. Using Peak 7 as a reference peak, the relative retention time of the control fingerprint was calculated. The results are shown in Table 7.

[0118] Table 6 Fingerprint similarity

[0119]

[0120] Table 7 Relative retention time of the control fingerprint

[0121]

[0122] Example 5: Application of high-throughput mass detection method for Qingfei Paidu granules

[0123] 1. Instruments and reagents: Same as those in Example 1 "1"

[0124] 2. Methods

[0125] 2.1 Chromatographic conditions: Same as those in "2.1" of Example 1

[0126] 2.2 Mass spectrometry conditions: Same as those in "2.2" of Example 1

[0127] 2.3 Solution conditions

[0128] 2.3.1 Preparation of Reference Solution: Accurately weigh appropriate amounts of ephedrine hydrochloride, pseudoephedrine hydrochloride, baicalin, and ammonium glycyrrhizate, and add methanol to prepare a mixed solution containing 40 μg of each per mL. Separately, accurately weigh appropriate amounts of aristolochic acid I, and add methanol to prepare a reference solution containing 4.97 ng of aristolochic acid I per mL.

[0129] 2.3.2 Preparation of test solution: the same as the preparation of test solution in "2.3.2" of Example 1.

[0130] 3. Application of high-throughput mass detection method for Qingfei Paidu granules

[0131] 3.1 Application of Identification

[0132] A sample of Qingfei Paidu Granules with batch number QFP200711 was taken, and the test solution was prepared according to the "2.3.2" method. The solution was determined according to the chromatography method "2.1" and the mass spectrometry method "2.2". With reference to the basic information of chemical components in Table 2 in Example 1, the primary parent ion m / z of the chemical components was extracted. Combined with the retention time and secondary fragment ion information, 19 medicinal flavors in the Qingfei Paidu Granules sample were identified, and the results met the requirements.

[0133] 3.2 Application of content determination

[0134] Take the Qingfei Paidu Granule sample with batch number QFP200711 and prepare the test solution according to the method in "2.3.2". Take ephedrine, pseudoephedrine hydrochloride, baicalin and glycyrrhizic acid and prepare the mixed reference solution according to the method in "2.3.1". Determine the corresponding peak area according to the chromatographic method in "2.1". Use the external standard method ( Among them A x The contents of ephedrine, pseudoephedrine hydrochloride, baicalin, and glycyrrhizic acid in the test sample were calculated using the formula (where Rf represents the peak area of ​​the analyte in the sample, Rf represents the correction factor, and m represents the sample weight) (the average correction factor for ephedrine hydrochloride is 21281.10, the average correction factor for pseudoephedrine hydrochloride is 21313.61, the average correction factor for baicalin is 2088.01, and the average correction factor for glycyrrhizic acid is 925.64). The results showed that the Qingfei Paidu Granules sample contained 0.35% ephedrine, 0.17% pseudoephedrine hydrochloride, 1.50% baicalin, and 0.21% glycyrrhizic acid, all meeting the requirements.

[0135] 3.3 Application of Inspection

[0136] Take the Qingfei Paidu Granule sample with batch number QFP200711 and prepare the test solution according to the method in "2.3.2". Take the aristolochic acid I reference substance and prepare the reference solution according to the method in "2.3.1". Determine according to the chromatography method in "2.1" and the mass spectrometry method in "2.2". Use the electrospray ionization source (ESI) in the positive ion mode to extract and compare the Qingfei Paidu Granule test solution and the aristolochic acid I reference substance solution with a concentration of 4.97 ng / mL [M+NH4] + The results of the primary ion map showed that no aristolochic acid I was detected in the Qingfei Paidu granules, and the results met the requirements.

[0137] 3.4 Application of Fingerprint

[0138] A sample of Qingfei Paidu Granules, batch number QFP200711, was prepared using the method in section 2.3.2. Chromatographic analysis was performed using the chromatographic method in section 2.1, with the chromatographic peak information recorded and imported into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System. The fingerprint of the test solution exhibited 10 characteristic peaks, corresponding to the retention times of the 10 chromatographic peaks in the control fingerprint in Example 4. Similarity matching was performed between the fingerprints of the test and control using these 10 shared peaks. The results showed a similarity of 0.997 between the fingerprints of the test and control, exceeding 0.95 and meeting the requirements.

[0139] The comparison between the technical solution provided by the present invention and the prior art is as follows:

[0140] Exclusivity Identify the number of medicinal flavors Fingerprint Contains tested ingredients Articles / Patents Difference 10 110 minutes 2 The present invention powerful 19 40min 5

[0141] The present invention establishes a high-throughput quality detection method for Qingfei Paidu Granules. The method has strong specificity and can realize the specific identification of 19 medicinal ingredients of Qingfei Paidu Granules, limit inspection of the toxic component aristolochic acid I, fingerprint analysis and content determination of four index components through a single analysis. It can quickly, comprehensively and accurately evaluate the quality of Qingfei Paidu Granules, and provide technical support for the research and development of new drugs for Qingfei Paidu Granules.

[0142] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Therefore, any equivalent changes or modifications made in accordance with the scope of the patent of the present invention shall fall within the scope of the patent claims of the present invention.

Claims

1. A high-throughput mass detection method for Qingfei Paidu granules, characterized in that: The detection method comprises the following steps: a) Preparation of reference solution: Separately prepare a reference solution containing ephedrine hydrochloride, pseudoephedrine hydrochloride, baicalin, and glycyrrhizic acid by adding methanol or acetonitrile solvent; b) Preparation of test solution: Add 50-100% methanol or 50-100% ethanol to Qingfei Paidu Granules at a solid-liquid ratio of 1:10-1:300 g / mL. Reflux or sonicate for 15-60 min. Cool to room temperature, add solvent to make up the loss, shake well, filter, and collect the filtrate to obtain the Qingfei Paidu Granule test solution. c) High Performance Liquid Chromatography-Tandem Mass Spectrometry: Accurately pipette the reference solution prepared in step a) and the test solution prepared in step b) into a high performance liquid chromatography-tandem mass spectrometer, and record the chromatogram and mass spectrum. The chromatographic column packing material is octadecylsilane bonded silica gel, the column length is 50-100 mm, the diameter is 2.1 mm, and the particle size is 1.7 μm. The mobile phase A is acetonitrile and the mobile phase B is an aqueous solution of formic acid containing an additive at a volume concentration of 0.1-0.5%. Gradient elution is used: 0-8 min, 2-5% A; 8-11 min, 5-13% A; 11-24 min, 13-20% A; 24-37 min, 20-42% A; 37-38 min, 42-90% A; 38-43 min, 90% A; the flow rate is 0.2 mL / min-0.5 mL / min, and the injection volume is 0.5-2 μL, the detection wavelength was 210-330 nm; the ionization modes used for mass spectrometry were electrospray positive ionization and negative ion ionization, curtain gas: 25-45 psi; Gas 1: 30-70 psi; Gas 2: 30-70 psi; temperature: 300-500 °C; ionization pressure: 4500-5500 V, -4500--5500 V; declustering voltage: 60-80 V; full scan range: m / z 50-1700; MS / MS scan range: m / z 50-1700; fragmentation voltage: 30-40 V; d) Identification: 19 single-ingredient negative prescription samples and Qingfei Paidu Granule samples were subjected to the method in step b) to prepare test solutions of the 19 single-ingredient negative prescriptions and Qingfei Paidu Granules. The compounds were assayed according to step c), and their compound information was recorded. This information was used to identify the compounds in Qingfei Paidu Granules. The primary precursor ions of the compounds were then used to extract the primary mass spectra of Qingfei Paidu Granules and the 19 single-ingredient negative prescriptions. The mass spectral peak areas of the compounds were recorded, and negative interferences were calculated. Compounds with negative interferences of less than 5% were considered specific components of the medicinal flavors. A method for identifying the 19 medicinal flavors in Qingfei Paidu Granules was established. e) Content determination: Prepare a mixed reference solution of ephedrine hydrochloride, pseudoephedrine hydrochloride, baicalin, and glycyrrhizic acid according to step a); prepare more than three batches of Qingfei Paidu Granule test solution according to step b); determine them in sequence according to step c); record the peak areas of ephedrine hydrochloride, pseudoephedrine hydrochloride, baicalin, and glycyrrhizic acid in the test solution and the reference solution; use the external standard method , where A x Indicates the peak area of ​​the component to be measured in the sample, Rf indicates the correction factor, m indicates the sample mass, , calculate the mass contents of ephedrine hydrochloride, pseudoephedrine hydrochloride, baicalin and glycyrrhizic acid in the Qingfei Paidu granules sample.

2. The detection method according to claim 1, wherein In step b), Qingfei Paidu Granules are taken and a methanol solvent with a volume concentration of 75-100% or an ethanol solvent with a volume concentration of 75-100% is added, with a material-liquid ratio of 1:100-1:250 g / mL. More than 10 batches of Qingfei Paidu Granule test solutions are prepared according to the method in step b). In step d), the compound with a negative interference of less than 1% is used as the specific component of the medicinal flavor.

3. The detection method according to claim 1, wherein Separately, an appropriate amount of aristolochic acid I reference substance is taken and accurately weighed. Methanol is added to prepare a reference substance solution containing 0.2-2 μg of aristolochic acid I per 1 mL, which is then diluted 50-200 times with methanol or acetonitrile solution. Separately, Qingfei Paidu Granules are taken and a test solution is prepared according to the method in step b). The test solution is determined according to step c), the primary ion map is recorded, and the signal-to-noise ratio S / N is calculated, where S is the signal measured from the aristolochic acid I reference substance solution of known concentration, and N is the signal measured from the methanol or acetonitrile solution. By comparing the primary ion maps of the aristolochic acid I reference substance solution and the Qingfei Paidu Granule test solution when the signal-to-noise ratio is 3-10, the mass spectrometric peak area of ​​the Qingfei Paidu Granule test solution at the position corresponding to the retention time of the aristolochic acid I reference substance solution should be 0. If it is greater than 0, it indicates that the toxic component aristolochic acid I is contained therein. A limit test method for the toxic component aristolochic acid I in Qingfei Paidu Granules is established.

4. The detection method according to claim 3, characterized in that Add methanol to prepare a reference solution containing 0.2-1 μg of aristolochic acid I per 1 mL, and dilute it 50-100 times with methanol or acetonitrile solution.

5. The detection method according to claim 1, wherein According to the method in step b), more than three batches of Qingfei Paidu Granule test solution were prepared, and the solutions were measured sequentially according to step c), and the chromatograms were recorded. All the chromatograms were imported into the "Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System Software (2012 Edition)" software of the Chinese Pharmacopoeia Commission, and the common peaks were matched, among which the chromatographic peaks with a peak area accounting for more than 5% were obtained to obtain the control fingerprint spectrum, and the fingerprint spectrum method of Qingfei Paidu Decoction was established.

6. The detection method according to claim 5, characterized in that Chromatographic peaks that account for 10% to 50% of the peak area.

7. The detection method according to claim 1, characterized in that In the step a), appropriate amounts of ephedrine hydrochloride, pseudoephedrine hydrochloride, baicalin, and glycyrrhizic acid reference substances are taken and accurately weighed, and methanol is added to prepare a mixed reference substance solution containing 20-100 μg of ephedrine hydrochloride, 20-100 μg of pseudoephedrine hydrochloride, 20-100 μg of baicalin, and 20-100 μg of glycyrrhizic acid per 1 mL.

8. The detection method according to claim 1, wherein Add methanol to prepare a mixed reference solution containing 20-50 μg of ephedrine hydrochloride, 20-50 μg of pseudoephedrine hydrochloride, 40-80 μg of baicalin, and 20-50 μg of glycyrrhizic acid per 1 mL.

9. The detection method according to claim 1, wherein In the step d), 37 chemical components in the Qingfei Paidu granules were identified, among which the ephedra-specific components were ephedrine and pseudoephedrine, the licorice-specific components were liquiritin, apigenin, and glycyrrhizic acid, the apricot-specific components were amygdalin and prunin, the cassia twig-specific components were protocatechuic aldehyde / parahydroxybenzoic acid, the oriental-specific component was alismatol A, the polyporus-specific component was polyporus ketone A, the Atractylodes-specific component was atractylodesin A, the Poria-specific component was tuckahoe acid A, the bupleurum-specific component was saikosaponin A / B2, the scutellaria-specific components were baicalin, baicalin, wogonin, baicalein, and scutellaria flavonoid II, the ginger-specific component was sericinine, the ginger-specific component was 4-shogaol, and the aster-specific component was asterin A / asterinin. A. The specific components of coltsfoot flower are chlorogenic acid, cryptochlorogenic acid, kelp-like alkaloids, and isochlorogenic acid C; the specific components of belamcanda ternata are belamcandaside, irisin, irisin, and irisin; the specific component of asarum is 3,4-dimethoxycinnamyl alcohol; the specific component of immature bitter orange is citric acid; the specific components of dried tangerine peel are synephrine, naringin, hesperidin, and neohesperidin; the specific component of patchouli is bigninoside II. The specific qualitative identification of 19 medicinal flavors in Qingfei Paidu Granules can be achieved by determining whether they contain mass spectrometry peaks of specific components.

10. The detection method according to claim 1, characterized in that In the step e), the mass content of ephedrine hydrochloride in the Qingfei Paidu granules should be 0.24% to 0.45%, the mass content of pseudoephedrine hydrochloride should be 0.13% to 0.23%, the mass content of baicalin should be 1.09% to 2.02%, and the mass content of glycyrrhizic acid should be 0.14% to 0.26%, ensuring that the content of the active ingredient and the main component in the Qingfei Paidu granules is uniform and stable.

11. The detection method according to claim 3, characterized in that: When the signal-to-noise ratio is equal to 3-10, the concentration of the aristolochic acid I reference solution is 1-20 ng / mL, and the mass spectrum peak area of ​​the test sample's primary ion map at the position corresponding to the retention time of the aristolochic acid I reference primary ion map should be 0, ensuring that the Qingfei Paidu Granules do not contain the toxic component aristolochic acid I.

12. The detection method according to claim 11, characterized in that When the signal-to-noise ratio is 3-10, the concentration of aristolochic acid I reference solution is 1-10 ng / mL.

13. The detection method according to claim 5, characterized in that The similarity calculation was performed based on 10 common peaks whose peak area accounted for more than 5%. The similarity between the test sample chromatogram and the control chromatogram was not less than 0.95, thus achieving the overall quality control of the fingerprint spectrum of Qingfei Paidu Granules.

14. The detection method according to claim 13, characterized in that The similarity was calculated based on 10 common peaks with peak area accounting for 10% to 50%.

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